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Endocrinology Vol. 142, No. 9 3764-3773
Copyright © 2001 by The Endocrine Society

ARTICLES

The Effect of GHRH on Somatotrope Hyperplasia and Tumor Formation in the Presence and Absence of GH Signaling

R. D. Kineman, L. T. Teixeira, G. V. Amargo, K. T. Coschigano, J. J. Kopchick and L. A. Frohman

Department of Medicine, University of Illinois (R.D.K., L.T.T., G.V.A., L.A.F.), Chicago, Illinois 60612; and Edison Biotechnology Institute (K.T.C., J.J.K.) and Department of Biochemical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701

Address all correspondence and requests for reprints to: Rhonda D. Kineman, Ph.D., Department of Medicine (M/C 640), University of Illinois, 1819 West Polk, Chicago, Illinois 60612.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Excessive GHRH stimulation leads to somatotrope hyperplasia and, ultimately, pituitary adenoma formation in the metallothionein promoter-driven human GHRH (hGHRH) transgenic mouse. This pituitary phenotype is similar to that observed in humans with ectopic production of GHRH. In both mice and man, GHRH hyperstimulation also results in dramatic increases in circulating GH and IGF-I. To determine whether GH/IGF-I modulates the development and growth rate of GHRH-induced pituitary tumors, pituitary growth and histology were evaluated in mice generated from cross-breeding metallothionein promoter-driven hGHRH transgenic mice with GH receptor binding protein (GHR) gene disrupted mice (GHR-/-). Expression of the hGHRH transgene in 2-month-old GHR intact (GHR+) mice resulted in the doubling of pituitary weight that was largely attributed to an increase in the number of GH-immunopositive cells. Pituitary weight of GHR+ hGHRH mice did not significantly change between 2 and 6 months of age, whereas at 12 months, weights increased up to 100-fold those of GHR+ pituitaries, and 70% of the glands contained grossly visible adenomas. All adenomas stained positively for GH, whereas some showed scattered PRL staining. Pituitaries of GHR-/- mice were half the size of those of GHR+ mice. Although reduced in size, the histological features of GHR-/- mouse pituitaries were suggestive of somatotrope hyperplasia. Despite evidence of somatotrope hyperplasia, pituitaries from GHR-/- mice as old as 28 months of age were similar in size to those of 2-month-old mice and did not show signs of adenoma formation. Expression of the hGHRH transgene in GHR-/- mice did not significantly increase pituitary size between 2 and 6 months of age. However, at 12 months the majority of GHR-/-, hGHRH pituitaries developed adenomas with mean pituitary weight and histological features similar to those of GHR+, hGHRH mice. These observations demonstrate that intact GH signaling is not required for GHRH tumor formation. Although the majority of GHR+, hGHRH and GHR-/-, hGHRH pituitaries developed tumors by 12 months of age, a small subset remained morphologically indistinct from those at 2 months of age. These observations taken together with the fact that overt tumor formation is preceded by a static pituitary growth phase between 2 and 6 months, indicates that protective mechanisms are in place to maintain pituitary mass despite hGHRH hyperstimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRH IS A hypothalamic neuropeptide that stimulates the release and synthesis of GH from pituitary somatotropes. There is also strong evidence supporting a role for GHRH in the proliferation of the somatotrope population. Spontaneous inactivating mutations in the GHRH receptor of both humans and mice (lit/lit) results in a decrease in pituitary size, which in the mouse is associated with a dramatic reduction in somatotrope numbers (1, 2, 3). These observations demonstrate that GHRH is required for expansion of the somatotrope population during early pituitary development. In addition, individuals with ectopic production of GHRH have enlarged pituitaries associated with an increase in somatotrope numbers (hyperplasia) (4, 5). In the majority of these cases, ectopic GHRH production does not lead to pituitary adenoma formation, although somatotropinomas have been observed in some (6, 7). A similar pituitary phenotype is observed in the metallothionein promoter-driven, human GHRH (MT-hGHRH) mouse, in which the hGHRH transgene is expressed throughout the body, including the pituitary and hypothalamus (8, 9, 10, 11, 12). The pituitary of the MT-hGHRH mouse is enlarged due to somatotrope hyperplasia, which is evident in early neonatal life. With advancing age (10–14 months) somatotropinomas develop in a subset of MT-hGHRH mice. Taken together, these observations indicate that GHRH is not only important for normal pituitary development, but can also exert a role in aberrant somatotrope growth and pituitary adenoma formation.

In support of a direct mitogenic action of GHRH on somatotrope proliferation, Billestrup et al. (13) reported that GHRH in the presence of serum selectively increased [3H]thymidine incorporation (a marker of DNA synthesis) in GH-immunopositive cells of adult rat pituitary cultures. Forskolin, a receptor-independent activator of adenylyl cyclase, also stimulated DNA synthesis to the same extent as did GHRH, indicating that the mitogenic actions of GHRH are mediated through a cAMP-signaling pathway. In addition, sustained activation of the cAMP pathway by constitutive activation of Gs{alpha}, induced by expression of the rat GH promoter-driven cholera toxin transgene in mice, results in enlarged pituitaries with histological features of somatotrope hyperplasia (14). Furthermore, 40% of spontaneous somatotropinomas in humans express a constituitively active form of Gs{alpha} (gsp) (15, 16).

GHRH hyperstimulation and signaling in vivo are inextricably linked to the presence of high circulating levels of GH and IGF-I. As elevated levels of GH/IGF-I have been associated with neoplastic transformation both in vivo and in vitro (17, 18), the possibility arises that the full mitogenic action of GHRH on the pituitary somatotrope population may be dependent on GH/IGF-I stimulation. A direct codependence of ligand-stimulated cAMP and IGF-I signal transduction pathways for cell division has been observed in primary thyrocytes and thyroid cell lines where the full proliferative actions of TSH is dependent on both cAMP and activation of the IGF-I receptor (19, 20, 21). A codependence of GHRH and GH/IGF-I on pituitary cell proliferation is also supported by the observation that pituitaries are half the normal size in rodent models where hypothalamic expression of GHRH is elevated and circulating IGF-I levels are low due to the absence of GH (spontaneous dwarf rat) (22, 23) or GH signaling (GH receptor/binding protein gene-disrupted mouse (GHR-/-) (24, 25, 26). However, the possibility cannot be excluded that the elevation in endogenous GHRH expression observed in spontaneous dwarf rats and GHR-/- mice may not be sufficient to elicit abnormal somatotrope proliferation and tumor formation.

It has also been argued that elevated GH/IGF-I levels may actually inhibit somatotrope proliferation and tumor formation by negative feedback regulation. IGF-I has been shown to inhibit both basal and GHRH-stimulated GH release and synthesis (27, 28). The inhibitory actions of IGF-I on GH production may be mediated by the suppression of Pit-1, a transcription factor required for GH gene expression (29). As Pit-1 expression is stimulated by GHRH and inhibited by IGF-I in vitro (30), and Pit-1 is required for the normal expansion of the somatotrope population in vivo (29), elevated levels of IGF-I may act to block the proliferative actions of GHRH. In support of this hypothesis is the observation that pituitaries of MT-bGH transgenic mice are reduced in size and contain decreased numbers of somatotropes in association with elevations in circulating IGF-I due to the expression of the bovine GH transgene (31). Alternatively, the negative correlation between elevated circulating GH/IGF-I levels and somatotrope numbers in the MT-bGH mouse may be indirectly related to the suppression of hypothalamic GHRH input by GH/IGF-I negative feedback mechanisms (32).

To determine whether GHRH hypersecretion can alter somatotrope proliferation and adenoma formation in the absence of GH signaling in vivo, the MT-hGHRH transgene was introduced into GHR-/- mice by cross-breeding the respective mouse strains, and pituitary growth and histology of the offspring were examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Evaluation of pituitary cell components of MT-hGHRH and GHR-/- mice
Pituitaries were collected from 2- to 3-month-old male MT-hGHRH mice (C57BL/6xSJL background), GHR-/- mice (129/OlaxBALBc background), and their respective wild-type littermates to determine the relative proportions of pituitary cell types in each mouse strain used in the cross-breeding studies (see below). Pituitaries were enzymatically dispersed into single cells and plated on poly-L-lysine-coated microscope slides as previously described (33). After a 1-h incubation, cells were fixed and processed for avidin-biotin-horseradish peroxidase immunocytochemistry using the following primary antisera: monkey antirat GH (1:100,000) (34), rabbit antirat PRL (Dr. A. F. Parlow, NIDDK, National Hormone and Pituitary Program (NHPP; 1:20,000), rabbit anti-TSH (NIDDK, NHPP; 1:10,000), or rabbit anti-ACTH(NIDDK, NHPP; 1:10,000). The relative proportion of each cell type in pituitary single cell preparations was determined by counting at least 500 cells on each of 3 slides/animal. All experiments were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the University of Illinois animal care committee.

Examination of pituitary phenotype in mice expressing the MT-hGHRH transgene in the presence or absence of the GHR
Heterozygote MT-hGHRH mice were cross-bred with GHR+/- mice for two successive generations to produce GHR+/+, GHR+/-, and GHR-/- mice, half of which expressed the MT-hGHRH transgene (hGHRH). Pups were weaned at 4 wk, and genotypes were determined by PCR of tail snip DNA (see below for details). Animals were killed by decapitation between 2–12 months of age. Trunk blood was collected for hormone determinations, and pituitaries were weighed and frozen for hGHRH determination or fixed in neutral buffered formalin for 24 h and subsequently rinsed and stored in 70% ethanol until paraffin embedding and sectioning for histological evaluation.

PCR genotyping
The GHR gene was disrupted by homologous recombination by inserting a neomycin resistance (neo) gene cassette into exon 4 of the GHR gene, which encodes a portion of the GH-binding domain (24). Therefore, PCR of genomic DNA was used to differentiate the wild-type GHR allele from the neo-disrupted allele by a sense primer specific for GHR gene intron 3 (5'-cct ccc aga gag act ggc tt-3') in combination with antisense primers specific for GHR intron 4 (5'-ccc tga gac ctc ctc agt tc-3') or the neo gene (5'-gct cga cat tgg gtg gaa aca t-3'). The presence of an intact GHR gene resulted in a 390-bp PCR product, whereas that of the neo-disrupted gene resulted in two PCR products of 220 and 290 bp (35). The MT-hGHRH transgenic mouse line was originally developed by transfecting a modified hGHRH minigene containing the 5'-flanking region of the hGHRH gene, exon 1, intron A, exon 2, and intron B and the cDNA sequence for exons 3–5 fused to the MT promoter (8). Therefore, the MT-hGHRH transgene was detected by PCR amplification using a human GHRH minigene exon 3 sense primer (5'-cgg tat gca gat gcc atc ttc-3') and exon 4 antisense primer (5'-ttt gtt ctg ccc aca tgc ctg-3'). The presence of the hGHRH transgene gave a single PCR product of 169 bp. The primer sequences were specific to the hGHRH transgene and did not amplify the native mouse GHRH gene. Details of the tail extraction, PCR reagents, and reaction conditions have been previously reported (35, 36).

Pituitary histology and immunocytochemistry
Pituitary sections (5 µm) from mice generated by cross-breeding MT-hGHRH and GHR-/- mice were stained with hematoxylin and eosin or processed for GH and PRL (33) and hGHRH (37) immunocytochemistry as previously described.

RIAs
Serum samples were assayed for GH by RIA using a homologous rat GH system (34), previously shown to have 100% cross-reactivity to mouse GH. Serum PRL concentrations were determined in a homologous mouse RIA using the reagents and protocol provided by NIDDK NHPP. Total serum IGF-I levels were assessed using the Nichols Institute Diagnostics human IGF-I RIA Kit (San Juan Capistrano, CA) after acid/ethanol extraction according to the manufacturer’s instructions. Serum and tissue hGHRH levels, were measured by RIA as previously reported (38, 39). The primary antiserum used in both the hGHRH RIA and hGHRH immunocytochemistry was highly specific for hGHRH and did not cross-react with other GHRH-like peptides or mouse GHRH.

Statistical analysis
The effects of genotype and age on mean pituitary weight and serum hormone levels were assessed by ANOVA using Duncan’s new multiple range test, and data were log transformed where appropriate. The effect of hGHRH transgene expression on circulating and tissue hGHRH concentrations in the presence and absence of the GHR was determined by t test. No significant differences were observed between GHR+/+ and GHR+/- mice for any parameter examined. Therefore, data from these genotypes were pooled and assigned the designation GHR+.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pituitary cell composition of pituitaries from GHR-/- and MT-hGHRH mouse strains and their respective normal littermates
Previous reports had demonstrated that pituitary weights of young adult GHR-/- mice are approximately half those of normal controls, whereas pituitary weights of MT-hGHRH mice are approximately 2-fold those of controls (25, 26). To determine whether the change in pituitary size is the result of a uniform change in all pituitary cell types or of a change in select pituitary cell types, single cell pituitary preparations from 2- to 3-month-old GHR-/- and MT-hGHRH mice and their respective wild-type littermates were immunostained for GH, PRL, TSH, and ACTH. The percentage of each pituitary cell type in each mouse strain is shown in Fig. 1Go. The percentage of GH-immunopositive cells was greater in pituitaries of GHR-/- mice compared with those of GHR-intact littermates, whereas the percentage of PRL-immunopositive cells was markedly reduced (Fig. 1AGo). In contrast, the percentages of TSH- and ACTH-immunopositive cells were similar between genotypes. The overall number of each cell type was estimated by multiplying the total number of cells recovered per pituitary by the percentage of each cell type. The total number of somatotropes per pituitary was similar in the presence and absence of the GHR, as shown in the inset to Fig. 1AGo, whereas the total number of lactotropes was markedly reduced in its absence. However, on a body weight basis, total GH cell numbers in the GHR-/- mouse pituitary were disproportionately increased, whereas PRL cell numbers were disproportionately decreased.



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  		Figure 1. Relative proportions of GH-, PRL-, TSH-, and ACTH-immunostained cells in pituitaries of 2-month-old male GHR-/- mice (A), MT-hGHRH mice (B), and their respective wild-type littermates. Dispersed pituitary cells were stained for each hormone using avidin-biotin-horseradish peroxidase immunocytochemistry. The data shown are the results from a single experiment repeated twice with similar results, where the proportions shown were derived from counting at least 500 cells on each of 3 slides for each hormone tested. Insets represent the total number of each cell type per pituitary, as calculated by multiplying the percentage of each cell type by the total number of pituitary cells recovered after enzymatic dispersion.

 
Examination of the hormone-specific cell types in the MT-hGHRH mouse pituitary also revealed an increase in the percentage of GH-immunopositive cells and a decrease in the percentage of PRL-immunopositive cells compared with nontransgenic littermates (Fig. 1BGo). In addition, the percentages of TSH- and ACTH-immunopositive cells were reduced in mice expressing the hGHRH transgene. In contrast to GHR-/- mice, the total number of GH cells in MT-hGHRH pituitaries was increased by more than 5-fold, as shown in the inset to Fig. 1BGo. The total numbers of PRL and TSH cells were also increased in MT-hGHRH pituitaries, although to a lesser extent.

The sum of the percentages of GH, PRL, TSH, and ACTH cells was greater than 100% in both groups of wild-type controls and in MT-hGHRH pituitaries. This can be explained by the fact that a subset of pituitary cells (mammosomatotropes) in normal and hyperplastic pituitaries produces both GH and PRL (40, 41, 42, 43). Therefore, these bihormonal cells would be counted twice, artificially raising the cumulative percentage of hormone-positive cells.

Effect of hGHRH transgene expression on the pituitary phenotype of 2-month-old GHR+ and GHR-/- mice
The effects of hGHRH transgene expression in the presence and absence of the GHR on circulating IGF-I, GH, body weight, and pituitary weight at 2 months of age are illustrated in Fig. 2Go. Serum IGF-I levels, body weight, and pituitary weight of GHR-/- mice were significantly less than those in GHR+ controls, whereas circulating GH levels were elevated, consistent with the original observations of Zhou et al. (24). Expression of the hGHRH transgene in GHR+ mice stimulated both GH and IGF-I levels and increased body and pituitary weight, consistent with the original reports of the MT-hGHRH mouse phenotype (8, 9). However, expression of the hGHRH transgene in GHR-/- mice did not significantly alter GH, IGF-I, body weight, or pituitary weight compared with those in GHR-/- mice not expressing the hGHRH transgene. A similar effect of genotype was observed in female mice (data not shown).



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  		Figure 2. Circulating hormone levels, body weights, and pituitary weights of 2-month-old male mice that expressed the MT-hGHRH transgene (hGHRH Tg) in the presence (GHR+) or absence (GHR-/-) of a functional GH receptor. Mice used in these studies were generated by cross-breeding MT-hGHRH and GHR-/- mice. Similar results were observed in female mice (data not shown). Within each graph, means with no letters (a, b, and c) in common are significantly different (P < 0.05).

 
GH immunocytochemical analysis of pituitary sections revealed that more cells stained positively for GH in GHR-/- (Fig. 3BGo), GHR+,hGHRH (Fig. 3CGo), and GHR-/-,hGHRH (Fig. 3DGo) pituitaries than in GHR+ controls (Fig. 3AGo). The somatotropes of GHR-/- mice (Fig. 3BGo) were smaller and more densely aggregated compared with those of GHR+ mice (Fig. 3AGo). In contrast, many somatotropes of GHR+,hGHRH mice (Fig. 3CGo) appeared larger (hypertrophied) than those of GHR+ controls. Somatotropes of GHR-/-, hGHRH mice (Fig. 3DGo), like those of GHR-/- mice, were densely packed, with some cells appearing enlarged relative to those of GHR-/- mice. To extend these observations, the number of pituitary cell nuclei were counted in a defined area (181 µm2) of hematoxylin/eosin-stained pituitary sections (two to five mice per genotype, three slides per pituitary spaced at 30-µm intervals). The number of nuclei per area in GHR-/- mice was 27% greater than that in GHR+ mice (Fig. 3Go, graph), suggesting that the mean pituitary cell size was decreased in the absence of the GHR. The number of nuclei per area in both GHR+ and GHR-/- mice expressing the hGHRH transgene was decreased by 20% and 17%, compared with their respective controls, providing additional evidence that hGHRH expression increased somatotrope cell size, i.e. hypertrophy, in both the presence and absence of the GHR. Although there were relatively more GH-positive cells in GHR-/-, GHR+,hGHRH, and GHR-/-,hGHRH pituitaries, examination of the cytoarchitecture of the pituitary in hematoxylin/eosin-stained sections revealed that cells formed branching cords and trabecular structures, as observed in normal pituitaries (data not shown). An increase in GH cell numbers with maintenance of normal pituitary cytoarchitecture is indicative of somatotrope hyperplasia.



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  		Figure 3. GH immunostaining of pituitary sections (top panel) taken from mice that did (A and C) or did not (B and D) express a functional GHR in the absence (A and B) or presence (C and D) of the MT-hGHRH transgene. Shown are representative pituitary sections from 8-wk-old female mice. Similar patterns of GH immunostaining were observed in male mice (data not shown). The graph illustrates the mean ± SEM of the number of pituitary cell nuclei per unit area (181 µm2) on hematoxylin/eosin-stained pituitary sections (three areas per pituitary, two to five pituitaries per genotype) from GHR+ and GHR-/- mice, with or without the MT-hGHRH transgene (hGHRH Tg).

 
hGHRH concentrations were measured in serum, liver, spleen, pituitary, and hypothalamus of male GHR+,hGHRH and GHR-/-,hGHRH mice to determine whether the lack of effect of hGHRH transgene expression on the overall size of the GHR-/-,hGHRH pituitary, as shown in Fig. 2Go, could be attributed to a decrease in the amount of the expressed transgene. As illustrated in Fig. 4AGo, immunoreactive hGHRH concentrations in the serum, liver, and spleen of GHR-/-, hGHRH mice were 40% of those in GHR+,hGHRH animals. As previously reported, the liver and spleen are the primary contributors to circulating hGHRH levels in the MT-hGHRH mouse, although most of this immunoreactivity, in contrast to that in the pituitary and hypothalamus, is biologically inactive (10). However, hGHRH concentrations in the pituitary and hypothalamus of GHR-/-,hGHRH mice did not significantly differ from those observed in GHR+,hGHRH mice (Fig. 4AGo). Immunocytochemical evaluation of hGHRH in pituitary sections from GHR+,hGHRH and GHR-/-, hGHRH mice demonstrated that the absence of the GHR did not reduce the relative number of cells expressing hGHRH. Taken together these results confirm that the local (pituitary and hypothalamic) production of hGHRH is not diminished in the absence of the GHR and therefore cannot explain the lack of effect of the hGHRH transgene on pituitary growth observed in the 2-month-old GHR-/-,hGHRH mouse.



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  		Figure 4. A, Serum, liver (Liv), spleen (Spl), pituitary (Pit), and hypothalamic (Htm) concentrations of immunoreactive hGHRH in GHR+,hGHRH and GHR-/-,hGHRH mice at 2 months of age. Shown are the mean ± SEM of determinations for five to eight animals per genotype. Note that the y-axis is in log scale. B, Representative examples of hGHRH immunostaining in pituitary sections from a GHR+,hGHRH and a GHR-/-,hGHRH mouse. Asterisks indicate hGHRH values of GHR-/-,hGHRH mice that significantly differ from those of GHR+, hGHRH mice (P < 0.05).

 
Effect of age and hGHRH transgene expression on the pituitary phenotype of GHR+ and GHR-/- mice
Pituitary weights of 2-, 4-, 6-, and 12-month-old GHR+ and GHR-/- mice that did or did not express the hGHRH transgene were compared to determine whether the effect of the hGHRH transgene on somatotrope proliferation in the GHR-/- mouse is age dependent. The results are summarized in Fig. 5Go. In the absence of the hGHRH transgene, pituitary weights remained relatively constant across all age groups in both GHR+ and GHR-/- male and female mice where pituitary weights of GHR-/- mice were consistently less than those of GHR+ controls (P < 0.05). Examination of pituitaries from female mice greater than 28 months of age from the original GHR-/- strain also confirmed that GHR-/- pituitaries remained smaller than those of their GHR intact littermates, and no GHR-/- pituitary showed visible signs of adenoma formation (data not shown).



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  		Figure 5. Mean pituitary weights of 2-, 4-, 6-, and 12-month (M)-old wild-type and hGHRH transgenic mice in the presence (GHR+) and absence (GHR-/-) of the GHR. Values are the mean ± SEM, and the number of animals per group is shown below each bar. Animal numbers were sufficient to test the effects of age and genotype on pituitary weights for male GHR+ and GHR+,hGHRH mice (top left panel) by two-way ANOVA. Limited numbers of animals only allowed for testing of the effects of age on male GHR-/-,hGHRH, female GHR+, hGHRH, and female GHR-/-, hGHR pituitary weights by one-way ANOVA followed by Duncan’s new multiple range test. Within each graph means with no letters (a, b, and c) in common are significantly different (P < 0.05).

 
Pituitary weights of GHR+,hGHRH male and female mice, although twice those of GHR+ controls, also remained relatively constant at 2, 4, and 6 months of age. However, at 12 months, the mean pituitary weight of GHR+,hGHRH mice markedly increased to greater than 10-fold that of GHR+ controls. A similar pattern was observed in GHR-/-, hGHRH mice, in which pituitary weights did not significantly change between 2 and 6 months of age, but exceeded those of GHR-/- mice by greater than 30-fold at 12 months. Although the mean pituitary weight was markedly increased at 12 months in mice expressing the hGHRH transgene, a subset (8%) of pituitaries exhibited gross morphology, histology, and weight similar to those observed at 2 months of age (Fig. 6Go, x). It should also be noted that although there was no statistical difference in mean pituitary weights of female GHR+,hGHRH and GHR-/-,hGHRH mice between 2 and 6 months of age, two of six and six of seven pituitaries, respectively, at 6 months were greater than twice the maximal size observed at 2 months (Fig. 6Go). This was not observed in pituitaries of male GHR+,hGHRH and GHR-/-,hGHRH suggesting that the female pituitary is more sensitive to the proliferative actions of hGHRH.



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  		Figure 6. Scatterplot of individual pituitary weights of 2-, 4-, 6-, and 12-month (M)-old male and female GHR+,hGHRH and GHR-/-, hGHRH mice. Open symbols indicate pituitaries with asymmetric morphology and grossly visible single or multiple adenomas. x, Pituitaries of 12-month-old mice that did not exceed weights observed at 2 months of age.

 
Histological evaluation of the enlarged pituitaries of 12-month-old GHR+,hGHRH and GHR-/-,hGHRH mice (Fig. 6Go, open symbols) revealed areas of hyperplasia interspersed with large areas containing closely packed homogeneous cells that had lost the normal pituitary cytoarchitecture, features indicative of an adenoma. There were also areas that had lost cellularity, resembling pseudo-alveolar and pseudo-cystic formations, in addition to hematoma formation. Within single pituitaries there were multiple adenomatous areas collectively representing more than half of the total pituitary mass. In each adenoma, all cells stained positively for GH, whereas in some, scattered PRL immunopositive cells were also present (data not shown). The relative intensity of GH staining in the adenomas was less than that observed in hyperplastic tissue. No clear genotype or sex differences were apparent in the intensity or extent of GH or PRL immunostaining within pituitary adenomas. The adenomatous features of the enlarged GHR+,hGHRH and GHR-/-,hGHRH pituitaries are virtually identical with those reported by us and others for the MT-hGHRH mouse (11, 41, 42)

Circulating GH levels in GHR+,hGHRH and GHR-/-, hGHRH mice were compared across age to determine whether the decrease in GH immunostaining in adenomas was indicative of a decrease in GH production or a decrease in GH storage and the results are presented in Fig. 7Go. Circulating GH levels were independent of sex and paralleled the increase in pituitary weight, with a marked increase at 12 months of age. Overall, GH levels in GHR-/-,hGHRH mice were higher than those in GHR+,hGHRH mice. When circulating GH levels of 12-month-old mice were adjusted for pituitary weight, the adenomatous pituitaries of both male and female GHR-/-,hGHRH mice released 6-fold more GH on a milligram wet weight basis than did those of GHR+,hGHRH mice (Fig. 8Go).



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  		Figure 7. Serum GH and PRL levels in male and female GHR+,hGHRH and GHR-/-,hGHRH mice at 2, 4, 6, and 12 months of age. Within each graph, means with no letters(a–g) in common are significantly different (P < 0.05).

 


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  		Figure 8. Circulating GH and PRL levels of 12-month-old GHR+, hGHRH and GHR-/-,hGHRH male and female mice adjusted by pituitary weight. Within each graph, means with no letters (a and b) in common are significantly different (P < 0.05).

 
Circulating PRL levels in GHR+ and GHR-/- were not significantly different at all ages tested (data not shown). Serum PRL levels in GHR+,hGHRH and GHR-/-,hGHRH mice, similar to those of GH, paralleled pituitary weight. However, serum PRL concentrations were greater in hGHRH transgenic females compared with transgenic males at all ages (Fig. 7Go). When circulating PRL levels of 12-month-old mice were adjusted for pituitary weight, the adenomatous pituitaries of female GHR+,hGHRH mice released 10-fold more PRL on a milligram wet weight basis than did those of their male counterparts (Fig. 8Go). However, these sex differences were not evident in GHR-/-,hGHRH mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current observations unequivocally demonstrate that GHR signaling and elevated IGF-I levels are not required for hGHRH transgene-induced somatotropinoma formation, as adenomas are evident in 70% of all transgenic mice by 12 months of age regardless of the presence of functional GHRs. However, hGHRH transgene expression did not appreciably alter pituitary growth of GHR-/- mice before tumor formation. In contrast, hGHRH expression in GHR-intact mice resulted in a doubling of pituitary size that was associated with a marked expansion of the somatotrope population. The inability of hGHRH to alter pituitary growth of the young mature GHR-/- mouse suggests that GHR signaling may be required for hGHRH-induced somatotrope hyperplasia. However, an alternative explanation is that endogenous GHRH levels are elevated in the GHR-/- mouse due to the lack of GH negative feedback regulation (26) and may be sufficient to maximally expand the somatotrope population, mimicking the early actions of the hGHRH transgene. This possibility is supported by our present observations and those of Asa et al. (25), who observed that pituitaries of GHR-/- mice are smaller than those of GHR-intact controls and have an increased proportion of GH-immunoreactive cells. In addition, Asa et al. (25) reported a distortion of the GHR-/- pituitary reticulin network, consistent with hyperplasia. Finally, we report herein that the absolute number of GH cells in the GHR-/- pituitary was disproportionately increased relative to body weight. Despite signs of hyperplasia, pituitary adenomas were not evident in GHR-/- mice, even in animals greater than 2 yr of age. Taken together these observations suggest the modest increase in GHRH expression, as occurs in the GHR-/- mouse in the absence of GH negative feedback, is sufficient to expand the somatotrope population, but chronic exposure to supraphysiological levels of GHRH is required to induce adenoma formation.

Although supraphysiological levels of hGHRH were present in pituitaries and hypothalami of both GHR+,hGHRH and GHR-/-,hGHRH mice, the appearance of pituitary adenomas was not preceded by a progressive increase in pituitary size. In fact, tumor formation was preceded by a static pituitary growth phase in which mean pituitary weight remained constant for a minimum of 4 months, and in 8% of 12-month-old mice expressing the hGHRH transgene, pituitary weights and gross morphology were indistinguishable from those observed at 2 months of age. Several possibilities could explain these observations. First, the expression of the hGHRH transgene during early pituitary development could result in expansion of the somatotrope stem cell population, but the adult somatotropes may become desensitized to the proliferative actions of GHRH. Alternatively, the normal somatotrope may be capable of only a finite number of divisions regardless of the time of onset and duration of GHRH hyperstimulation. Finally, GHRH hyperstimulation could result in an increase in somatotrope proliferation independently of age, but the increase in the rate of proliferation is counterbalanced by an increase in the rate of cell death in the adult gland. Although the exact mechanisms involved in the maintenance of pituitary mass in the presence of elevated GHRH remains to be determined, it is clear that sustained supraphysiological levels of GHRH secondary to expression of the hGHRH transgene ultimately led to a loss of these protective mechanisms in select cells (presumably caused by somatic mutations), thereby allowing for clonal expansion and adenoma formation.

If elevation in endogenous GHRH leads to the expansion of the somatotrope population in GHR-/- mice, why is pituitary size half that observed in GHR-intact controls? First, there was a decrease in mean pituitary cell size of the GHR-/- mouse relative to GHR-intact controls. The reduction in cell size may be in part explained by a decrease in GH storage, as a consequence of a decrease in IGF-I inhibition, which is known to block both basal and GHRH-stimulated GH release (27). Second, the number of PRL cells is markedly reduced in the GHR-/- pituitary. This decrease may be related to the decrease in circulating IGF-I levels. The requirement of IGF-I for normal lactotrope proliferation is supported by the observation that pituitaries of adult IGF-I-/- mice are approximately half the size of those of IGF-I intact controls (44). In the absence of IGF-I, the proportion of lactotropes is reduced by 50%, whereas the proportion of somatotropes is maintained. IGF-I also promotes proliferation of lactotropes in primary rat pituitary cultures as measured by [3H]thymidine incorporation (45).

Despite the decrease in the size of the lactotrope population, circulating PRL levels have been reported to be elevated in both the GHR-/- mouse (35) and in patients with Laron dwarfism (46), suggesting that an intact GHR signaling system is required to maintain normal PRL levels, potentially by suppressing hypothalamic dopaminergic input. We did not observe any significant differences between circulating PRL levels in GHR-/- mice and those in GHR+ controls in the present study. These divergent results may be related to the sampling technique, as in the present study trunk blood was collected after decapitation in unanesthetized mice, whereas in the previous report, blood of GHR-/- and GHR+ mice was collected after exposure to ether (35). Of interest in this study was the observation that the sexual dimorphic pattern of PRL levels was accentuated in GHR+,hGHRH mice bearing adenomatous pituitaries, whereas no gender differences were evident in tumor-bearing GHR-/-,hGHRH mice. As both estrogens (47) and IGF-I (45) have been shown to stimulate lactotrope proliferation, the possibility exists that the combined actions of hGHRH and estrogen on the pituitary lactotrope population may be augmented by IGF-I. Therefore, in the female GHR-/- mouse, low serum IGF-I may not allow for maximal lactotrope stimulation. We also cannot exclude the possibility that gonadal function may be compromised in the female, as it is in the male GHR-/- mouse (35), and these changes may account for the lower levels of PRL observed in GHR-/-,hGHRH female mice.

Overall, circulating GH levels in the presence of the hGHRH transgene were greater in GHR-/- mice than in GHR+ mice. These observations demonstrate that GH/IGF-I negative feedback exerts an important role in suppression of GHRH-induced GH secretion and that this counterregulatory mechanism remains intact in somatotropinomas. IGF-I inhibits both basal and GHRH-stimulated GH release as well as GH gene expression in primary rat pituitary cell cultures (27). IGF-I has also been shown to decrease GH production in human somatotropinoma cultures (48, 49). Interestingly, tumors responsive to the inhibitory actions of IGF-I in vitro were shown to produce lower levels of circulating GH in vivo (49). Despite the inhibitory effect of an intact GH signaling system on GHRH-induced GH production, this protective mechanism does not extend to the proliferative effects of GHRH, because the time of appearance and size of pituitary adenomas were similar in GHR-intact and GHR-/- mice.

In conclusion, our results demonstrate that hGHRH- induced somatotrope hyperplasia and tumor formation are independent of GH signaling and elevated circulating IGF-I. These observations also demonstrate that the appearance of GH- producing tumors in the presence and absence of GH signaling is not preceded by a progressive increase in pituitary size, demonstrating that protective mechanisms are in place to maintain pituitary mass despite hGHRH hyperstimulation.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by USPHS Grant DK-30667 (to R.D.K.), the Bane Foundation (to L.A.F.), Sensus Corp. and the State of Ohio’s Eminent Scholars Program, which includes a gift from Milton and Lawrence Goll (to J.J.K.).

Abbreviations: GHR, GH receptor; GHR+, GHR intact mice; GHR-/-, GHR/binding protein gene-disrupted mice; hGHRH, human GHRH; MT-hGHRH, metallothionein promoter-driven, hGHRH; NHPP, National Hormone and Pituitary Program.

Received February 22, 2001.

Accepted for publication May 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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