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In Vivo Response-Based Identification of Direct Hormone Target Cell Populations Using High-Density Tissue Arrays

Kimmel Cancer Center, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Address all correspondence and requests for reprints to: Matthew LeBaron, Ph.D., Thomas Jefferson University, Kimmel Cancer Center, Department of Cancer Biology, 233 South 10th Street, 330 BLSB, Philadelphia, Pennsylvania 19107-5541. E-mail: mlebaron{at}kimmelcancercenter.org.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion Circulatory system Musculoskeletal system Nervous system Urinary system Respiratory system Connective tissue Conclusion References

 
To identify cell populations directly responsive to prolactin (PRL), GH, erythropoietin, or granulocyte-colony stimulating factor within the physiological setting of an intact mammal, we combined in situ detection of hormone-activated signal transducer and activator of transcription (Stat)-5 in rats with high-throughput tissue array analysis using cutting-edge matrix assembly (CEMA). Inducible activation of Stat5a/b, as judged by levels of nuclear-localized, phosphoTyr694/699-Stat5a/b, served as an immediate and sensitive in situ marker of receptor signaling in rat tissues after injection into male and female rats of a single, receptor-saturating dose of hormone for maximal receptor activation. CEMA tissue arrays facilitated analysis of most tissues, including architecturally complex, thin-walled, and stratified tissues such as gut and skin. In 40 tissues analyzed, 35 PRL-responsive and 32 GH-responsive cell types were detected, of which 22 cell types were responsive to both hormones. Interestingly, PRL but not GH activated Stat5 in nearly all of the endocrine glands. In mammary glands, PRL activated Stat5 in a majority of luminal epithelial cells but not myoepithelial cells, stromal fibroblasts, or adipocytes, whereas GH activated Stat5 in a significant fraction of myoepithelial cells, fibroblasts, and adipocytes but only in a minority of luminal cells. Finally, the organism-wide screening revealed a yet-to-be identified erythropoietin-responsive cell type in connective tissue. CEMA tissue arrays provide cost-effective in situ analysis of large numbers of tissues. Biomarker-based identification of cell populations responsive to individual hormones may shed new light on endocrine disease as well as improve understanding of effects and side effects of hormones and drugs.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion Circulatory system Musculoskeletal system Nervous system Urinary system Respiratory system Connective tissue Conclusion References

 
IDENTIFICATION OF HORMONE- and cytokine-responsive cell populations in the physiological context of the intact animal is needed for a comprehensive understanding of the physiological roles of individual hormones. Identification of hormone target cells also may lead to new insight into the roles of hormones in disease processes. For instance, mapping of prolactin (PRL)- and GH-responsive cells in experimental animals may help explain the many symptoms and manifestations of the endocrine overproduction syndromes, hyperprolactinemia and acromegaly. Likewise, improved mapping of hormonal target cells may be useful for anticipating or elucidating therapy-related side effects of GH, erythropoietin (EPO), or granulocyte-colony stimulating factor (GCSF), hormones that are currently widely administered to humans in clinical practice or taken for athletic performance enhancement or antiaging purposes.

Key insight into the main functions of polypeptide hormones can be gleaned from gene knockout studies of the individual hormones or their receptors in mice. Such genetic models, however, are limited to providing information about gene functions that are critical and therefore cannot be compensated for during development by redundant mechanisms. Furthermore, it is difficult to distinguish between direct and indirect effects of gene loss on tissue and cell function. For instance, infertility of female PRL receptor (PRLR)-null mice is caused by blastocyst implantation failure that is secondary to ovarian progesterone deficiency (1, 2). Likewise, in chronic hormonal overexpression or hyperstimulation models, biological effects also may be direct or indirect. Alternatively, ligand binding assays and mRNA or protein expression studies of receptors have been used to identify candidate hormone target cells at the organ, tissue, or cell level. Complicating these analyses is the existence of multiple isoforms or splice variants of transmembrane receptors that differ in signaling competence or may even be dominant-negative variants. For instance, truncated splice forms of PRLRs have been shown to act as dominant-negative suppressors of at least some functions of the full-length receptor (3) and are expressed at high levels in certain organs such as liver (4). Other factors, including the absence of coreceptors or presence of inhibitors, also may affect cellular responsiveness. Finally, gene transcript levels may not reflect protein expression levels (5). Therefore, the existence of receptors in a given tissue, either at the protein or transcript level, does not prove signaling competence.

To identify direct target cells responsive to PRL, GH, EPO, and GCSF within the physiological setting of the whole animal, we combined in situ detection of rapid receptor-mediated signal transducer and activator of transcription (Stat)-5a/b tyrosine-phosphorylation with high-throughput tissue array analysis using cutting-edge matrix assembly (CEMA) (6, 7). Because these hormones have the capacity to activate both or either of the highly homologous Stat5a and Stat5b gene products (8) and the phospho-Stat5a/b(Y694/699) antibody used does not discriminate between phosphorylated Stat5a and Stat5b (9), the term Stat5 in this text refers to both or either protein. Inducible tyrosine phosphorylation of Stat5 is an immediate, sensitive, and common marker of signaling by receptors for these hormones. CEMA allows arraying of most tissue types, including architecturally complex, thin-walled, and stratified tissues such as gut and skin. Furthermore, analysis of tissue arrays is cost effective, less labor intensive, and limits interassay and interslide variability in sample staining and analysis (10). The experimental protocol involved in situ analysis of nuclear-localized, tyrosine-phosphorylated Stat5 in formalin-fixed rat tissues harvested 30 min after injection of a single, receptor-saturating dose of hormone for maximal receptor activation.

The data provide a high-resolution map of PRL, GH, EPO, and GCSF-responsive cell types in male and female rat tissues based on rapid Stat5 activation as the readout. In general, the map revealed new insight into the broad arrays of target cells for PRL and GH and the more restricted target cell populations of EPO and GCSF. In addition to providing new information on hormone-responsive target cells, the present work also provides proof of concept of in situ biomarker-based mapping of cell types responsive to hormones or drugs. In vivo response-based detection of receptor-mediated signaling at single cell resolution is expected to facilitate understanding of effects and side effects of drugs and hormones.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion Circulatory system Musculoskeletal system Nervous system Urinary system Respiratory system Connective tissue Conclusion References

 
Animals and tissue preparation
Adult Sprague Dawley rats (Taconic, Hudson, NY) approximately 9 wk of age were used for the study, with the males weighing approximately 300 g and the females 225 g. Animals were housed and used in accordance with institutional guidelines for animal care. Before organ harvest the animals were injected ip with vehicle, ovine (o) PRL [4 µg/g body weight (bw)], oGH (4 µg/g bw), oPRL + oGH (4 µg/g bw each hormone), human GCSF (1 µg/g bw), or human EPO (10,000 U/rat). Previous publications used similar doses of PRL and GH to analyze activated Stat5 in vivo (6, 9, 11, 12). Highly purified, pituitary oPRL (lot no. AFP9220A) and oGH (lot no. AFP10692C) were provided by Dr. A. F. Parlow (under sponsorship of the National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program, Torrance, CA), whereas recombinant human EPO (Epogen; Amgen Inc., Thousand Oaks, CA) and recombinant human GCSF (Neupogen; Amgen) were purchased commercially. We did not measure circulating levels of injected hormones in the rats at time the animals were killed. Using the standard formula for calculation of blood volume for the rat (75 ml/kg bw), we estimated the theoretically maximal circulating concentrations after injection as approximately 2500 nM for oPRL and oGH, approximately 100 nM for EPO (444,444 mU/ml), and approximately 800 nM for GCSF. By using these supraphysiological doses (high nanomolar), our intent was to rapidly achieve receptor-saturating doses (low nanomolar) at peripheral cells and tissues to reach all potential target cells for the hormones.

Experiments were performed on both male and female rats, with four to six rats included in vehicle injected control groups to establish baseline levels of Stat5 activation, and at least two rats were included for each injection parameter for each gender. This relatively large number of vehicle-injected rats used to establish baseline Stat5 activation levels was especially critical in male rats due to the pulsatile nature of GH secretion (13). In the most GH-responsive tissues (i.e. liver and connective tissues), however, we did not detect basal Stat5 activation in control animals, indicating that Stat5 activation in the GH target tissues under baseline conditions did not reach detectable levels in the six males analyzed. To minimize tissue harvesting time, the injections were staggered so that only one rat was dissected at a time. Thirty minutes after injection, rats were killed, rapidly dissected by three investigators, and the respective tissues and organs collected and immersed in 10% phosphate-buffered formalin at 4 C within 10 min for 18 h and then transferred to 70% ethanol until paraffin infiltration and embedding. Preliminary testing included harvesting of tissues at 20, 30, and 40 min after injection of PRL and GH, and no difference was noted in Stat5 activation status; we therefore concluded that 30 min was representative of maximal activation of Stat5. The same 30-min time point was used for Stat5 activation by EPO and GCSF, which is also consistent with published in vitro studies (14, 15). Furthermore, in a previous study we plotted the dephosphorylation of Stat5 in rat liver over time and noted that phosphorylation was relatively unchanged within the first hour postmortem, indicating the stability of the phosphoepitope for immunohistochemistry (IHC) (6). Samples were sectioned and placed on standard microscope slides; alternate slides were stained with hematoxylin and eosin (H & E) for histological analysis or used for IHC.

CEMA tissue array construction
Where possible, tissues were constructed into CEMA tissue arrays, as previously described (6, 7). Tissues and organs needed to be of sufficient size to be used as plates in the construction process. Briefly, paraffin-embedded tissues were trimmed to the desired thickness (typically 250–300 µm) to form primary plates. In the case of thin-walled tissues such as skin or gut, the tissue was directly used as a primary plate, as previously described (6, 7). The primary plates were bonded into a primary stack and sectioned transversely at the appropriate thickness (typically 250–300 µm) to form a secondary plate. These secondary plates were then stacked and bonded into a secondary stack. This bonded tissue array block contained elements of each of the original samples and, when transversely sectioned to micrometer-thin array sections, allowed transfer to support slides for analysis.

IHC
Immunostaining using AX1 antiphospho-Stat5 antibody (Advantex BioReagents, Conroe, TX) detected activated, tyrosine-phosphorylated Stat5, as previously described (9). Briefly, tissues were deparaffinized and rehydrated in ethanol. For antigen retrieval, the samples were microwave treated in a pressure cooker in AR-10 antigen retrieval solution (Advantex BioReagents). Endogenous peroxidase activity was blocked by pretreating the slides with hydrogen peroxide and normal goat serum was added to decrease nonspecific binding. The antiphosphotyrosine Stat5 monoclonal antibody was diluted in BSA and PBS and added to the sample. The secondary antibody was a biotinylated goat-antimouse IgG followed by streptavidin-horseradish-peroxidase complex with the chromogen 3,3'-diaminobenzidine (DAB) used to visualize the antigen-antibody complexes. As a positive control, Stat5 activation status was normalized with lactating mouse mammary gland for each experiment, and all samples were lightly counterstained with Mayer hematoxylin.

IHC scoring
Three separate reviewers (M.J.L., M.T.N., and H.R.) independently scored the tissues using a standard scale: – (negative), + (low staining), 2+ (medium staining), or 3+ (high staining). Where applicable, the cell types or specific subsets of cells within a tissue were identified and scored. All staining stronger than the minimum criteria for 3+ staining was normalized to 3+. Reported scores are representative of the compiled results from the three reviewers on each of the rats used in each treatment. In cases of discrepancy, the samples were reanalyzed by all three reviewers simultaneously and a consensus was agreed on. Throughout this manuscript, the terms activated Stat5 and Stat5 activation refer to nuclear localized Stat5a/b phosphorylated on the conserved residue Stat5a/b-Tyr694/9 as detected by IHC (16). In this work, hormone-inducible Stat5 is used as a readout of hormone-induced receptor activation, and future work will need to determine effects of Stat5 activation in various target cells. There may be instances in which nuclear localized Stat5a/b phosphorylated on the conserved residue Stat5a/b-Tyr694/9 is not functionally active due to presence of inhibitory cofactors such as PIAS-3 (17).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion Circulatory system Musculoskeletal system Nervous system Urinary system Respiratory system Connective tissue Conclusion References

 
To provide a detailed map of cells directly responsive to PRL, GH, EPO, or GCSF in rat tissues, we used in situ activation of Stat5 as a rapid and sensitive response marker of cellular receptor activation. By analyzing the Stat5 activation status in situ after an injection of hormone, we were able to identify subpopulations of hormone-responsive cells within tissues and organs, thus providing organism-wide localization of direct target cells of these hormones. Collectively, this work provides a high resolution map of hormone-responsive cells in tissues throughout the entire rat body. Previous work had demonstrated that an ip injection is rapidly taken up (10 sec) into the blood via the portal circulation and within 5 min is completely equilibrated with blood circulation (18). For the present study, the dose of hormone administered was selected to result in acute and maximal receptor activation to facilitate detection of hormone-responsive cells. It should be noted that some of the responses detected after injection of the high-hormone doses used may not be observed at physiological hormone levels but possibly under pharmacological or pathological conditions. Previous in vivo studies used similar hormone dosage (see Materials and Methods), whereas others studied hormone-responsive Stat5 activation in the liver using approximately 100-fold lower hormone levels (19, 20). These considerations should be noted when interpreting the data. The 30-min hormone exposure period was selected based on preliminary data (see Materials and Methods).

A limitation of this study is that activation of receptors for PRL, GH, EPO, and GCSF is measured solely by Stat5 activation. Although each of these receptors is thought to signal primarily through the receptor-associated Janus kinase (Jak)-2 leading to Stat5 activation, alternative and/or additional signaling pathways may be activated. Therefore, a cell still may be responsive to a given hormone, even if no Stat5 activation is detected, and hence, a negative result does not rule out receptor signaling. Nonetheless, the use of an in situ, immunohistochemical approach provides the best possible readout of receptor signaling in the intact organism because it provides spatial information at the single cell level within tissues. Future approaches with the use of more sensitive, fluorescent-based detection (vs. DAB) may detect additional target cells. It will also be important to expand this work to include other biomarkers of receptor activation. For instance, EPO has been demonstrated to stimulate cardiac and vascular cells through a phospholipase C/protein kinase C pathway (21), but it is unclear whether this is Jak2 independent. Likewise, Jak2-independent signaling through Src tyrosine kinase Fyn (22) and MAPK (23, 24, 25) by PRLR has been proposed, and PRL and GH are capable of activating Stat transcription factors other than Stat5 (26, 27). Although rapid Stat5 activation by hormones in a given cell indicates functional receptors and direct responsiveness, absence of a Stat5 response in a cell type does not rule out hormone responsiveness via alternative pathways. The purity and specificity of these highly purified hormone preparations was supported by distinct target cell responsiveness. For example, a large number of tissues were exclusively responsive to either GH or PRL, indicating that contamination with the other hormone in the ovine pituitary derived GH and PRL preparations was not a critical issue. Furthermore, EPO and GCSF were clinical-grade, recombinant hormone preparations. Whereas it cannot be excluded that some endotoxin and lipopolysaccharide remained in these solutions, endotoxin and lipopolysaccharide are not direct activators of the Stats, (reviewed in Ref. 28).

In addition to performing injections of PRL and GH independently, PRL and GH were also coinjected in an effort to identify additive hormone effects in target cells. In some cases, the combination of the two hormones activated distinct target cells that when combined resulted in an overall increased level Stat5 responsiveness within the tissue (e.g. mammary gland). In other cases both hormones affected overlapping target cells (e.g. liver) and additive effect on staining intensity was assessed. Whereas lack of detectable additive effect in such cases may reflect rate-limiting effects of the joint Jak2-Stat5 pathways when coactivated by PRL and GH, it may also be due to limitations in sensitivity and dynamic range of the DAB chromogen. Lack of additive effects of PRL and GH should therefore be interpreted with caution.

CEMA tissue arraying technology (6, 7) was used to facilitate parallel analysis of hundreds of tissue features, including multilayered tissues such as skin and intestines (Fig. 1). In parallel, we also used sections from standard tissue blocks for staining and scoring of all samples. Tissues that were highly heterogeneous (e.g. vertebrae or kidneys) or too small for effective arraying (e.g. ovaries or adrenals) were analyzed exclusively in the standard manner. Below follows a presentation and discussion of the data categorized by organ system. For ease of presentation in the text, unless otherwise noted, there was no detectable response to EPO or GCSF in cells of the respective tissue or organ.

View larger version (75K): [in this window] [in a new window]   FIG. 1. CEMA array of sheet-like tissues with stratified geometry. Thin-walled tissues were arrayed using CEMA technology, and alternate slides were stained for phosphorylated Stat5 or H & E in high throughput analysis. Representative H & E-stained, low-magnification images are presented for better image contrast and presentation. A, CEMA skin array. Columns represent tissues from the same location in replicate rats (ventral skin, just right of the midline) and rows represent different treatments. Scale bar, 200 µm. B, CEMA gut array. Columns represent progression though the GI tract of a single rat at 10 mm resolution (this image represents approximately 10 cm distal from the gastroduodenal junction), and rows represent similar GI locations between replicate animals and treatments. Scale bar, 200 µm.

View larger version (149K): [in this window] [in a new window]   FIG. 2. Stat5 activation status in response to PRL and GH administration in endocrine organs. Animals were injected with vehicle (basal), PRL, GH, or PRL + GH, and tissues were harvested 30 min after injection and fixed in formalin. Paraffin-embedded tissue sections were stained with standard IHC procedures for anti-PY Stat5, DAB (brown color) was used as a chromogen to detect activated Stat5, and samples were counterstained with hematoxylin (blue color). Hormone-responsive target tissues and cell types were as follows: Adrenal gland, cells from all three layers of the adrenal cortex were highly responsive to PRL (3+); pancreas, cells within the islets of Langerhans were strongly responsive to PRL (3+); testis, testosterone-producing Leydig cells had a low basal activation (+) and were strongly induced by PRL (3+); thyroid, luminal epithelial cells had a low basal Stat5 activation (+), which were strongly induced by PRL (3+) and moderately induced by GH (2+). Scale bar, 30 µm.

Pancreatic islets of Langerhans.
The pancreas is involved in digestion and nutrient uptake by both endocrine and exocrine functions. Interspersed between exocrine pancreatic acini are distinct endocrine cell masses termed the islets of Langerhans. These islets were another endocrine tissue component in which PRL induced marked Stat5 tyrosine phosphorylation (Fig. 2). Previous work demonstrated direct effects of PRL on many aspects of islet cell biology, including the release of insulin into the circulation (41, 42, 43), and PRLR mRNA expression has been generally localized to the cells of the islets (4). In contrast, in our study GH did not stimulate Stat5 phosphorylation higher than basal levels in any of the subpopulations of cells within the islets, indicating a significant difference between cell responsiveness to PRL and GH. Others also have shown an enhanced effect of PRL over GH in activating Stat5 in rat islet cells; however, the difference was attributed to desensitization by continued exposure to GH, beginning after 1 h of treatment, whereas PRL had a biphasic activation that resulted in prolonged Stat5 activation (44). Several laboratories are investigating PRLR signaling within the islets of Langerhans to gain further insight that could lead to better treatment for type 2 diabetes in humans.

Testis.
The testis produces sperm cells in the seminiferous tubules and androgens in the interstitial cells or Leydig cells. It is well established that PRL promotes the maintenance of testicular Leydig cell morphology (45) and generally promotes androgen production (46, 47, 48, 49, 50) in many mammals. Consistent with a direct effect of PRL on Leydig cells, PRLR mRNA has been identified in the Leydig cells (51, 52) and PRL binding to isolated Leydig cells has been reported (53). PRL^–/– (54) and PRLR^–/– (55) males, however, are fertile and have no histological abnormalities of the testis, and no alterations of plasma testosterone were detected. Thus, PRL signaling in Leydig cells is not critical for fertility of male mice but may be compensated for by other pathways. Our study indicated that PRLR in a majority of rat Leydig cells are responsive to PRL in vivo (Fig. 2). Although GH or GCSF did not induce Stat5 activation in Leydig cells, marked EPO-induced activation of Stat5 also was detected (see Fig. 5). Previous work on isolated rat Leydig cells has shown that EPO can stimulate testosterone production in these cells (56). The direct effect of EPO on testosterone production was reinforced by work from Foresta et al. (57), which indicated that gonadotropins did not mediate EPO’s androgen-producing effect. It is intriguing that the seemingly diverse hormones, PRL and EPO, acting through separate receptors, share Stat5 as a common target in Leydig cells. It will be of interest to determine whether EPO signaling may compensate for PRLR or PRL loss in the respective gene knockout mice and whether PRL and EPO induce the same subset of genes in Leydig cells. In addition, are other signaling pathways than Stat5 coactivated by these two hormones? Based on our analysis of Stat5 activation, no other cells of the testes were responsive to PRL, GH, EPO, or GCSF.

View larger version (84K): [in this window] [in a new window]   FIG. 5. Stat5 activation status in response to EPO administration in rat tissues. Animals were injected with vehicle (basal) or EPO, and tissues were harvested 30 min after injection and fixed in formalin. Paraffin-embedded tissue sections were stained with standard IHC procedures for anti-PY Stat5, DAB was used as a chromogen to detect activated Stat5, and tissues were counterstained with hematoxylin. Hormone-responsive target tissues and cell types were as follows: testis, testosterone-producing Leydig cells had a low basal Stat5 activation (+) and were strongly responsive to EPO (3+); spleen, cells in the red pulp showed strong Stat5 activation in response to EPO (3+); bone marrow, hematopoietic precursors in vertebra marrow were highly responsive to EPO (3+); quadriceps epimysium, round, yet-to-be-identified cell type dispersed within the muscular connective tissue was highly responsive to EPO (3+). Scale bar, 30 µm.

Ovary.
The ovaries undergo a cyclic maturation process in which egg cells are released and rising levels of the ovarian steroids progesterone and estrogen are produced by luteal cells and interstitial cells, respectively. The surface of the ovary is covered by an epithelial cell layer, germinal epithelium, from which the majority of ovarian malignancies originate. In rodents, PRL has both luteotropic and luteolytic effects (34). Receptors for PRL have been described on progesterone-producing luteal cells at the mRNA (69, 70) and protein binding levels (71). mRNA analysis of rat tissues indicated that PRLR levels are among the highest in the ovary and predominantly express of the long receptor form, but specific localization to cell type within the ovary was not made (29). Furthermore, GH also has been reported to affect ovarian function in rats by influencing ovarian follicle development (72), but a direct target cell for GH has not been identified. Interestingly, highly expressing bovine GH transgenic female mice are infertile as a result of deficiency of luteal function (73), an effect that has been suggested to be associated with a loss of the PRL surges (74), whereas rat PRL transgenic mice (75) and hyperprolactinemic mice (76) have less pronounced fertility problems.

After injection of PRL into rats, luteal cells in the ovaries showed a marked and uniformed Stat5 activation response (Table 1). A consistent but moderate Stat5 activation also was detected in estrogen-producing interstitial cells in response to PRL, whereas granulosa, egg, or ovarian epithelial cells did not respond. These observations support the view that luteal cells and interstitial cells are direct targets for PRL, which is consistent with established data regarding PRL stimulating progesterone and estrogen production in rat ovaries (77, 78, 79), such as the conversion of estrone to estradiol by 17ß-hydroxysteroid dehydrogenase (80) and PRL-induced inhibition of progesterone catabolism (81). Furthermore, PRL-induced Stat5 activation has been shown to be involved in estrogen receptor gene regulation (82) as related to corpus luteum formation. Our study did not find evidence of direct GH target cells in the endocrine-associated cell populations of the ovary in rats; however, although the germinal epithelium of the ovary was unresponsive to PRL, GH induced marked Stat5 activation in the germinal epithelium. Likewise, epithelial cells of the ovarian fimbriae showed moderate response to GH but were unresponsive to PRL.

Pituitary.
The pituitary consists of the anterior, intermediate, and posterior lobes and produces at least seven major peptide hormones, including PRL and GH. It is remarkable that the rat anterior pituitary has among the highest levels of expression of PRLR mRNA for any of the organs tested (29), suggesting a sensitive autoregulatory mechanism for synthesis and release of the hormone. Based on transcript analysis, the long form of the PRLR is predominant, with nearly a 100:1 ratio to the short form. Likewise, GHR mRNA was localized to the anterior lobe of the pituitary by in situ hybridization (33). We did not, however, detect PRLR inducible activation of Stat5 in the anterior pituitary by PRL or GH administration above the low constitutive basal levels (Table 1) in either male or female rats. In fact, of the endocrine organs analyzed, the pituitary was the only tissue in which we did not detect PRL-inducible Stat5 activation. The lack of effect may be a result of desensitization by high local hormone levels.

In summary, we documented that a wide array of endocrine cells in the rat are directly responsive to PRL and activate Stat5, whereas the same cells generally are unresponsive to GH. The selective induction of PRLR, but not GHR, signaling in these endocrine-related cell types support the concept that PRL may act as a master coordinator of endocrine regulation (Table 1). In fact, throughout pregnancy, serum PRL levels rise until the onset of labor (85), consistent with the need for a coordinated endocrine response during the increased hormonal and metabolic demands of pregnancy and lactation. Furthermore, it is known that PRL, but not GH, is rapidly released during acute stress in both males and females, and PRL is considered a stress hormone (37, 38, 39, 40). PRL therefore also may help provide a coordinated regulation of endocrine glands during stress, similar to the immunomodulatory effects of PRL documented in burn-stressed animals (86). Finally, in humans, some of the many symptoms of hyperprolactinemia may be related to endocrine dysregulation.

Female reproductive system
Mammary gland.
Development and function of the mammary gland is an intricate process that is regulated by many hormones and growth factors, including estrogen, progesterone, insulin, glucocorticoids, GH, and PRL. The terminally differentiated function of the mammary gland, however, depends on the lobuloalveolar development of secretory alveoli during pregnancy and lactation, and this process is critically dependent on PRLR-Jak2-Stat5 signaling (87, 88, 89, 90). Anatomically, rodent mammary glands consist of branching ducts and distal lobuloalveolar compartments embedded in a mammary stromal compartment (91). The mammary epithelia contain centrally located luminal cells and peripherally located myoepithelial cells. Collaborative work from the Kelly, Kopchick, and Hennighausen laboratories (11) used gene deletion mouse models, mammary transplants, and mammary stromal/parenchymal dissection to investigate the roles of PRL and GH in the mammary gland. Based on these studies of dissected tissue extracts, PRL was reported to mainly activate Stat5 in the epithelial compartment, whereas GH had effects in both epithelial and stromal cells. Furthermore, GHRs have been reported in myoepithelial cells of human and canine mammary glands (92, 93). By using an in situ IHC approach to detect nuclear-localized, tyrosine-phosphorylated Stat5, we determined in the present studies that in nonpregnant rats, PRL markedly activated Stat5 in a large proportion (80%) of the luminal mammary epithelial cells over the low basal constitutive Stat5 activation (Fig. 3). This finding is in agreement with previous work from our laboratory, in which we reported low but consistent basal activation of Stat5 in luminal epithelial cells of nonpregnant mouse epithelium. This activation was lost rapidly after hypophysectomy but could be recovered by injection of PRL but only partially by GH (9). Furthermore, basal Stat5 activation remained active throughout the estrus and luminal mammary epithelial tissue from proestrus, estrus, postestrus, and diestrus showed equivalent levels of basal phosphorylation of Stat5. PRL did not activate Stat5 in myoepithelial cells, stromal fibroblasts, or mammary adipocytes, which have been suggested to resemble brown adipose tissue (94), whereas adipocytes from other tissues composed of white adipose tissue were responsive to PRL.

View larger version (122K): [in this window] [in a new window]   FIG. 3. Stat5 activation status in response to PRL and GH administration in reproductive tissues. Animals were injected with vehicle (basal), PRL, GH, or PRL + GH, and tissues were harvested 30 min after injection and fixed in formalin. Paraffin-embedded tissue sections were stained with standard IHC procedures for anti-PY Stat5, DAB was used as a chromogen to detect activated Stat5, and tissues were counterstained with hematoxylin. Hormone-responsive target tissues and cell types were as follows: mammary gland (female), luminal epithelial cells of the ducts and alveoli exhibited a low basal Stat5 activation (+) and were strongly activated by PRL (3+). GH induced Stat5 activation in the myoepithelial cells and in a minority of ductal luminal epithelial cells (2+). Together PRL and GH activated essentially all epithelial cells of ducts and alveoli (3+); preputial gland (female), sebaceous glandular epithelial acini were moderately responsive to GH (2+); prostate, the dorsal lobe epithelial cells had a low basal Stat5 activation (+) and were strongly activated by PRL (3+); preputial gland (male), sebaceous glandular epithelial acini were strongly responsive to PRL (3+) and GH (3+). Scale bar, 30 µm.

Uterus.
The uterus has been identified as a site of extrapituitary PRL production involving both the endometrial and myometrial cells (102, 103, 104, 105, 106). In addition, studies have suggested that uterine PRL is directly involved in decidualization (107) and maintenance of full-term pregnancy (2) as well as water-electrolyte balance of the amnion (108) and modulating local immune reactions to prevent rejection of the implanting fetus (107). In the nonpregnant rat uterus, PRLR mRNA levels were about the median of tissues tested for total transcript levels; however, the ratio of full length to short isoform was among the highest of any tissue, with only 2% of the population as the short form (29). Little is known about GH effects on the rat uterus. Injection of PRL or GH did not activate Stat5 in the myocytes of the uterine myometrium or the endometrial cells (Table 1), nor did we detect a response in the endometrium to any of the hormones administered. Our study material included endometrial tissue from proliferative and secretory phases, all with negative results for Stat5 activation. PRL is responsible for the maintenance of full-term pregnancy and circulating PRL levels are dramatically elevated during gestation. It would be of interest to determine whether uterine cell types become hormone responsive with respect to Stat5 activation during pregnancy.

Other female reproductive tissues.
We detected a moderate GH-selective response in the deep layers of the stratified squamous epithelium of the vagina. Additionally, analysis of the vaginal preputial sebaceous glands displayed no basal Stat5 activation, and the epithelial acini were unresponsive to PRL treatment. Administration of GH, however, induced moderate Stat5 activation throughout the gland (Fig. 2). This result is consistent with previous work that has identified an effect of GH on female rat preputial glands (109) and the presence of GHR on rat sebaceous glands (110, 111, 112).

Taken together, these data on the female reproductive system indicate an endocrine cell-centric role of PRL, with the notable exception of PRL’s well-documented function as a growth and differentiation factor in the epithelial cells of the mammary gland in rodents. Interestingly, in contrast to the well-documented role of GH in stromal cell types throughout the body, many of the epithelial cells of the female reproductive tissues were responsive to GH.

Male reproductive system
Prostate.
The rodent prostate is an accessory sex gland that consists of three distinct paired lobes and the coagulating glands, which jointly surround the urethra and contribute enzymes, ions, and other molecules to the seminal fluid. Effects of PRL on rat prostate gland physiology have been well documented, including a proliferative effect in conjunction with androgens, especially in the dorsal and lateral lobes (113, 114, 115), citrate production in the differentiated epithelium of the ventral lobe (116, 117), polyamine biosynthesis (118), and numerous other prostate-specific alterations (116). PRL is produced locally in the rat and human prostate gland and probably also signals in an autocrine or paracrine manner to affect prostate physiology (119, 120). For the rat coagulating gland, long-term overexpression of PRL has indicated an increase in gland size (121), but definitive signaling characteristics of this gland have yet to be described. Furthermore, little work has been done with respect to identifying GH signaling in the rat prostate complex, although in humans a few studies have correlated acromegaly with benign prostate hyperplasia (122, 123).

Analysis of Stat5 activation revealed that the basal Stat5 activation state varied between the lobes of the rat prostate (Table 1). The ventral prostate and the coagulating gland displayed no basal staining, whereas the dorsal and lateral prostates showed low staining (+) and moderate (2+) staining, respectively. After PRL injection, the dorsal (Fig. 3) and lateral lobes of the prostate exhibited strong (3+) staining in essentially all of the luminal epithelial cells, whereas the ventral prostate was induced to a moderate intensity of Stat5 activation, indicating that the dorsal and lateral rat prostate lobes are more sensitive to PRL, compared with the ventral prostate. Moreover, these data are consistent with previous work from our laboratories demonstrating PRL-induced Stat5 activation in ex vivo organ cultures of rat prostate (124). Furthermore, the coagulating glands did not respond to either PRL or GH. In fact, GH did not activate Stat5 in cells of of the prostate lobes.

Seminal vesicle.
Another male sex organ of the rat with a documented response to PRL is the seminal vesicle, which secretes fructose and prostaglandins into the semen. Similar to the prostate, PRL and androgens coordinately stimulate the secretory function and morphology of the seminal vesicles (113, 114, 115) and specifically alter the chemical composition of the secretions in both rodents and primates (125, 126, 127). Furthermore, human GH transgenic mice had dramatically enlarged seminal vesicles; however, this effect may have been mediated through the PRLR and not GHR because mice with transgenic expression of the nonlactogenic bovine GH did not show similar hypertrophy (128). Injection of PRL activated Stat5 in luminal epithelial cells of the seminal vesicles to a maximal level (3+), from a low (+) basal activation state. Importantly, acute high-dose GH did not induce Stat5 activation in the seminal vesicle, which supports the concept that hyperplasia in human GH overexpressing transgenic mice is mediated by PRL receptors.

Epididymis.
The epididymis represents a key segment of the excurrent duct system for spermatozoa and serves the dual functions of absorbing fluid from the seminiferous tubules, defective sperm, and other bodies and secreting factors that promote maturation of sperm. PRL has been implicated as a regulator of rat epididymal function, including stimulating energy metabolism (129) and affecting the chemical composition of the secretions (130, 131, 132). A role for GH in epididymal function has not been well defined; however, evidence from rats suggested improved sperm maturation and increased motility after GH treatment (133). In the present study, rat epididymis displayed a low (+) baseline of Stat5 activity, and after PRL administration the luminal epithelial cells became markedly activated (3+). In contrast, GH did not affect Stat5 activation levels in cells of the epididymis. The reported effect of GH on the viability of spermatozoa in the absence of inducible Stat5 activation in GH-injected rats may reflect an indirect biological effect of GH mediated by IGF-I or other secondary factors.

Preputial gland.
The male preputial gland is a sebaceous gland that is well developed in rats, and its secretion is mixed with urine to mark territory and identify members of a group. Previous studies have identified effects of PRL and GH on the male preputial gland in rats. Transplantation of a pituitary under the renal capsule elevated circulating PRL and increased the weight of the male preputial gland (121). Furthermore, GH-mediated signaling effects were differentiation related and not proliferative and resulted in increased secretion when assayed in a rat preputial cell culture model (134). Our analyses revealed no basal Stat5 activation; however, treatment with either PRL or GH induced a strong (3+) and uniform activation of Stat5 in the epithelium throughout the sebaceous gland acini (Fig. 3). These observations suggest that previously reported biological effects of PRL and GH on the male preputial gland are direct.

Other male reproductive tissues.
Although not uniquely related to the reproductive functions of the male rat, the urethral transitional epithelium is involved critically in transporting semen from the reproductive organs during ejaculation. There was no basal or inducible Stat5 activation in the urethral epithelium in response to either PRL or GH injection.

In general, sex steroid-producing cells of gonads in both male and female rats were responsive to PRL but not GH, consistent with the noted broad responsiveness of endocrine cells to PRL. In contrast, nonsteroid producing reproductive tissues in male and female rats showed a divergent responsiveness to PRL and GH. With the exception of the skin-derived preputial glands and mammary glands, male reproductive tissues were preferentially responsive to PRL and not GH, whereas female reproductive tissues were preferentially responsive to GH and not PRL. This differential responsiveness of male and female reproductive tissues to PRL and GH perhaps is related to differences in GH secretory patterns of male and female rats. In male rats, GH secretion is pulsatile and, acting through Stat5, is responsible for sexual dimorphic growth patterns (13, 135). On the other hand, intrinsic differences in male and female reproductive tissues (e.g. gene expression levels) may be a more likely explanation of the differences in responsiveness to PRL and GH.

Digestive system
Liver.
The liver is the largest internal organ and also is the largest glandular tissue mass in mammals. This complex organ has both endocrine and exocrine functions and hence is involved in many physiological processes, including metabolism, digestion, serum protein production, and IGF-I secretion. Absolute PRLR mRNA levels in rat liver are among the highest of any tissue tested (29), and numerous pathways and growth-related genes have been identified that are activated by PRL (34). It should be noted, however, that the majority of PRLR transcripts in rat liver encode the short form of the receptor (29). Additional reports have shown that PRL has a somatogenic effect in the liver, including stimulation of IGF-I production (136, 137). Others have reported that PRL treatment is not able to activate Stat5 in the liver (138), possibly because of the dominant-negative effect of the short PRLR on Stat5 activation by the long PRLR (3, 139). The differences in results between the studies also may be a function of the sensitivity of the assays used. The report that did not detect PRL-induced Stat5 activation in rat liver relied on immunoblotting and EMSAs (138). By way of comparison, previous studies identified basal Stat5 activation in the nonpregnant mammary gland by IHC (9), whereas immunoblotting or EMSA was not sensitive enough to detect the activated Stat5 (140, 141, 142, 143). Although it is generally accepted that Stat5 tyrosine phosphorylation is required for DNA binding, it may not be sufficient, depending on the specific nucleotide sequence of the probe used in an EMSA assay and the presence of additional cofactors that may modulate DNA binding (17). Furthermore in the liver, a very recent study (144) identified low but specific expression of the long form of the PRLR on the cholangiocytes of the intrahepatic bile ducts at the mRNA and protein levels and that regulation of the PRL isoforms was opposite of that detected within hepatocytes. Lastly, a large body of work identified GH-induced activation of Stat5 in the liver and stimulation of IGF-I, which has been established as a mediator of cell and organism growth, and GH-induced liver secretion of IGF-I forms the basis of the long-standing somatomedin hypothesis (145).

In the present study, no Stat5 activation was detectable in hepatocytes of either male or female rats under basal conditions. After PRL or GH injection, however, the majority of hepatocytes exhibited strong Stat5 activation (Fig. 4), and there was no difference in responsiveness between hepatocytes of male and female rats. No additive effect was detected by Stat5-pY staining intensity with the coinjection of PRL and GH in the hepatocytes. The marked activation of Stat5 by PRL was unexpected in light of the predominant expression in rat liver of the short PRLR isoform as determined by RT-PCR. We did not detect responsiveness to PRL or GH in any of the other cell types of the liver, including cholangiocytes in which low levels of the PRLR were recently reported (144). In that report, the authors indicated that the long form of the PRLR was up-regulated by cholestasis, suggesting that under normal conditions, the receptor levels are below threshold levels for detectable response.

View larger version (133K): [in this window] [in a new window]   FIG. 4. Stat5 activation status in response to PRL and GH administration in rat tissues. Animals were injected with vehicle (basal), PRL, GH, or PRL + GH, and tissues were harvested 30 min after injection and fixed in formalin. Paraffin-embedded tissue sections were stained with standard IHC procedures for anti-PY Stat5, DAB was used as a chromogen to detect activated Stat5, and tissues were counterstained with hematoxylin. Hormone-responsive target tissues and cell types were as follows: submaxillary gland, serous acinar cells had Stat5 moderately activated by PRL (2+) and to a low degree by GH (+), with no response detected in the ductal epithelium (–); liver, hepatocytes were strongly activated by PRL (3+) or GH (3+); choroid plexus, epithelial cells were strongly responsive to PRL (3+); cerebral meninges, cells of both the pia and arachnoid were strongly responsive to GH administration (3+); abdominal adipose tissue, adipocytes from mesocolon-associated intraabdominal adipose tissue were moderately responsive to PRL (2+) and strongly responsive to GH (3+); quadriceps adipose tissue, muscle-associated adipose tissue was moderately responsive to PRL (2+) and strongly responsive to GH (3+); dermal adipose tissue, sc adipocytes were moderately responsive to PRL (2+) and strongly responsive to GH (3+). Scale bar, 30 µm.

Exocrine pancreas.
Like the liver, the pancreas is both an exocrine and endocrine organ. The exocrine pancreas consists of serous acini and a ductal tree that secrete digestive enzymes into the gastrointestinal (GI) tract. The responsiveness of endocrine islets of Langerhans to PRL and GH as measured by in situ Stat5 activation was presented under the endocrine system above (Fig. 2). mRNA for PRLR is detectable in homogenates of pancreas tissue (29). Although the effects of PRL and GH on the endocrine pancreas have been studied in great detail, relatively little work has been reported on the exocrine pancreas. In one study, chronic hyperprolactinemia induced by a pituitary graft under the renal capsule was associated with increased proliferation of pancreatic acinar cells and an increase in pancreatic secretions; however, this increase was transient and reverted back to control levels over time (151). In the present study, injection of PRL or GH revealed that exocrine pancreas acinar cells showed a low but direct response to PRL and GH in both male and female rats (Table 1). Furthermore, a combination of the two hormones did not have an additive effect on Stat5-pY staining intensity in the exocrine pancreas.

Alimentary canal.
The alimentary canal is made up of the esophagus plus the GI tract and is responsible for digestion, absorption, movement, and excretion of nutrients and waste. PRL has been reported to induce hyperplasia, increased villus height, and mass of the intestinal mucosa (152, 153). Furthermore, a protective effect against gastric ulcers was observed in chronic hyperprolactinemic rats (154). Effects on growth and changes in metabolism within the intestine by PRL and GH have been reported (155). Also, acromegalic patients have relatively longer overall colon length, and these patients have increased incidence of colon polyp formation, colon cancer incidence, and higher colon cancer mortality (156). Lastly, studies involving EPO also have identified some GI tract-specific effects in the gastric mucosa (157) and small intestine (158); however, in the present study, we did not detect EPO-induced activation of Stat5 in the alimentary canal (Table 1).

Throughout the entire length of the alimentary canal, we detected a low to moderate activation of Stat5 by PRL and/or GH in the mucosal epithelial cells (Table 1). We did not detect basal levels of Stat5 activation in the GI tract, with the exception of low levels in the epithelial cells in the base of the crypts of the colon. Specifically, moderate levels of inducible Stat5 activation were observed in the basal layers of the stratified squamous epithelium of the esophagus in response to GH or PRL treatment. No additive effect on Stat5-pY staining intensity of PRL and GH was detected in these target cells. We also noted a low, but detectable, activation of Stat5 in response to PRL, but not to GH, in the gastric epithelium. The different regions of the small intestine appeared to be somewhat differentially responsive to PRL treatment but uniform in the response to GH. The CEMA tissue arraying technology was particularly effective for systematic analysis of the entire GI tract at high resolution. In particular, Stat5 activation was scored as low in the duodenum in response to both PRL and GH, whereas in the ileum and jejunum PRL induced moderate activation and GH induced low levels of Stat5 activation (Table 1).

Integumentary system
Skin.
As the largest organ in the body, the skin provides a barrier and is important in sensory perception. PRL is known to play a role in various skin functions in many vertebrates. In mammals, PRL has been shown to be involved in the control of proliferation of melanocytes (159) and keratinocytes (160), hair loss in nesting animals (161), and hair growth (162, 163). Absolute PRLR mRNA expression levels in the skin ranked in the middle of the organs tested but notably had among the highest ratio of the full-length variant of the receptor to the short form (29), presumably resulting in more efficient intracellular signaling. GH also is known to have a growth-promoting effect on the skin, and a significant pharmacological benefit is seen in elderly patients receiving supplemental GH (164). Consistent with this, GHR has been identified in the epidermis of rat skin at the transcript level (33) and the protein level (110).

We detected low baseline levels of Stat5 activation in a subset of the basal epidermis cells and marked Stat5 activation in response to PRL throughout this basal layer of the epidermis. GH had a moderate effect in activating Stat5 in the basal epithelial cells (Table 1). We noted a strong (3+) basal activation of Stat5 in the dermal papilla of the hair follicle, but these cells did not show further activation of Stat5 in response to either PRL or GH (Table 1). Local production of PRL and PRLR has been reported in murine and human hair follicle, specifically in the inner and outer root sheath and matrix keratinocytes (165, 166, 167) but not the dermal papilla, suggesting the mechanism of Stat5 activation in the dermal papilla is independent of the autocrine/paracrine PRL-PRLR signaling proposed to exist within the hair follicle. Reduced hair growth, however, was noted in Stat5b-null mice (135), indicating PRL-Stat5b signaling may be involved in growth regulation of hair, presumably by an alternate mechanism than PRL-induced apoptosis of the follicle (167). Clearly, additional work is needed to understand the role of Stat5 and its regulation in hair follicle biology.

Hematopoietic system
Effects of PRL on many aspects of the hematopoietic system have been well documented and reviewed extensively and include effects on lymphoid cells, monocytes, macrophages, and thymic epithelial cells (34, 168, 169). The effects of PRL on these cells have been categorized as redundant and overlapping with other cytokines because PRL and PRLR knockout mice generally have normal immunity and hematopoietic status (1, 170, 171, 172). Similarly, GHR transcript and protein expression also has been identified in both B and T lymphocytes and monocytes (173), and polymorphonuclear leukocytes have been shown to express EPO receptor (EPOR) (174) and GCSF receptor (175) mRNA and specifically bind EPO. Our study did not examine responsiveness of circulating leukocytes to PRL, GH, EPO, or GCSF but included examination of hematopoietic cells in lymphatic organs and bone marrow.

Spleen.
The spleen is the largest lymphatic organ and acts to filter the blood for antigens or defective blood cells. The spleen contains aggregations of B and T lymphocytes, erythrocytes, plasma cells, macrophages, and granulocytes and in rodents also megakaryocytes for platelet production. The spleen has been shown to express PRLR mRNA (29) and GHR mRNA (33), and studies have implicated an important role for PRL signaling in the organ (176, 177). We did not, however, detect definitive activation of Stat5 after PRL treatment, but GH stimulated Stat5 to a low, but detectable, level in an estimated 10% of cells in the white pulp of the spleen (Table 1). EPO strongly activated Stat5 in an estimated 30% of cells in the red pulp of the spleen, presumably erythroblasts (Fig. 5), whereas GCSF had a moderate induction of Stat5 activation in neutrophils in the red pulp and lymphocytes associated with the periarterial lymphatic sheath. Further work is needed to identify which subpopulations of cells within the spleen respond to the individual hormones.

Thymus.
In the thymus, stem lymphocytes are transformed into T lymphocytes, which are important for cell-mediated immunity as well as development of B lymphocytes. Several lines of evidence have suggested a significant role for PRL and GH in the thymus. For example, as reviewed by De Mello-Coelho et al. (178), expression of PRLR and GHR has been identified on both thymocytes and thymic epithelial cells, and both cell types can produce PRL and GH. Furthermore, differentiating T cells are responsive to PRL and GH, and GH administration increases total thymocyte numbers (178). The present study revealed a subset of thymic cells that were responsive to PRL and GH (Table 1). The data suggested that the same cells were responsive to the two hormones because roughly the same percentage (1–3%) and localization (cortex and medulla) of lymphocytes were responsive to either PRL or GH, and no further Stat5-pY staining intensity was detected after combined injection of both hormones.

Bone marrow.
The spaces within long or spongy bones are filled with red bone marrow if there is active hematopoiesis and yellow marrow if there is no active hematopoiesis. The hematopoietic cells mature within the marrow and must actively migrate into the circulation. The role of PRL and GH on bone marrow cells is not clearly defined, although a recent study in human tissue demonstrated that GH can directly and indirectly increase the hematopoietic activity of bone marrow by several mechanisms (179). We analyzed the bone marrow from a long bone (femur) and an irregular bone (vertebra). Although a subpopulation of cells (5%) in the red marrow of femur displayed a moderate response to PRL, no PRL-responsive cells were detected in red bone marrow of the vertebra (Table 1). In contrast, subpopulations of cells from either femur or vertebra bone marrow were equally responsive to GH, displaying a moderate response in approximately 5% of cells. We identified an additive effect of PRL and GH on Stat5-pY signal intensity of responsive cells in the femur but not on cell numbers, suggesting that the same cells were responsive to both PRL and GH. No additional Stat5-pY staining intensity above that induced by GH stimulation alone was detected in the vertebral marrow when PRL and GH were coinjected. Furthermore, vertebral marrow cells showed a marked activation of Stat5 in response to EPO, presumably in erythroblast cells (Fig. 5). A small population of cells in the vertebral marrow exhibited low Stat5 activation in response to GCSF, consistent with granulocyte/monocyte colony-forming cells (data not shown).

Gut-associated lymphatic tissue.
One component of the gut-associated lymphatic tissue is aggregates of lymphatic nodules located in the distal small intestine (ileum) called Peyer’s patches. These nonencapsulated aggregations of lymphocytes are highly reactive when presented with antigens and form germinal centers. Peyer’s patches had a moderate (2+) basal activation status that was enhanced to 3+ with PRL administration, demonstrating the presence of PRL-responsive cells within Peyer’s patches (Table 1). GH treatment did not induce a discernable increase in Stat5 phosphorylation status above basal levels in any population of lymphocytes in Peyer’s patches. Likewise, EPO and GCSF treatment also did not stimulate Stat5 activation above basal levels in the lymphatic aggregations of the ileum and jejunum. Another type of gut-associated lymphatic tissue (GALT) is classified as diffuse lymphatic tissue and is present in the lamina propria. Interestingly, lymphocytes present within in the small intestinal villi generally had consistent moderate levels of basal Stat5 activation but were unresponsive to PRL or GH treatment (Table 1). Because Stat5 activation is associated with general activation of lymphocytes (180), the observations suggest that lymphocytes in the lamina propria are activated.

	Circulatory system

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Previous studies identified functional PRLR in the rat heart (29) and established an involvement of GH in acromegalic patients on cardiac function, including hypertrophy (181) and energy metabolism (182). In addition, previous work has identified a proliferative effect of EPO on smooth muscle vasculature, an effect that was suggested to be mediated by an undetermined tyrosine kinase, phospholipase C, and protein kinase C (21). Other work has identified constitutively associated Jak2 with EPOR in an erythroid cell line (183). The role of EPO-induced Stat5 in hematopoietic cell proliferation is controversial, with some reports of active Stat5 positively correlated with proliferation (184, 185), whereas other reports have not shown such a correlation (186, 187). In the present study, we did not detect basal or inducible Stat5 activation in cardiac cells in responsiveness to PRL or GH (Table 1). The endothelial cells of the aorta, however, displayed a moderate response to GH but not PRL. No inducible activation of Stat5 was detected in cardiac cells or cardiac vascular cells in response to EPO or GCSF. Nevertheless, further work is needed to localize EPO-responsive cardiac cells through Stat5-independent pathways.

	Musculoskeletal system

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GH influences skeletal muscle indirectly through release of growth-promoting IGF-I but also may have direct local actions (188, 189). The complex biology of GH and IGF-I and their interaction is beyond the scope of this work; however, the identification of specific GH-responsive cells within muscle may provide some insight into this process. Furthermore, PRLR mRNA has been detected in skeletal muscle, albeit at the lowest level of any of the tissues examined, and no function for PRL has been associated with skeletal muscle (29). With respect to GH, cultured mouse skeletal muscle myoblast cells (190) and in vivo analysis of rat skeletal muscle extracts showed that GH induced Jak2 and Stat5 tyrosine phosphorylation (191); however, contributions from the PRLR could not be excluded because human GH was used. Furthermore, recent work investigated the effect of an iv bolus of GH on human skeletal muscle by analyzing extracts of biopsies (192). GH-induced activation of Stat5 was detected by Western blot but did not determine the cellular targets within muscles. In rats, the present study localized a moderate GH-induced activation of Stat5 in myocytes of the quadriceps (Table 1). Furthermore, although we did not detect an in situ Stat5 response to PRL in the skeletal muscle myocytes, our analysis identified a low response to PRL in the cells of the muscular fascia and moderate Stat5 activation in these same cells in response to GH (Table 1), as detailed in the Connective tissue section of this manuscript.

Previous work has established that the effect of GH in bone is consistent with growth-promoting roles of GH and IGF-I on this target organ (193, 194, 195), and PRLR mRNA has been identified in osteoblasts (196). Furthermore, PRLR^–/– animals show reduced ossification (196), suggesting an important, uncompensatable role for PRL signaling in bone homeostasis. We identified a low but specific responsiveness to PRL of chondrocytes and osteocytes of the femur (Table 1), indicating that at least some of the effects of PRL on bone are direct. GH also induced low but detectable activation of Stat5 in femoral chondrocytes, whereas osteocytes showed moderate Stat5 activation in response to GH. Our study indicated that chondrocytes and osteocytes in male and female rats were comparably responsive to hormone treatment. Additional studies will be needed to determine which osteocytes are responsive to GH and PRL and determine age-dependent differences of relevance for osteoporosis and other diseases of the bone.

	Nervous system

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PRL and GH have been shown to have direct effects on brain function, including behavior, emotion, and stress response (189, 197). For PRL, the behavioral effects are associated primarily with reproduction and have been recognized since the 1930s (198). Consistent with this, PRLR-null mice show diminished maternal behavior (199). Transcript analysis by the group of Nagano and Kelly (29) identified the choroid plexus of the brain as having the highest levels of PRLR message per milligram of tissue of any tissue examined. Evidence suggests that PRLRs in the choroid plexus are involved in binding and transporting functional PRL from the blood to the cerebrospinal fluid (200). Although a long-standing dogma stated that PRL and GH were too large to cross the blood-brain barrier (201), more recent studies identified several peptide cytokines and hormones capable of specific transbarrier passage, including several ILs (202, 203, 204), TNF (205), and interferons (206, 207). Transport of PRL (208, 209, 210) and GH (211, 212) into the central nervous system presumably plays a significant role in brain physiology because exogenous administration of both hormones have many documented effects on behavior. Furthermore, studies of radiolabeled GH binding to different tissues in both human and rat identified the choroid plexus as having the strongest binding, suggesting that the choroid plexus has high levels of GHR (213, 214). In the present study, we detected marked activation of Stat5 in the epithelial cells of the choroid plexus by PRL (Fig. 3). Intriguingly, GH did not induce Stat5 tyrosine phosphorylation, suggesting either a defective or alternate signaling axis. As one such possible mechanism, it has been suggested that a substantial portion of GH binding in the choroid plexus is attributable to GH binding protein (213).

EPO has been well studied with respect to the brain, and EPOR has been documented as mRNA in brain capillary endothelial cells (215), mRNA and protein in neurons of hippocampal and cortical origin (216), and mRNA in astrocytes and microglia (217). Furthermore, patients receiving EPO have shown an increase in cognition that is probably not entirely attributable to an increase in hematocrit (158). Our initial investigation did not detect inducible Stat5 activation by either PRL, GH, EPO, or GCSF within the cortex or medulla of rat brain (Table 1), but higher resolution mapping of different regions of the brain will be needed. Receptors for PRL and GH have been reported in the cerebral cortex, hippocampus, and hypothalamus in both rodents and man (29, 218). It also is possible that intrathecal or intraventricular injection of these hormones will be more effective for detection of responsive cell types within the brain, especially because local production has been reported for PRL, GH, and EPO. Of the cerebral meninges, cells of both the pia and arachnoid were strongly responsive to GH (Fig. 5) but not to PRL.

In the peripheral nervous system, we detected a response to PRL (2+) and GH (3+) in the satellite cells of the spinal ganglia (Table 1). These satellite cells form a complete layer around the neuron cell body and function to control the microenvironment of the neuron, providing a key point of influence over the nervous system.

	Urinary system

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The kidneys are the main functional units in the urinary system and are responsible for filtration of blood and retention and excretion of solutes. The kidneys also produce EPO as an endocrine function. PRL’s most primitive function in vertebrates may be as a regulator of water and electrolyte balance. In fact, in several nonmammalian vertebrates, including fish and reptiles, the most prominent role of PRL appears to be osmoregulation (219, 220, 221). Therefore, it is not surprising that PRL has been implicated in regulation of renal sodium and potassium excretion in mammals (222) and as a stimulator of renal sodium-potassium ATPase in rats (223). Local production of PRL protein and expression of PRLR message and protein in the kidney have been demonstrated in rats and mice (29, 224). In the present study, we detected a marked PRL-induced activation of Stat5 in the epithelial cells of the ducts and tubules in the cortex of the kidney but not in the glomeruli or medullary tubules or ducts (Table 1). GHR has also been identified in the kidney (188), and GHR mRNA was specifically localized to the proximal tubules of the cortex by in situ hybridization (33). We noted a moderate activation in response to GH in the same cell types that were responsive to PRL. Although EPO is produced in the kidney, we did not detect responsiveness to EPO or GCSF treatment in the kidney. Furthermore, PRL or GH did not activate Stat5 in the cells of the bladder, an organ not thought to be involved in regulating salt balance.

	Respiratory system

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Together with the kidneys and liver, the lungs function to retrieve essential components and rid the body of waste. The key lung function is the exchange of gasses across the cell membranes in the alveoli. The present study failed to detect any PRL or GH-responsive cells in the lung (Table 1). Because PRLR mRNA expression has been reported in the lung (29) and PRL affects surfactant production in the fetal lung (225, 226), however, it is possible that other mediators than Stat5 are involved in lung cells.

	Connective tissue

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White adipose tissue.
Adipocytes represent a differentiated and specialized cell type within loose connective tissue and provide energy storage, insulation, and protection of vital organs. Recent studies documented expression of the PRLR transcript on adipocytes and have begun to determine the regulatory role of PRL in adipose tissue (227, 228). GH is known to induce the secretion of IGF-I in adipocytes as well as exert other direct effects (188, 189). Furthermore, a recent study characterized GH responsiveness in extracts of biopsies taken from human adipose tissue (192), demonstrating GH-inducible Stat5 activation in abdominal adipose tissue, and correlated this with downstream gene activation (IGF-I) and DNA binding of Stat5 by EMSA. In the present study, we investigated cellular responsiveness to PRL, GH, EPO, and GCSF within white adipose tissue from three separate anatomical locations within the rat. Interestingly, each adipose compartment showed similar response patterns, even though the microenvironments and associated tissues were different. In particular, abdominal adipose tissue from mesocolon (Fig. 4), quadriceps-associated muscular adipose tissue (data not shown), and sc adipose tissue (data not shown) all showed moderate responsiveness to PRL and strong responsiveness to GH.

Dense irregular connective tissue.
Connective tissue that is identified by relatively low cellularity and a large component of fibers that are oriented in various directions is termed dense irregular connective tissue. Cell types present in this type of connective tissue generally are restricted to fibroblasts involved in synthesis of the protective fibers. We compared the responsiveness of cells within the connective tissue from multiple locations to PRL and GH. Similar to adipose tissue, Stat5 activation was moderate in response to PRL and high in response to GH in connective tissue associated with subcutis or quadriceps muscle (Table 1). One key difference between the connective tissue locations was marked activation of Stat5 in response to EPO in a round cell type dispersed throughout skeletal muscle connective tissue but not in sc connective tissue (Fig. 5). Intriguingly, recent studies identified the expression of EPOR on myoblast and primary satellite cells (14), which are thought to be a reservoir of muscle stem cells. Furthermore, a more recent study tracked bone marrow-derived stem cells to muscular connective tissue and locations of satellite cells, suggesting that such cells may play a role in muscular stem cell biology (229). Ongoing work aims to identify the described EPO responsive cells of connective tissue to determine whether they represent erythroblasts or possible muscle stem cells.

	Conclusion

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In summary, the present analysis presents a detailed map of cell populations in rat tissues that are directly responsive to either PRL, GH, EPO, or GCSF based on rapid Stat5 activation as a readout of cellular receptor activation. Several new insights were gained. First, hormone-producing cells of endocrine glands of male and female rats were consistently responsive to PRL but not GH. Second, in mammary gland epithelia, PRL responsiveness was restricted to luminal epithelial cells of ducts and alveolar structures, whereas GH activated Stat5 in a significant portion of myoepithelial cells and only a minority of luminal epithelial cells. Third, an EPO-responsive cell type that, to our knowledge, has not previously been described was detected in connective tissue. Fourth, in addition to mammary gland and prostate, marked responsiveness of choroid plexus, adrenal cortex, liver, and preputial glands to PRL may point to physiological and disease-associated effects of PRL in these relatively understudied PRL target tissues. Finally, adipocytes in white fat tissue taken from three distinct anatomical locations in both male and female rats were strongly responsive to GH and moderately responsive to PRL. Intriguingly, adipocytes in the mammary gland were not responsive to PRL, possibly due to the proposed resemblance of mammary fat to brown fat (94).

The new data may stimulate further investigation into the distinct target cells of GH and PRL in mammary gland development and disease. Because of the lactogenic activity of human GH, however, further work is needed to determine to what extent human GH and PRL serve overlapping functions in human breast epithelia. The detection of an EPO-responsive cell type unique to the connective tissue of skeletal muscle is intriguing, and ongoing work seeks to identify this cell type. The integrated, system-wide approach to map hormone-responsive cell types may be readily extended to other hormones, cytokines, or drugs, provided a rapid in situ readout is available. Cost-effective analysis using tissue arrays will be critical for practical undertaking of such large efforts. New ideas for improved understanding of hormonal overproduction syndromes, as well as understanding of drug effects, may be generated.

	Acknowledgments

 
We thank Neil Agarwal, Jennifer Johnson, and Mike Messuri for expert immunohistochemical technical assistance.

	Footnotes

 
This work was supported by National Institutes of Health Grants R01-DK52013, R01-CA101841, and R01-CA83813 (to H.R.); Department of Defense Grant W81XWH-05-01-0062; American Cancer Society Grant RSG-04-196-01-MGO and R01-CA113580 (to M.T.N.); and National Cancer Institute Support Grant 1P30CA56036-08 (to the Kimmel Cancer Center). This project is funded, in part, under a grant with the Pennsylvania Department of Health. The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions.

Disclosure Statement: T.J.A. and M.T.N. have nothing to declare. M.J.L. is an inventor on CEMA arraying technology, patent pending. H.R. is an inventor on CEMA arraying technology, patent pending. H.R. has equity interests in and consults for Advantex BioReagents, L.L.P. (Conroe, TX).

First Published Online November 30, 2006

Abbreviations: bw, Body weight; CEMA, cutting-edge matrix assembly; DAB, 3,3'-diaminobenzidine; EPO, erythropoietin; EPOR, EPO receptor; GCSF, granulocyte-colony stimulating factor; GHR, GH receptor; GI, gastrointestinal; H & E, hematoxylin and eosin; IHC, immunohistochemistry; Jak, Janus kinase; o, ovine; PRL, prolactin; PRLR, PRL receptor; Stat, signal transducer and activator of transcription.

Received September 6, 2006.

Accepted for publication November 20, 2006.

	References

Top Abstract Introduction Materials and Methods Results and Discussion Circulatory system Musculoskeletal system Nervous system Urinary system Respiratory system Connective tissue Conclusion References

 

1. Ormandy CJ, Camus A, Barra J, Damotte D, Lucas B, Buteau H, Edery M, Brousse N, Babinet C, Binart N, Kelly PA 1997 Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev 11:167–178[Abstract/Free Full Text]
2. Reese J, Binart N, Brown N, Ma WG, Paria BC, Das SK, Kelly PA, Dey SK 2000 Implantation and decidualization defects in prolactin receptor (PRLR)-deficient mice are mediated by ovarian but not uterine PRLR. Endocrinology 141:1872–1881[Abstract/Free Full Text]
3. Perrot-Applanat M, Gualillo O, Pezet A, Vincent V, Edery M, Kelly PA 1997 Dominant negative and cooperative effects of mutant forms of prolactin receptor. Mol Endocrinol 11:1020–1032[Abstract/Free Full Text]
4. Ouhtit A, Kelly PA, Morel G 1994 Visualization of gene expression of short and long forms of prolactin receptor in rat digestive tissues. Am J Physiol 266:G807–G815
5. Gygi SP, Rochon Y, Franza BR, Aebersold R 1999 Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730[Abstract/Free Full Text]
6. LeBaron MJ, Crismon HR, Utama FE, Neilson LM, Sultan AS, Johnson KJ, Andersson EC, Rui H 2005 Ultrahigh density microarrays of solid samples. Nat Methods 2:511–513[CrossRef][Medline]
7. Rui H, LeBaron MJ 2005 Creating tissue microarrays by cutting-edge matrix assembly. Expert Rev Med Devices 2:673–680[CrossRef][Medline]
8. Grimley PM, Dong F, Rui H 1999 Stat5a and Stat5b: fraternal twins of signal transduction and transcriptional activation. Cytokine Growth Factor Rev 10:131–157[CrossRef][Medline]
9. Nevalainen MT, Xie J, Bubendorf L, Wagner KU, Rui H 2002 Basal activation of transcription factor signal transducer and activator of transcription (Stat5) in nonpregnant mouse and human breast epithelium. Mol Endocrinol 16:1108–1124[Abstract/Free Full Text]
10. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP 1998 Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4:844–847[CrossRef][Medline]
11. Gallego MI, Binart N, Robinson GW, Okagaki R, Coschigano KT, Perry J, Kopchick JJ, Oka T, Kelly PA, Hennighausen L 2001 Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects. Dev Biol 229:163–175[CrossRef][Medline]
12. Ke Y, Lesperance J, Zhang EE, Bard-Chapeau EA, Oshima RG, Muller WJ, Feng GS 2006 Conditional deletion of Shp2 in the mammary gland leads to impaired lobulo-alveolar outgrowth and attenuated Stat5 activation. J Biol Chem 281:34374–34380[Abstract/Free Full Text]
13. Waxman DJ, Ram PA, Park SH, Choi HK 1995 Intermittent plasma growth hormone triggers tyrosine phosphorylation and nuclear translocation of a liver-expressed, Stat 5-related DNA binding protein. Proposed role as an intracellular regulator of male-specific liver gene transcription. J Biol Chem 270:13262–13270[Abstract/Free Full Text]
14. Ogilvie M, Yu X, Nicolas-Metral V, Pulido SM, Liu C, Ruegg UT, Noguchi CT 2000 Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts. J Biol Chem 275:39754–39761[Abstract/Free Full Text]
15. Chakraborty A, Dyer KF, Tweardy DJ 2000 Delineation and mapping of Stat5 isoforms activated by granulocyte colony-stimulating factor in myeloid cells. Blood Cells Mol Dis 26:320–330[CrossRef][Medline]
16. Nevalainen MT, Xie J, Torhorst J, Bubendorf L, Haas P, Kononen J, Sauter G, Rui H 2004 Signal transducer and activator of transcription-5 activation and breast cancer prognosis. J Clin Oncol 22:2053–2060[Abstract/Free Full Text]
17. Rycyzyn MA, Clevenger CV 2002 The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer. Proc Natl Acad Sci USA 99:6790–6795[Abstract/Free Full Text]
18. Lukas G, Brindle SD, Greengard P 1971 The route of absorption of intraperitoneally administered compounds. J Pharmacol Exp Ther 178:562–564[Abstract/Free Full Text]
19. Miquet JG, Sotelo AI, Dominici FP, Bonkowski MS, Bartke A, Turyn D 2005 Increased sensitivity to GH in liver of Ames dwarf (Prop1df/Prop1df) mice related to diminished CIS abundance. J Endocrinol 187:387–397[Abstract/Free Full Text]
20. Ram PA, Park SH, Choi HK, Waxman DJ 1996 Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liver. Differential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation. J Biol Chem 271:5929–5940[Abstract/Free Full Text]
21. Wald MR, Borda ES, Sterin-Borda L 1996 Mitogenic effect of erythropoietin on neonatal rat cardiomyocytes: signal transduction pathways. J Cell Physiol 167:461–468[CrossRef][Medline]
22. Clevenger CV, Medaglia MV 1994 The protein tyrosine kinase P59fyn is associated with prolactin (PRL) receptor and is activated by PRL stimulation of T-lymphocytes. Mol Endocrinol 8:674–681[Abstract/Free Full Text]
23. Clevenger CV, Torigoe T, Reed JC 1994 Prolactin induces rapid phosphorylation and activation of prolactin receptor-associated RAF-1 kinase in a T-cell line. J Biol Chem 269:5559–5565[Abstract/Free Full Text]
24. Erwin RA, Kirken RA, Malabarba MG, Farrar WL, Rui H 1995 Prolactin activates Ras via signaling proteins SHC, growth factor receptor bound 2, and son of sevenless. Endocrinology 136:3512–3518[Abstract]
25. Rao YP, Buckley DJ, Buckley AR 1995 Rapid activation of mitogen-activated protein kinase and p21ras by prolactin and interleukin 2 in rat Nb2 node lymphoma cells. Cell Growth Differ 6:1235–1244[Abstract]
26. DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba MG, Hackett RH, Larner AC, Farrar WL 1996 Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580. Mol Cell Endocrinol 117:131–140[CrossRef][Medline]
27. Smit LS, Meyer DJ, Billestrup N, Norstedt G, Schwartz J, Carter-Su C 1996 The role of the growth hormone (GH) receptor and JAK1 and JAK2 kinases in the activation of Stats 1, 3, and 5 by GH. Mol Endocrinol 10:519–533[Abstract/Free Full Text]
28. Kovarik P, Stoiber D, Novy M, Decker T 1998 Stat1 combines signals derived from IFN- and LPS receptors during macrophage activation. EMBO J 17:3660–3668[CrossRef][Medline]
29. Nagano M, Kelly PA 1994 Tissue distribution and regulation of rat prolactin receptor gene expression. Quantitative analysis by polymerase chain reaction. J Biol Chem 269:13337–13345[Abstract/Free Full Text]
30. Higuchi K, Nawata H, Maki T, Higashizima M, Kato K, Ibayashi H 1984 Prolactin has a direct effect on adrenal androgen secretion. J Clin Endocrinol Metab 59:714–718[Abstract/Free Full Text]
31. Glasow A, Breidert M, Haidan A, Anderegg U, Kelly PA, Bornstein SR 1996 Functional aspects of the effect of prolactin (PRL) on adrenal steroidogenesis and distribution of the PRL receptor in the human adrenal gland. J Clin Endocrinol Metab 81:3103–3111[Abstract/Free Full Text]
32. Albertson BD, Sienkiewicz ML, Kimball D, Munabi AK, Cassorla F, Loriaux DL 1987 New evidence for a direct effect of prolactin on rat adrenal steroidogenesis. Endocr Res 13:317–333[Medline]
33. Mertani HC, Morel G 1995 In situ gene expression of growth hormone (GH) receptor and GH binding protein in adult male rat tissues. Mol Cell Endocrinol 109:47–61[CrossRef][Medline]
34. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA 1998 Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19:225–268[Abstract/Free Full Text]
35. Fernandez-Ruiz J, Cebeira M, Agrasal C, Tresguerres JA, Esquifino AI, Ramos JA 1987 Effect of elevated prolactin levels on the synthesis and release of catecholamines from the adrenal medulla in female rats. Neuroendocrinology 45:208–211[Medline]
36. Fernandez-Ruiz JJ, Martinez-Arrieta R, Hernandez ML, Ramos JA 1988 Possible direct effect of prolactin on catecholamine synthesis and release in rat adrenal medulla: in vitro studies. J Endocrinol Invest 11:603–608[Medline]
37. Meites J, Clemens JA 1972 Hypothalamic control of prolactin secretion. Vitam Horm 30:165–221[Medline]
38. Noel GL, Suh HK, Stone JG, Frantz AG 1972 Human prolactin and growth hormone release during surgery and other conditions of stress. J Clin Endocrinol Metab 35:840–851[Abstract/Free Full Text]
39. Siegel RA, Conforti N, Chowers I 1980 Neural pathways mediating the prolactin secretory response to acute neurogenic stress in the male rat. Brain Res 198:43–53[CrossRef][Medline]
40. Drago F, D’Agata V, Iacona T, Spadaro F, Grassi M, Valerio C, Raffaele R, Astuto C, Lauria N, Vitetta M 1989 Prolactin as a protective factor in stress-induced biological changes. J Clin Lab Anal 3:340–344[Medline]
41. Sorenson RL, Brelje TC, Hegre OD, Marshall S, Anaya P, Sheridan JD 1987 Prolactin (in vitro) decreases the glucose stimulation threshold, enhances insulin secretion, and increases dye coupling among islet B cells. Endocrinology 121:1447–1453[Abstract/Free Full Text]
42. Curry DL, Bennett LL, Li CH 1975 Dynamics of insulin release by perfused hamster (Mesocricetus auratus) pancreases: effects of hypophysectomy, bovine and human growth hormone, and prolactin. J Endocrinol 65:245–251[Abstract/Free Full Text]
43. Nielsen JH 1982 Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets. Endocrinology 110:600–606[Abstract/Free Full Text]
44. Brelje TC, Stout LE, Bhagroo NV, Sorenson RL 2004 Distinctive roles for prolactin and growth hormone in the activation of signal transducer and activator of transcription 5 in pancreatic islets of Langerhans. Endocrinology 145:4162–4175[Abstract/Free Full Text]
45. Nag S, Sanyal S, Ghosh KK, Biswas NM 1981 Prolactin suppression and spermatogenic developments in maturing rats. A quantitative study. Horm Res 15:72–77[Medline]
46. Dombrowicz D, Sente B, Closset J, Hennen G 1992 Dose-dependent effects of human prolactin on the immature hypophysectomized rat testis. Endocrinology 130:695–700[Abstract/Free Full Text]
47. Purvis K, Clausen OP, Olsen A, Haug E, Hansson V 1979 Prolactin and Leydig cell responsiveness to LH/hCG in the rat. Arch Androl 3:219–230[Medline]
48. Papadopoulos V, Drosdowsky MA, Carreau S 1986 In vitro effects of prolactin and dexamethasone on rat Leydig cell aromatase activity. Andrologia 18:79–83[Medline]
49. Rubin RT, Poland RE, Tower BB 1976 Prolactin-related testosterone secretion in normal adult men. J Clin Endocrinol Metab 42:112–116[Abstract/Free Full Text]
50. Gunasekar PG, Kumaran B, Govindarajulu P 1988 Prolactin and Leydig cell steroidogenic enzymes in the bonnet monkey (Macaca radiata). Int J Androl 11:53–59[Medline]
51. Ouhtit A, Morel G, Kelly PA 1993 Visualization of gene expression of short and long forms of prolactin receptor in rat reproductive tissues. Biol Reprod 49:528–536[Abstract]
52. Aragona C, Friesen HG 1975 Specific prolactin binding sites in the prostate and testis of rats. Endocrinology 97:677–684[Abstract/Free Full Text]
53. Charreau EH, Attramadal A, Torjesen PA, Purvis K, Calandra R, Hansson V 1977 Prolactin binding in rat testis: specific receptors in interstitial cells. Mol Cell Endocrinol 6:303–307[CrossRef][Medline]
54. Steger RW, Chandrashekar V, Zhao W, Bartke A, Horseman ND 1998 Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 139:3691–3695[Abstract/Free Full Text]
55. Binart N, Melaine N, Pineau C, Kercret H, Touzalin AM, Imbert-Bollore P, Kelly PA, Jegou B 2003 Male reproductive function is not affected in prolactin receptor-deficient mice. Endocrinology 144:3779–3782[Abstract/Free Full Text]
56. Mioni R, Gottardello F, Bordon P, Montini G, Foresta C 1992 Evidence for specific binding and stimulatory effects of recombinant human erythropoietin on isolated adult rat Leydig cells. Acta Endocrinol (Copenh) 127:459–465[Abstract/Free Full Text]
57. Foresta C, Mioni R, Bordon P, Miotto D, Montini G, Varotto A 1994 Erythropoietin stimulates testosterone production in man. J Clin Endocrinol Metab 78:753–756[Abstract]
58. Cameron DF, Murray FT, Drylie DD 1984 Ultrastructural lesions in testes from hyperprolactinemic men. J Androl 5:283–293[Abstract/Free Full Text]
59. Franks S, Jacobs HS, Martin N, Nabarro JD 1978 Hyperprolactinaemia and impotence. Clin Endocrinol (Oxf) 8:277–287[Medline]
60. Carter JN, Tyson JE, Tolis G, Van Vliet S, Faiman C, Friesen HG 1978 Prolactin-screening tumors and hypogonadism in 22 men. N Engl J Med 299:847–852[Abstract]
61. Segal S, Polishuk WZ, Ben-David M 1976 Hyperprolactinemic male infertility. Fertil Steril 27:1425–1427[Medline]
62. Saidi K, Wenn RV, Sharif F 1977 Bromocriptine for male infertility. Lancet 1:250–251[CrossRef][Medline]
63. Hermanns U, Hafez ES 1981 Prolactin and male reproduction. Arch Androl 6:95–125[Medline]
64. Hondo E, Kurohmaru M, Sakai S, Ogawa K, Hayashi Y 1995 Prolactin receptor expression in rat spermatogenic cells. Biol Reprod 52:1284–1290[Abstract]
65. Magrini G, Iselin H, Ebiner JR, Felber JP 1979 New aspects of androgens, prolactin, and ACTH interaction in men. Arch Androl 2:141–155[Medline]
66. Waeber C, Reymond O, Reymond M, Lemarchand-Beraud T 1983 Effects of hyper- and hypoprolactinemia on gonadotropin secretion, rat testicular luteinizing hormone/human chorionic gonadotropin receptors and testosterone production by isolated Leydig cells. Biol Reprod 28:167–177[Abstract]
67. Leonard MP, Nickel CJ, Morales A 1989 Hyperprolactinemia and impotence: why, when and how to investigate. J Urol 142:992–994[Medline]
68. Colao A, Vitale G, Cappabianca P, Briganti F, Ciccarelli A, De Rosa M, Zarrilli S, Lombardi G 2004 Outcome of cabergoline treatment in men with prolactinoma: effects of a 24-month treatment on prolactin levels, tumor mass, recovery of pituitary function, and semen analysis. J Clin Endocrinol Metab 89:1704–1711[Abstract/Free Full Text]
69. Shirota M, Banville D, Ali S, Jolicoeur C, Boutin JM, Edery M, Djiane J, Kelly PA 1990 Expression of two forms of prolactin receptor in rat ovary and liver. Mol Endocrinol 4:1136–1143[Abstract/Free Full Text]
70. Zhang R, Buczko E, Tsai-Morris CH, Hu ZZ, Dufau ML 1990 Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species. Biochem Biophys Res Commun 168:415–422[CrossRef][Medline]
71. Poindexter AN, Buttram Jr VC, Besch PK, Smith RG 1979 Prolactin receptors in the ovary. Fertil Steril 31:273–277[Medline]
72. Slot KA, Kastelijn J, Bachelot A, Kelly PA, Binart N, Teerds KJ 2006 Reduced recruitment and survival of primordial and growing follicles in GH receptor-deficient mice. Reproduction 131:525–532[Abstract/Free Full Text]
73. Cecim M, Kerr J, Bartke A 1995 Infertility in transgenic mice overexpressing the bovine growth hormone gene: luteal failure secondary to prolactin deficiency. Biol Reprod 52:1162–1166[Abstract]
74. Cecim M, Fadden C, Kerr J, Steger RW, Bartke A 1995 Infertility in transgenic mice overexpressing the bovine growth hormone gene: disruption of the neuroendocrine control of prolactin secretion during pregnancy. Biol Reprod 52:1187–1192[Abstract]
75. Wennbo H, Gebre-Medhin M, Gritli-Linde A, Ohlsson C, Isaksson OG, Tornell J 1997 Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J Clin Invest 100:2744–2751[Medline]
76. Huseby RA, Soares MJ, Talamantes F 1985 Ectopic pituitary grafts in mice: hormone levels, effects on fertility, and the development of adenomyosis uteri, prolactinomas, and mammary carcinomas. Endocrinology 116:1440–1448[Abstract/Free Full Text]
77. Munabi AK, Mericq V, Koppelman MC, Gelato MC, Macher AM, Albertson BD, Loriaux DL, Cassorla F 1984 The effects of prolactin on rat ovarian function. Steroids 43:631–637[CrossRef][Medline]
78. Matsuyama S, Shiota K, Takahashi M 1990 Possible role of transforming growth factor-ß as a mediator of luteotropic action of prolactin in rat luteal cell cultures. Endocrinology 127:1561–1567[Abstract/Free Full Text]
79. Frasor J, Gibori G 2003 Prolactin regulation of estrogen receptor expression. Trends Endocrinol Metab 14:118–123[CrossRef][Medline]
80. Duan WR, Parmer TG, Albarracin CT, Zhong L, Gibori G 1997 PRAP, a prolactin receptor associated protein: its gene expression and regulation in the corpus luteum. Endocrinology 138:3216–3221[Abstract/Free Full Text]
81. Albarracin CT, Parmer TG, Duan WR, Nelson SE, Gibori G 1994 Identification of a major prolactin-regulated protein as 20-hydroxysteroid dehydrogenase: coordinate regulation of its activity, protein content, and messenger ribonucleic acid expression. Endocrinology 134:2453–2460[Abstract/Free Full Text]
82. Frasor J, Park K, Byers M, Telleria C, Kitamura T, Yu-Lee LY, Djiane J, Park-Sarge OK, Gibori G 2001 Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ER and ERß transcription. Mol Endocrinol 15:2172–2181[Abstract/Free Full Text]
83. Kedzia C, Lacroix L, Ameur N, Ragot T, Kelly PA, Caillou B, Binart N 2005 Medullary thyroid carcinoma arises in the absence of prolactin signaling. Cancer Res 65:8497–8503[Abstract/Free Full Text]
84. Mullis PE, O’Donovan N, Eble A, Marti U, Diem P, Burgi U, Peter HJ 2000 Growth hormone regulates growth hormone receptor gene transcription in primary human thyroid cells. Mol Cell Endocrinol 166:111–119[CrossRef][Medline]
85. Le Moli R, Endert E, Fliers E, Mulder T, Prummel MF, Romijn JA, Wiersinga WM 1999 Establishment of reference values for endocrine tests. II: Hyperprolactinemia. Neth J Med 55:71–75[CrossRef][Medline]
86. Dugan AL, Thellin O, Buckley DJ, Buckley AR, Ogle CK, Horseman ND 2002 Effects of prolactin deficiency on myelopoiesis and splenic T lymphocyte proliferation in thermally injured mice. Endocrinology 143:4147–4151[Abstract]
87. Wagner KU, Krempler A, Triplett AA, Qi Y, George NM, Zhu J, Rui H 2004 Impaired alveologenesis and maintenance of secretory mammary epithelial cells in Jak2 conditional knockout mice. Mol Cell Biol 24:5510–5520[Abstract/Free Full Text]
88. Ormandy CJ, Binart N, Kelly PA 1997 Mammary gland development in prolactin receptor knockout mice. J Mammary Gland Biol Neoplasia 2:355–364[CrossRef][Medline]
89. Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A, Hennighausen L 1997 Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186[Abstract/Free Full Text]
90. Harris J, Stanford PM, Sutherland K, Oakes SR, Naylor MJ, Robertson FG, Blazek KD, Kazlauskas M, Hilton HN, Wittlin S, Alexander WS, Lindeman GJ, Visvader JE, Ormandy CJ 2006 Socs2 and elf5 mediate prolactin-induced mammary gland development. Mol Endocrinol 20:1177–1187[Abstract/Free Full Text]
91. Hennighausen L, Robinson GW 1998 Think globally, act locally: the making of a mouse mammary gland. Genes Dev 12:449–455[Free Full Text]
92. Mertani HC, Garcia-Caballero T, Lambert A, Gerard F, Palayer C, Boutin JM, Vonderhaar BK, Waters MJ, Lobie PE, Morel G 1998 Cellular expression of growth hormone and prolactin receptors in human breast disorders. Int J Cancer 79:202–211[CrossRef][Medline]
93. van Garderen E, van der Poel HJ, Swennenhuis JF, Wissink EH, Rutteman GR, Hellmen E, Mol JA, Schalken JA 1999 Expression and molecular characterization of the growth hormone receptor in canine mammary tissue and mammary tumors. Endocrinology 140:5907–5914[Abstract/Free Full Text]
94. Master SR, Hartman JL, D’Cruz CM, Moody SE, Keiper EA, Ha SI, Cox JD, Belka GK, Chodosh LA 2002 Functional microarray analysis of mammary organogenesis reveals a developmental role in adaptive thermogenesis. Mol Endocrinol 16:1185–1203[Abstract/Free Full Text]
95. Tanaka T, Shiu RP, Gout PW, Beer CT, Noble RL, Friesen HG 1980 A new sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum. J Clin Endocrinol Metab 51:1058–1063[Abstract/Free Full Text]
96. Walden PD, Ruan W, Feldman M, Kleinberg DL 1998 Evidence that the mammary fat pad mediates the action of growth hormone in mammary gland development. Endocrinology 139:659–662[Abstract/Free Full Text]
97. Plaut K, Ikeda M, Vonderhaar BK 1993 Role of growth hormone and insulin-like growth factor-I in mammary development. Endocrinology 133:1843–1848[Abstract/Free Full Text]
98. Flint DJ, Vernon RG 1998 Effects of food restriction on the responses of the mammary gland and adipose tissue to prolactin and growth hormone in the lactating rat. J Endocrinol 156:299–305[Abstract]
99. Zebrowska T, Siadkowska E, Zwierzchowski L, Gajewska A, Kochman K 1997 In vivo effect of growth hormone on DNA synthesis and expression of milk protein genes in the rabbit mammary gland. J Physiol Pharmacol 48:825–837[Medline]
100. Emerman JT, Leahy M, Gout PW, Bruchovsky N 1985 Elevated growth hormone levels in sera from breast cancer patients. Horm Metab Res 17:421–424[Medline]
101. Gebre-Medhin M, Kindblom LG, Wennbo H, Tornell J, Meis-Kindblom JM 2001 Growth hormone receptor is expressed in human breast cancer. Am J Pathol 158:1217–1222[Abstract/Free Full Text]
102. Riddick DH, Luciano AA, Kusmik WF, Maslar IA 1978 De novo synthesis of prolactin by human decidua. Life Sci 23:1913–1921[CrossRef][Medline]
103. DiMattia GE, Gellersen B, Duckworth ML, Friesen HG 1990 Human prolactin gene expression. The use of an alternative noncoding exon in decidua and the IM-9-P3 lymphoblast cell line. J Biol Chem 265:16412–16421[Abstract/Free Full Text]
104. Gellersen B, Bonhoff A, Hunt N, Bohnet HG 1991 Decidual-type prolactin expression by the human myometrium. Endocrinology 129:158–168[Abstract/Free Full Text]
105. Gellersen B, Kempf R, Telgmann R, DiMattia GE 1994 Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma. Mol Endocrinol 8:356–373[Abstract/Free Full Text]
106. Stewart EA, Jain P, Penglase MD, Friedman AJ, Nowak RA 1995 The myometrium of postmenopausal women produces prolactin in response to human chorionic gonadotropin and -subunit in vitro. Fertil Steril 64:972–976[Medline]
107. Handwerger S, Richards R, Markoff E 1991 Autocrine/paracrine regulation of prolactin release from human decidual cells. Ann NY Acad Sci 622:111–119[CrossRef][Medline]
108. Tyson JE 1982 The evolutionary role of prolactin in mammalian osmoregulation: effects on fetoplacental hydromineral transport. Semin Perinatol 6:216–228[Medline]
109. Ozegovic B, Milkovic S 1972 Effects of adrenocorticotrophic hormone, growth hormone, prolactin, adrenalectomy and corticoids upon the weight, protein and nucleic acid content of the female rat preputial glands. Endocrinology 90:903–908[Abstract/Free Full Text]
110. Lobie PE, Breipohl W, Lincoln DT, Garcia-Aragon J, Waters MJ 1990 Localization of the growth hormone receptor/binding protein in skin. J Endocrinol 126:467–471[Abstract/Free Full Text]
111. Oakes SR, Haynes KM, Waters MJ, Herington AC, Werther GA 1992 Demonstration and localization of growth hormone receptor in human skin and skin fibroblasts. J Clin Endocrinol Metab 75:1368–1373[Abstract]
112. Lobie PE, Garcia-Aragon J, Wang BS, Baumbach WR, Waters MJ 1992 Cellular localization of the growth hormone binding protein in the rat. Endocrinology 130:3057–3065[Abstract/Free Full Text]
113. Thomas JA, Manandhar MS, Keenan EJ, Edwards WD, Klase PA 1976 Effects of prolactin and dihydrotestosterone upon the rat prostate gland. Urol Int 31:265–271[Medline]
114. Negro-Vilar A, Saad WA, McCann SM 1977 Evidence for a role of prolactin in prostate and seminal vesicle growth in immature male rats. Endocrinology 100:729–737[Abstract/Free Full Text]
115. Wennbo H, Kindblom J, Isaksson OG, Tornell J 1997 Transgenic mice overexpressing the prolactin gene develop dramatic enlargement of the prostate gland. Endocrinology 138:4410–4415[Abstract/Free Full Text]
116. Costello LC, Franklin RB 1994 Effect of prolactin on the prostate. Prostate 24:162–166[Medline]
117. Costello LC, Liu Y, Zou J, Franklin RB 2000 Mitochondrial aconitase gene expression is regulated by testosterone and prolactin in prostate epithelial cells. Prostate 42:196–202[CrossRef][Medline]
118. Rui H, Purvis K 1987 Prolactin selectively stimulates ornithine decarboxylase in the lateral lobe of the rat prostate. Mol Cell Endocrinol 50:89–97[CrossRef][Medline]
119. Nevalainen MT, Valve EM, Makela SI, Blauer M, Tuohimaa PJ, Harkonen PL 1991 Estrogen and prolactin regulation of rat dorsal and lateral prostate in organ culture. Endocrinology 129:612–622[Abstract/Free Full Text]
120. Nevalainen MT, Valve EM, Ingleton PM, Nurmi M, Martikainen PM, Harkonen PL 1997 Prolactin and prolactin receptors are expressed and functioning in human prostate. J Clin Invest 99:618–627[Medline]
121. Tripathi Y, Mukhopadhyay AK 1988 Acid phosphatase activity and fresh tissue weights of accessory sex organs in adult male rats following pituitary transplantation. Indian J Physiol Pharmacol 32:271–277[Medline]
122. Colao A, Marzullo P, Ferone D, Spiezia S, Cerbone G, Marino V, Di Sarno A, Merola B, Lombardi G 1998 Prostatic hyperplasia: an unknown feature of acromegaly. J Clin Endocrinol Metab 83:775–779[Abstract/Free Full Text]
123. Colao A, Marzullo P, Spiezia S, Ferone D, Giaccio A, Cerbone G, Pivonello R, Di Somma C, Lombardi G 1999 Effect of growth hormone (GH) and insulin-like growth factor I on prostate diseases: an ultrasonographic and endocrine study in acromegaly, GH deficiency, and healthy subjects. J Clin Endocrinol Metab 84:1986–1991[Abstract/Free Full Text]
124. Ahonen TJ, Harkonen PL, Rui H, Nevalainen MT 2002 PRL signal transduction in the epithelial compartment of rat prostate maintained as long-term organ cultures in vitro. Endocrinology 143:228–238[Abstract/Free Full Text]
125. Arunakaran J, Balasubramanian K, Srinivasan N, Aruldhas MM, Govindarajulu P 1989 Effects of prolactin and androgens on seminal vesicular lipids of castrated mature bonnet monkeys, Macaca radiata. Indian J Exp Biol 27:329–333[Medline]
126. Arunakaran J, Balasubramanian K, Srinivasan N, Aruldhas MM, Govindarajulu P 1988 Effects of androgens, prolactin and bromocriptine on seminal vesicular enzymes of the pyruvate malate cycle involved in lipogenesis in castrated mature monkeys, Macaca radiata. Int J Androl 11:133–139[Medline]
127. Tam CC, Wong YC, Tang F 1992 Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig. Cell Tissue Res 270:105–112[CrossRef][Medline]
128. Prins GS, Cecim M, Birch L, Wagner TE, Bartke A 1992 Growth response and androgen receptor expression in seminal vesicles from aging transgenic mice expressing human or bovine growth hormone genes. Endocrinology 131:2016–2023[Abstract/Free Full Text]
129. Reddy YD, Reddy KV, Govindappa S 1985 Effect of prolactin and bromocriptine administration on epididymal function—a biochemical study in rats. Indian J Physiol Pharmacol 29:234–238[Medline]
130. Gautam R, Pereira BM 1992 The effect of ovine prolactin on the epididymal sialic acid concentration in male rats. Clin Exp Pharmacol Physiol 19:495–501[Medline]
131. Gautam R, Pereira BM 1993 Modulation in activity of some epididymal glycosidases by prolactin. Indian J Exp Biol 31:410–413[Medline]
132. Ray B, Gautam R, Gaur M, Srivastava N, Pereira BM 1994 Impact of prolactin on epididymal lipid profile in castrated rats. Indian J Exp Biol 32:299–303[Medline]
133. Gravance CG, Breier BH, Vickers MH, Casey PJ 1997 Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf (dw/dw) rats. Anim Reprod Sci 49:71–76[CrossRef][Medline]
134. Deplewski D, Rosenfield RL 1999 Growth hormone and insulin-like growth factors have different effects on sebaceous cell growth and differentiation. Endocrinology 140:4089–4094[Abstract/Free Full Text]
135. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:7239–7244[Abstract/Free Full Text]
136. Murphy LJ, Tachibana K, Friesen HG 1988 Stimulation of hepatic insulin-like growth factor-I gene expression by ovine prolactin: evidence for intrinsic somatogenic activity in the rat. Endocrinology 122:2027–2033[Abstract/Free Full Text]
137. Strain AJ, Ingleton PM 1990 Growth hormone- and prolactin-induced release of insulin-like growth factor by isolated rat hepatocytes. Biochem Soc Trans 18:1206[Medline]
138. Jahn GA, Daniel N, Jolivet G, Belair L, Bole-Feysot C, Kelly PA, Djiane J 1997 In vivo study of prolactin (PRL) intracellular signalling during lactogenesis in the rat: JAK/STAT pathway is activated by PRL in the mammary gland but not in the liver. Biol Reprod 57:894–900[Abstract]
139. Berlanga JJ, Garcia-Ruiz JP, Perrot-Applanat M, Kelly PA, Edery M 1997 The short form of the prolactin (PRL) receptor silences PRL induction of the ß-casein gene promoter. Mol Endocrinol 11:1449–1457[Abstract/Free Full Text]
140. Kazansky AV, Raught B, Lindsey SM, Wang YF, Rosen JM 1995 Regulation of mammary gland factor/Stat5a during mammary gland development. Mol Endocrinol 9:1598–1609[Abstract/Free Full Text]
141. Liu X, Robinson GW, Hennighausen L 1996 Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol Endocrinol 10:1496–1506[Abstract/Free Full Text]
142. Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a and Stat5b homodimers and heterodimers and their association with the glucocortiocoid receptor in mammary cells. Mol Cell Biol 18:1783–1792[Abstract/Free Full Text]
143. Yang J, Kennelly JJ, Baracos VE 2000 Physiological levels of Stat5 DNA binding activity and protein in bovine mammary gland. J Anim Sci 78:3126–3134[Abstract/Free Full Text]
144. Bogorad RL, Ostroukhova TY, Orlova AN, Rubtsov PM, Smirnova OV 2006 Long isoform of prolactin receptor predominates in rat intrahepatic bile ducts and further increases under obstructive cholestasis. J Endocrinol 188:345–354[Abstract/Free Full Text]
145. Salmon Jr WD, Daughaday WH 1957 A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825–836[Medline]
146. Royster M, Driscoll P, Kelly PA, Freemark M 1995 The prolactin receptor in the fetal rat: cellular localization of messenger ribonucleic acid, immunoreactive protein, and ligand-binding activity and induction of expression in late gestation. Endocrinology 136:3892–3900[Abstract]
147. Nagy E, Berczi I, Sabbadini E 1992 Endocrine control of the immunosuppressive activity of the submandibular gland. Brain Behav Immun 6:418–428[CrossRef][Medline]
148. Lis M, Boucher R, Chretien M, Genest J 1977 Dependence of tonin activity in rat submaxillary gland on growth hormone and testosterone. Am J Physiol 232:E522–E525
149. Lobie PE, Waters MJ 1997 Growth hormone (GH) regulation of submandibular gland structure and function in the GH-deficient rat: upregulation of haptocorrin. J Endocrinol 154:459–466[Abstract/Free Full Text]
150. Hiramatsu M, Kashimata M, Takayama F, Minami N 1994 Developmental changes in and hormonal modulation of epidermal growth factor concentration in the rat submandibular gland. J Endocrinol 140:357–363[Abstract/Free Full Text]
151. Matsuda M, Mori T, Park MK, Kawashima S 1995 Modification of pancreatic digestive function by pituitary grafting in mice. Eur J Endocrinol 133:221–226[Abstract/Free Full Text]
152. Mainoya JR 1978 Possible influence of prolactin on intestinal hypertrophy in pregnant and lactating rats. Experientia 34:1230–1231[CrossRef][Medline]
153. Muller E, Dowling RH 1981 Prolactin and the small intestine. Effect of hyperprolactinaemia on mucosal structure in the rat. Gut 22:558–565[Abstract/Free Full Text]
154. Drago F, Continella G, Conforto G, Scapagnini U 1985 Prolactin inhibits the development of stress-induced ulcers in the rat. Life Sci 36:191–197[CrossRef][Medline]
155. Bates RW, Miller RA, Garrison MM 1962 Evidence in the hypophysectomized pigeon of a synergism among prolactin, growth hormone, thyroxine and prednisone upon weight of the body, digestive tract, kidney and fat stores. Endocrinology 71:345–360[Medline]
156. Orme SM, McNally RJ, Cartwright RA, Belchetz PE 1998 Mortality and cancer incidence in acromegaly: a retrospective cohort study. United Kingdom Acromegaly Study Group. J Clin Endocrinol Metab 83:2730–2734[Abstract/Free Full Text]
157. Okada A, Kinoshita Y, Maekawa T, Hassan MS, Kawanami C, Asahara M, Matsushima Y, Kishi K, Nakata H, Naribayashi Y, Chiba T 1996 Erythropoietin stimulates proliferation of rat-cultured gastric mucosal cells. Digestion 57:328–332[Medline]
158. Lappin TR, Maxwell AP, Johnston PG 2002 EPO’s alter ego: erythropoietin has multiple actions. Stem Cells 20:485–492[CrossRef][Medline]
159. Duncan MJ, Goldman BD 1985 Physiological doses of prolactin stimulate pelage pigmentation in Djungarian hamster. Am J Physiol 248:R664–R667
160. Girolomoni G, Phillips JT, Bergstresser PR 1993 Prolactin stimulates proliferation of cultured human keratinocytes. J Invest Dermatol 101:275–279[CrossRef][Medline]
161. Nicoll CS 1977 Physiological actions of prolactin. In: Handbook of physiology. Section 7. Endocrinology. Bethesda, MD: American Physiological Society
162. Curlewis JD, Loudon AS, Milne JA, McNeilly AS 1988 Effects of chronic long-acting bromocriptine treatment on liveweight, voluntary food intake, coat growth and breeding season in non-pregnant red deer hinds. J Endocrinol 119:413–420[Abstract/Free Full Text]
163. Ibraheem M, Galbraith H, Scaife J, Ewen S 1994 Growth of secondary hair follicles of the Cashmere goat in vitro and their response to prolactin and melatonin. J Anat 185(Pt 1):135–142
164. Rudman D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L, Rudman IW, Mattson DE 1990 Effects of human growth hormone in men over 60 years old. N Engl J Med 323:1–6[Abstract/Free Full Text]
165. Foitzik K, Krause K, Nixon AJ, Ford CA, Ohnemus U, Pearson AJ, Paus R 2003 Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen. Am J Pathol 162:1611–1621[Abstract/Free Full Text]
166. Craven AJ, Ormandy CJ, Robertson FG, Wilkins RJ, Kelly PA, Nixon AJ, Pearson AJ 2001 Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice. Endocrinology 142:2533–2539[Abstract/Free Full Text]
167. Foitzik K, Krause K, Conrad F, Nakamura M, Funk W, Paus R 2006 Human scalp hair follicles are both a target and a source of prolactin, which serves as an autocrine and/or paracrine promoter of apoptosis-driven hair follicle regression. Am J Pathol 168:748–756[Abstract/Free Full Text]
168. Yu-Lee LY 1997 Molecular actions of prolactin in the immune system. Proc Soc Exp Biol Med 215:35–52[CrossRef][Medline]
169. Rui H 2000 Prolactin. In: Oppenheim J, Feldman M, eds. Cytokine reference, a compendium of cytokines and other mediators of host defense Vol. 1–2. St. Louis: Elsevier Science Academic Press
170. Goffin V, Bouchard B, Ormandy CJ, Weimann E, Ferrag F, Touraine P, Bole-Feysot C, Maaskant RA, Clement-Lacroix P, Edery M, Binart N, Kelly PA 1998 Prolactin: a hormone at the crossroads of neuroimmunoendocrinology. Ann NY Acad Sci 840:498–509[CrossRef][Medline]
171. Horseman ND, Zhao W, Montecino-Rodriguez E, Tanaka M, Nakashima K, Engle SJ, Smith F, Markoff E, Dorshkind K 1997 Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 16:6926–6935[CrossRef][Medline]
172. Bouchard B, Ormandy CJ, Di Santo JP, Kelly PA 1999 Immune system development and function in prolactin receptor-deficient mice. J Immunol 163:576–582[Abstract/Free Full Text]
173. Hull KL, Thiagarajah A, Harvey S 1996 Cellular localization of growth hormone receptors/binding proteins in immune tissues. Cell Tissue Res 286:69–80[CrossRef][Medline]
174. Sela S, Shurtz-Swirski R, Sharon R, Manaster J, Chezar J, Shkolnik G, Shapiro G, Shasha SM, Merchav S, Kristal B 2001 The polymorphonuclear leukocyte—a new target for erythropoietin. Nephron 88:205–210[CrossRef][Medline]
175. Tsuruta T, Tani K, Shimane M, Ozawa K, Takahashi S, Tsuchimoto D, Takahashi K, Nagata S, Sato N, Asano S 1996 Effects of myeloid cell growth factors on alkaline phosphatase, myeloperoxidase, defensin and granulocyte colony-stimulating factor receptor mRNA expression in haemopoietic cells of normal individuals and myeloid disorders. Br J Haematol 92:9–22[CrossRef][Medline]
176. Kelly PA, Ali S, Rozakis M, Goujon L, Nagano M, Pellegrini I, Gould D, Djiane J, Edery M, Finidori J, Postel-Vinay MC 1993 The growth hormone/prolactin receptor family. Recent Prog Horm Res 48:123–164[Medline]
177. Pellegrini I, Lebrun JJ, Ali S, Kelly PA 1992 Expression of prolactin and its receptor in human lymphoid cells. Mol Endocrinol 6:1023–1031[Abstract/Free Full Text]
178. De Mello-Coelho V, Savino W, Postel-Vinay MC, Dardenne M 1998 Role of prolactin and growth hormone on thymus physiology. Dev Immunol 6:317–323[Medline]
179. Hanley MB, Napolitano LA, McCune JM 2005 Growth hormone-induced stimulation of multilineage human hematopoiesis. Stem Cells 23:1170–1179[CrossRef][Medline]
180. Nagy ZS, Ross J, Cheng H, Stepkowski SM, Kirken RA 2004 Regulation of lymphoid cell apoptosis by Jaks and Stats. Crit Rev Immunol 24:87–110[CrossRef][Medline]
181. Lombardi G, Colao A, Ferone D, Marzullo P, Orio F, Longobardi S, Merola B 1997 Effect of growth hormone on cardiac function. Horm Res 48(Suppl 4):38–42
182. Lombardi G, Colao A, Cuocolo A, Longobardi S, Di Somma C, Orio F, Merola B, Nicolai E, Salvatore M 1997 Cardiological aspects of growth hormone and insulin-like growth factor-I. J Pediatr Endocrinol Metab 10:553–560[Medline]
183. Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN 1993 JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74:227–236[CrossRef][Medline]
184. Damen JE, Wakao H, Miyajima A, Krosl J, Humphries RK, Cutler RL, Krystal G 1995 Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation. EMBO J 14:5557–5568[Medline]
185. Chretien S, Varlet P, Verdier F, Gobert S, Cartron JP, Gisselbrecht S, Mayeux P, Lacombe C 1996 Erythropoietin-induced erythroid differentiation of the human erythroleukemia cell line TF-1 correlates with impaired STAT5 activation. EMBO J 15:4174–4181[Medline]
186. Klingmuller U, Bergelson S, Hsiao JG, Lodish HF 1996 Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5. Proc Natl Acad Sci USA 93:8324–8328[Abstract/Free Full Text]
187. Quelle FW, Wang D, Nosaka T, Thierfelder WE, Stravopodis D, Weinstein Y, Ihle JN 1996 Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. Mol Cell Biol 16:1622–1631[Abstract]
188. Kelly PA, Djiane J, Postel-Vinay MC, Edery M 1991 The prolactin/growth hormone receptor family. Endocr Rev 12:235–251[Abstract/Free Full Text]
189. Kopchick JJ, Andry JM 2000 Growth hormone (GH), GH receptor, and signal transduction. Mol Genet Metab 71:293–314[CrossRef][Medline]
190. Sadowski CL, Wheeler TT, Wang LH, Sadowski HB 2001 GH regulation of IGF-I and suppressor of cytokine signaling gene expression in C2C12 skeletal muscle cells. Endocrinology 142:3890–3900[Abstract/Free Full Text]
191. Chow JC, Ling PR, Qu Z, Laviola L, Ciccarone A, Bistrian BR, Smith RJ 1996 Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5, but not insulin receptor substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo. Endocrinology 137:2880–2886[Abstract]
192. Jorgensen JO, Jessen N, Pedersen SB, Vestergaard E, Gormsen L, Lund SA, Billestrup N 2006 Growth hormone receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus. Am J Physiol Endocrinol Metab 291:E899–E905
193. Isaksson OG, Jansson JO, Gause IA 1982 Growth hormone stimulates longitudinal bone growth directly. Science 216:1237–1239[Abstract/Free Full Text]
194. Madsen K, Friberg U, Roos P, Eden S, Isaksson O 1983 Growth hormone stimulates the proliferation of cultured chondrocytes from rabbit ear and rat rib growth cartilage. Nature 304:545–547[CrossRef][Medline]
195. Sandstedt J, Tornell J, Norjavaara E, Isaksson OG, Ohlsson C 1996 Elevated levels of growth hormone increase bone mineral content in normal young mice, but not in ovariectomized mice. Endocrinology 137:3368–3374[Abstract]
196. Clement-Lacroix P, Ormandy C, Lepescheux L, Ammann P, Damotte D, Goffin V, Bouchard B, Amling M, Gaillard-Kelly M, Binart N, Baron R, Kelly PA 1999 Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice. Endocrinology 140:96–105[Abstract/Free Full Text]
197. Rui H, Nevalainen MT 2003 Prolactin. In: Thomson A, Lotze MT, eds. Cytokine handbook. 4th ed. London: Elsevier Science, Ltd.; 113–146
198. Riddle O, Lahr E, Bern H 1935 Maternal behaviour induced in virgin rats by prolactin. Proc Soc Exp Biol Med 32:730–734[CrossRef]
199. Lucas BK, Ormandy CJ, Binart N, Bridges RS, Kelly PA 1998 Null mutation of the prolactin receptor gene produces a defect in maternal behavior. Endocrinology 139:4102–4107[Abstract/Free Full Text]
200. Walsh RJ, Slaby FJ, Posner BI 1987 A receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid. Endocrinology 120:1846–1850[Abstract/Free Full Text]
201. Walsh RJ, Posner BI, Kopriwa BM, Brawer JR 1978 Prolactin binding sites in the rat brain. Science 201:1041–1043[Abstract/Free Full Text]
202. Banks WA, Kastin AJ, Durham DA 1989 Bidirectional transport of interleukin-1 across the blood-brain barrier. Brain Res Bull 23:433–437[CrossRef][Medline]
203. Banks WA, Ortiz L, Plotkin SR, Kastin AJ 1991 Human interleukin (IL) 1, murine IL-1 and murine IL-1ß are transported from blood to brain in the mouse by a shared saturable mechanism. J Pharmacol Exp Ther 259:988–996[Abstract/Free Full Text]
204. Banks WA, Kastin AJ, Gutierrez EG 1994 Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci Lett 179:53–56[CrossRef][Medline]
205. Gutierrez EG, Banks WA, Kastin AJ 1993 Murine tumor necrosis factor is transported from blood to brain in the mouse. J Neuroimmunol 47:169–176[CrossRef][Medline]
206. Pan W, Banks WA, Kastin AJ 1997 Permeability of the blood-brain and blood-spinal cord barriers to interferons. J Neuroimmunol 76:105–111[CrossRef][Medline]
207. Pan W, Kastin AJ 1999 Penetration of neurotrophins and cytokines across the blood-brain/blood-spinal cord barrier. Adv Drug Deliv Rev 36:291–298[CrossRef][Medline]
208. Belchetz PE, Ridley RM, Baker HF 1982 Studies on the accessibility of prolactin and growth hormone to brain: effect of opiate agonists on hormone levels in serial, simultaneous plasma and cerebrospinal fluid samples in the rhesus monkey. Brain Res 239:310–314[CrossRef][Medline]
209. Dubey AK, Herbert J, Martensz ND, Beckford U, Jones MT 1983 Differential penetration of three anterior pituitary peptide hormones into the cerebrospinal fluid of rhesus monkeys. Life Sci 32:1857–1863[CrossRef][Medline]
210. Akmal M, Tuma S, Goldstein DA, Pattabhiraman R, Bernstein L, Massry SG 1984 Intact and carboxyterminal PTH do not cross the blood-cerebrospinal fluid barrier. Proc Soc Exp Biol Med 176:434–437[CrossRef][Medline]
211. Pan W, Yu Y, Cain CM, Nyberg F, Couraud PO, Kastin AJ 2005 Permeation of growth hormone across the blood-brain barrier. Endocrinology 146:4898–4904[Abstract/Free Full Text]
212. Yoshizato H, Fujikawa T, Soya H, Tanaka M, Nakashima K 1998 The growth hormone (GH) gene is expressed in the lateral hypothalamus: enhancement by GH-releasing hormone and repression by restraint stress. Endocrinology 139:2545–2551[Abstract/Free Full Text]
213. Nyberg F, Burman P 1996 Growth hormone and its receptors in the central nervous system—location and functional significance. Horm Res 45:18–22[Medline]
214. Lai Z, Roos P, Zhai O, Olsson Y, Fholenhag K, Larsson C, Nyberg F 1993 Age-related reduction of human growth hormone-binding sites in the human brain. Brain Res 621:260–266[CrossRef][Medline]
215. Yamaji R, Okada T, Moriya M, Naito M, Tsuruo T, Miyatake K, Nakano Y 1996 Brain capillary endothelial cells express two forms of erythropoietin receptor mRNA. Eur J Biochem 239:494–500[Medline]
216. Morishita E, Masuda S, Nagao M, Yasuda Y, Sasaki R 1997 Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 76:105–116[Medline]
217. Nagai A, Nakagawa E, Choi HB, Hatori K, Kobayashi S, Kim SU 2001 Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture. J Neuropathol Exp Neurol 60:386–392[Medline]
218. Lai ZN, Emtner M, Roos P, Nyberg F 1991 Characterization of putative growth hormone receptors in human choroid plexus. Brain Res 546:222–226[CrossRef][Medline]
219. Bern H, Nicoll C 1968 The comparative endocrinology of prolactin. Recent Prog Horm Res 24:681–720[Medline]
220. Nicoll CS 1980 Ontogeny and evolution of prolactin’s functions. Fed Proc 39:2563–2566[Medline]
221. Ben-Jonathan N, Mershon JL, Allen DL, Steinmetz RW 1996 Extrapituitary prolactin: distribution, regulation, functions, and clinical aspects. Endocr Rev 17:639–669[Abstract/Free Full Text]
222. Richardson BP 1973 Evidence for a physiological role of prolactin in osmoregulation in the rat after its inhibition by 2-bromo-ergokryptine. Br J Pharmacol 47:623P–624P
223. Pippard C, Baylis PH 1986 Prolactin stimulates Na+-K+-ATPase activity located in the outer renal medulla of the rat. J Endocrinol 108:95–99[Abstract/Free Full Text]
224. Sakai Y, Hiraoka Y, Ogawa M, Takeuchi Y, Aiso S 1999 The prolactin gene is expressed in the mouse kidney. Kidney Int 55:833–840[CrossRef][Medline]
225. Hamosh M, Hamosh P 1977 The effect of prolactin on the lecithin content of fetal rabbit lung. J Clin Invest 59:1002–1005[Medline]
226. Hauth JC, Parker Jr CR, MacDonald PC, Porter JC, Johnston JM 1978 A role of fetal prolactin in lung maturation. Obstet Gynecol 51:81–88[Medline]
227. Ling C, Billig H 2001 PRL receptor-mediated effects in female mouse adipocytes: PRL induces suppressors of cytokine signaling expression and suppresses insulin-induced leptin production in adipocytes in vitro. Endocrinology 142:4880–4890[Abstract/Free Full Text]
228. Ling C, Kindblom J, Wennbo H, Billig H 2001 Increased resistin expression in the adipose tissue of male prolactin transgenic mice and in male mice with elevated androgen levels. FEBS Lett 507:147–150[CrossRef][Medline]
229. Dreyfus PA, Chretien F, Chazaud B, Kirova Y, Caramelle P, Garcia L, Butler-Browne G, Gherardi RK 2004 Adult bone marrow-derived stem cells in muscle connective tissue and satellite cell niches. Am J Pathol 164:773–779[Abstract/Free Full Text]

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