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Endocrinology Vol. 141, No. 10 3527-3533
Copyright © 2000 by The Endocrine Society

ARTICLES

Hepatic Growth Hormone Signaling in the Late Gestation Fetal Rat1

Chanika Phornphutkul, G. Peter Frick, H. Maurice Goodman, Susan A. Berry and Philip A. Gruppuso

Department of Pediatrics, Brown University and Rhode Island Hospital (C.P., P.A.G.), Providence, Rhode Island 02903; Department of Physiology, University of Massachusetts Medical School (G.P.F., H.M.G.), Worcester, Massachusetts 01655; and Department of Pediatrics, University of Minnesota (S.A.B.), Minneapolis, Minnesota 55455

Address all correspondence and requests for reprints to: Philip A. Gruppuso, M.D., Department of Pediatrics, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: philip_gruppuso{at}brown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of GH in the developing fetus is poorly understood. Several studies have demonstrated a limited role for GH in late fetal life. In fact, few data are available regarding GH signal transduction in the late gestation fetus. We therefore focused on a comparison of hepatic GH signaling in near-term fetal rats [embryonic day 19 (E19)] and adult rats using a combination of in vitro studies employing hepatocytes in primary culture and in vivo studies. We found that GH receptor (GHr) binding was comparable in fetal liver and adult liver. The long isoform of the GHr underwent tyrosine phosphorylation in response to GH stimulation of E19 fetal hepatocytes in a manner similar to that seen in cultured adult hepatocytes. Furthermore, downstream signaling via the Janus kinase-2 tyrosine kinase, STAT1 (signal transducer and activator of transcription), and STAT5 was also intact in both, as demonstrated by the tyrosine phosphorylation of these signaling proteins. To confirm the relevance of these findings to the in vivo situation, GH was directly administered by ip injection to E19 fetal and adult rats. In both cases, tyrosine phosphorylation of STAT5 was markedly and rapidly induced. Finally, transfection of E19 fetal hepatocytes with GH-responsive reporter elements [Spi2.1(-275/+85)-CAT and 8xGHRE-TKCAT] demonstrated intact transcriptional regulation. Our data indicate that GHr abundance and activity as well as downstream GH signaling are similar in the late gestation fetal rat and in the adult and that these mechanisms appear capable of supporting physiological GH functions in the developing liver.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL established that GH has a critical role in mediating postnatal growth. However, the role of GH during late fetal life is not well established. GH-deficient rats are normal size at birth, and the onset of growth retardation is not usually apparent until day 15 of postnatal life (1). Birth weight and body size of GH receptor/GH-binding protein (GHr/GHBP) knockout mice are not different from those of wild-type mice (2). GH mediates somatic growth by promoting the production of insulin-like growth factor I in the liver and in other target tissues (3). However, insulin-like growth factor I messenger RNA (mRNA) has been reported to be low or undetectable in fetal liver from embryonic day 19 (E19) fetal rats (4, 5). Levels remain low in newborn rats until after day 15 of life (5), coinciding with the apparent emergence of GH-dependent somatic growth.

GHr and GHBP mRNA abundance have been reported to be low or undetectable in E19 fetal liver (6, 7, 8). Berry et al. (4) reported a level of hepatic GHr mRNA in late gestation fetal rats that approximated 10% of that seen in the adult. Mathews et al. (8) reported similar findings. Taken together with the aforementioned studies, these observations led to the idea that there exists a relative resistance to GH action in late gestation mammalian fetuses, and that this limited GH responsiveness can be attributed to low GHr abundance. However, relatively few studies have been performed that indicate the presence of intact GH signaling during late gestation. Ymer et al. (9) showed that GHBP is present in serum from fetal rabbits despite the low level of GHr binding. Of note, two serine protease inhibitor genes that are subject to transcriptional regulation by GH, Spi 2.1 and Spi 2.3, were found by Berry and co-workers to be expressed in fetal liver on E20 (4). This expression pattern correlated temporally with a substantial increase in plasma GH seen in late fetal and early neonatal life (10, 11). These available data suggest that GH may be an active metabolic hormone in late fetal life. The possibility that GH action may be limited by GHr abundance, although inferred from available data, has not been directly addressed, in that GHr binding and postreceptor signaling have not been assessed in the late gestation mammalian fetus.

The mechanism of signal transduction in response to GH has been partly elucidated in recent years. The binding of GH to its specific cell surface receptor results in the activation of multiple signal transduction pathways, including the Janus kinase-2 (JAK2)-STAT (signal transducer and activator of transcription) pathway (12, 13, 14). JAK2 has been shown to associate with the GHr (12). Ligation of the receptor stimulates tyrosine phosphorylation of JAK2, JAK2-mediated tyrosine phosphorylation of the GHr, and tyrosine phosphorylation of multiple signaling proteins belonging to the STAT family (12, 14). Although involved in GH signaling, transcriptional regulation by STAT proteins is a key event in signaling by numerous cytokines (15) as well as growth factors such as epidermal growth factor (EGF) (16). In the case of GH, receptor activation has been shown to stimulate the tyrosine phosphorylation and activation of multiple STAT proteins (no. 1, 3 and 5) in various cell lines (14). Furthermore, GH administration to hypophysectomized rats has been shown to stimulate tyrosine phosphorylation and activation of STAT1, -3, and -5 in the liver (17). The tyrosine phosphorylation of specific STAT proteins may differ depending on the dose of GH. Ram et al. (17) showed that although low dose GH administration to intact rats produced STAT5 tyrosine phosphorylation, only high dose GH promoted the tyrosine phosphorylation of STAT1.

To assess the status and potency of GH signaling in the late gestation fetus, we undertook a comparative analysis of the abundance and responsiveness of several components of the signal transduction pathway in E19 fetal and adult rats. Fetal studies were focused on day E19, because more than 80% of fetal growth in the rat occurs during the last 4 days of gestation, and because this represents the start of a period of rapid enzymic differentiation (18, 19). Given the established physiological significance and well characterized nature of hepatic GH signaling, we have focused our in vivo and in vitro studies on fetal and adult liver and hepatocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human GH (hGH; Protropin), used for receptor binding analyses, was obtained from Genentech, Inc. (South San Francisco, CA). All other studies used recombinant rat GH (rGH; NIDDK rGH-14; AFP-3699A), provided by Dr. P. A. Parlow at the National Hormone and Pituitary Program. Recombinant human EGF (hEGF) was obtained from PeproTech, Inc. (Rocky Hill, NJ). The production of antibodies directed toward the long form of the GHr (GHrL) has been described previously (20). Antibodies directed against other GH-signaling proteins were obtained from the following commercial sources: JAK2 (antibody 06–255), Upstate Biotechnology, Inc. (Lake Placid, NY); STAT1 (sc-346), STAT3 (sc-482), and STAT5 (sc-835), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antiphosphotyrosine antibody 4G10, Upstate Biotechnology, Inc.; phospho-specific STAT1, Upstate Biotechnology, Inc.; and phospho-specific STAT3, New England Biolabs, Inc. (Beverly, MA). Reagents for hepatocyte isolation and culture were described by Curran et al. (21).

Animals
Pregnant Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) of known gestation (term specified as E21) were delivered by cesarean section under pentobarbital anesthesia (50 mg/kg by ip injection) on E19. Male Sprague Dawley rats, weighing 150–175 g, were used for adult liver samples and adult hepatocyte isolations.

For in vivo experiments, fetuses were given in situ ip injections of rGH (1.5 µg/g estimated BW) or hEGF (0.5 µg/g BW) and were delivered 20 or 15 min later by cesarean section, respectively. Where noted, adult rats were also given ip injections of rGH (1.5 µg/g BW) or hEGF (0.5 µg/g), then killed 20 or 15 min later by decapitation under pentobarbital anesthesia. This procedure was carried out between 0930–1030 h, based on studies by Tannenbaum and Martin (22) who showed that this time of day is associated with attenuated basal GH secretion. All animal studies were approved by the institutional animal care and use committee at Rhode Island Hospital.

Hepatocytes isolation and primary culture
Fetal hepatocytes were isolated on E19 by collagenase digestion as described previously (21). Adult hepatocytes were isolated by collagenase perfusion (23). Fetal and adult cell suspensions were diluted for plating at 2 x 106 cells/100-mm Primaria tissue culture plate. Initial plating was carried out using MEM supplemented as described previously (21) and containing 5% FBS. After a 2-h attachment period, the medium was removed and replaced with supplemented MEM without serum but with 0.1 mg/ml BSA.

Sample preparation for immunoprecipitation and receptor binding studies
For immunoprecipitations, hepatocytes were incubated with rGH (22 ng/ml; 1 nM) or hEGF (1 µg/ml; 167 nM) at 37 C for 20 min. Cells were placed on ice, washed twice with PBS, and lysed in 1 ml (per 100-mm plate) ice-cold extraction buffer [10 mM Tris-base (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 µM sodium orthovanadate, 1% Triton X-100, and protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, and 20 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride) added just before use]. After incubation on ice for 30 min and centrifugation at 40,000 x g for 20 min, cell extracts were frozen and stored at -70 C until use. Protein content was determined using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL).

Liver homogenates were prepared using the above extraction buffer at 0.1 ml/mg tissue. After homogenization, samples were incubated on ice for 30 min and centrifuged at 40,000 x g for 20 min. The resulting supernatant was retained for analysis.

GHr binding assays
Analysis of GHr binding in liver homogenates was performed as described previously (20). Briefly, samples (50–100 µl) were incubated overnight at 23 C in 1.5-ml microfuge tubes containing 1 ml 10 mM Tris (pH 8.0), 0.14 M sodium chloride, 0.1% Triton X-100, 0.1% hemoglobin, 0.02% sodium azide, 20 ng/ml [125I]hGH, and anti-GHrL. Immune complexes were recovered by adding 25 µl of a 1:3 suspension of protein A-agarose beads (Sigma, St. Louis, MO) and gently agitating the samples on a rocker platform for 2 h at 23 C. The beads were collected by centrifugation at 16,000 x g for 1 min and washed twice with 1 ml Tris saline plus 0.1% Triton X-100 before counting.

GHr cross-linking analyses
Fetal and adult hepatocytes were incubated with [125I]hGH (6.5 ng/ml) at 27 C for 30 min with or without unlabeled hGH (5 µg/ml), then washed three times with MEM. Freshly prepared, water-soluble cross-linking reagent, 2.5 mM bis-(sulfosuccinimidyl)suberate (Pierce Chemical Co.), dissolved in MEM was added to cells. After incubation for 30 min at 4 C, cells were lysed using 1 ml/100-mm plate of 0.25 M sucrose and 1 mM EDTA containing the protease inhibitors described above. The lysates were centrifuged at 15,000 x g for 10 min. Pellets were solubilized using SDS-PAGE sample buffer and subjected to electrophoresis under reducing conditions in a 7.5% gel. After electrophoresis, dried gels were exposed to x-ray film.

Immunoprecipitation and Western immunoblotting
For all JAK and STAT immunoprecipitations, antibodies were cross-linked to protein A-Sepharose CL-4B (Pharmacia Biotech, Piscataway, NJ) before immunoprecipitation (24). Transfer to polyvinylidene difluoride (PVDF) membranes and immunological detection were performed as described previously (24). Detection employed an enhanced chemiluminescent method (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL).

Transfection and chloramphenicol acetyltransferase (CAT) assay
Fetal hepatocytes were isolated and cultured as described above. After an 18-h attachment period, transfection with Spi (serine protease inhibitor) 2.1(-275/+85; inserted into the HindIII/PstI sites of pCAT) (25) and 8xGHRE (GH-responsive element; inserted into the BamHI site of TKCAT) (25) was performed using GenePorter transfecting reagent (Gene Therapy Systems, Inc., San Diego, CA) in MEM for 18 h. Cells were then cultured for an additional 24 h in the presence or absence of 20 ng/ml rGH. At the end of 24 h, the cells were harvested for determination of CAT activity. Cell extracts (125 µl) were incubated for 3 h at 37 C with n-butyryl coenzyme A, then with [14C]chloramphenicol at 37 C for 90 min. Incubation was followed by xylene extraction. Results were expressed as counts per min incorporated into acetylated chloramphenicol as determined by direct scintillation counting (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GHr binding in liver (Fig. 1Go) was measured by immunoprecipitation-binding analysis. Results showed that the levels of GHr binding are comparable in fetal liver and adult liver (by t test, combined results for all fetal ages vs. adult, P = 0.10). Of note, we observed a modest decline in GHr binding between E17 and term (E21). Linear regression analysis showed r2 = 0.69 and P < 0.001. As our intention was to carry out signaling studies in vitro, the ability of GH to bind to GHr in E19 fetal and adult hepatocytes cultures was also examined. These studies used cross-linking of [125I]hGH to the GHr. The GH-GHr complex was detected as a 140-kDa labeled band that was sensitive to the presence of a 1000-fold excess of unlabeled GH (Fig. 2Go). Of note, negligible binding was detected in cultured fetal hepatocytes 2 h after plating. However, a marked increase in GHr was apparent by the end of the first day in culture. A similar pattern of GHr labeling was obtained using adult hepatocytes in primary culture (not shown). Our interpretation that [125I]rGH cross-linking represents GHrL (110 kDa) and not the PRL receptor (35–73 kDa) is based on the sizes of the two receptors (27).



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  		Figure 1. Abundance of GHr estimated by the amount of [125I]hGH bound after immunoprecipitation from liver extracts. Tissue homogenates were prepared with buffer containing 1% Triton X-100 and were immunoprecipitated with anti-GHrL in the presence of 20 ng/ml [125I]GH. The immunoprecipitates were recovered using protein A immobilized on agarose beads and washed to remove unbound ligand before measuring bound [125I]GH. Fetal analyses were performed in quadruplicate and are shown as individual data points. Results for adult samples are shown as the mean ± SD (n = 5).

 


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  		Figure 2. Cross-linking of GHr to cultured fetal hepatocytes. Fetal hepatocytes (3 x 106 cells/plate) were incubated with [125I]GH (6.5 ng/ml) after 0, 1, 2, or 3 days in culture (Cx). Incubation was carried in the absence (-) or presence (+) of unlabeled hGH. After 30 min, cells were cross-linked with 2.5 mM bis-(sulfosuccinimidyl) suberate, solubilized, and subjected to PAGE. The position of the 140-kDa [125I]GH/GHr complex is indicated to the right of the resulting autoradiogram. Molecular mass markers in kilodaltons are indicated to the left of the autoradiogram.

 
As an initial approach to studying GH signal transduction, we examined the tyrosine phosphorylation of GHr in response to GH treatment. Fetal and adult hepatocytes were exposed to GH or EGF. EGF, which activates STAT signaling through its own receptor tyrosine kinase, was used as a control in anticipation of additional comparative studies. The resulting cell lysates (~0.8 mg protein) were subjected to immunoprecipitation with GHrL antibody. This was followed by Western immunoblot with antiphosphotyrosine antibody. A signal at 110 kDa, the expected size for GHr, was detected in lysates from fetal hepatocytes treated with GH, but not in the control lysates (Fig. 3Go). EGF did not induce the appearance of the 110-kDa tyrosine phosphorylated band. In these initial experiments we did not see any response to GH in the adult hepatocyte samples (Fig. 3Go). However, when we increased the amount of protein to 4 mg/sample, we were able to see tyrosine phosphorylation at 110 kDa (not shown). This was interpreted as consistent with a lower level of GHr in cultured adult hepatocytes, a lower activity of the GHr kinase, or both. This discrepancy between fetal and adult hepatocyte GHr phosphorylation was observed in repeat experiments. Under all conditions, we detected a second tyrosine-phosphorylated band at approximately 48 kDa. This coincides with the size of the GHr-derived GHBP (20). However, our experiments do not confirm the identity of this band.



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  		Figure 3. Tyrosine phosphorylation of GHrL in fetal and adult hepatocytes. Hepatocytes maintained in culture for 48 h were treated with either rGH or EGF for 20 min, after which cells were lysed in extraction buffer containing 1% Triton X-100. The lysates (0.8 mg protein) were subjected to immunoprecipitation with GHrL antibody cross-linked to protein A beads. The resulting immunoprecipitates were resolved by PAGE, transferred to PVDF, and immunoblotted with phosphotyrosine antibody. The positions of the 110-kDa tyrosine-phosphorylated GHr and a second phosphorylated band (48 kDa) are indicated to the right of the autoradiogram. Molecular mass markers in kilodaltons are indicated to the left of the autoradiogram.

 
When these Western blots were stripped and reprobed with anti-GHrL, we could not detect any signal from either fetal or adult samples. We attributed this to the low content of GHr and the limited sensitivity of the Western immunoblotting procedure. To confirm that immunoreactive GHr derived from rat liver migrated at an apparent molecular mass of 110 kDa, we analyzed a liver homogenate from a pregnant rat, which is known to possess an elevated GHr content (20). The immunoreactive 110-kDa GHr could indeed be detected in this sample (not shown). In addition, the apparent increase in GHr abundance that was seen in cross-linking studies was confirmed by examining immunoprecipitated cell lysates from fetal hepatocytes that were in culture for 24 or 48 h. A modest time-dependent increase in the intensity of the tyrosine-phosphorylated GHr was observed (not shown).

To confirm the functional significance of the GHr tyrosine phosphorylation, we examined the signal immediately downstream from the GHr, JAK2 phosphorylation. E19 fetal hepatocytes and adult hepatocytes were exposed to rGH after being maintained in culture for 48 h. Cell lysates were analyzed using anti-JAK2 immunoprecipitation followed by antiphosphotyrosine immunoblotting, stripping, and reprobing with anti-JAK2 antibodies. The results (Fig. 4Go) showed that GH stimulated the tyrosine phosphorylation of JAK2 in both E19 fetal and adult hepatocytes. In this and two other experiments, fetal hepatocytes showed a greater phosphotyrosine-JAK2 signal than adult hepatocytes even though JAK2 contents were comparable. Again, the GH-induced tyrosine phosphorylation of JAK2 in fetal hepatocytes increased with time in culture (not shown), correlating with the cross-linking and GHr tyrosine phosphorylation results.



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  		Figure 4. Tyrosine phosphorylation of JAK2 in fetal and adult hepatocytes. Hepatocytes were maintained in culture for 48 h, then studied after no additions (C; control) or after exposure to rGH for 20 min. Cells were lysed in extraction buffer containing 1% Triton X-100. Lysates (0.8 mg protein) were immunoprecipitated with JAK2 antibody beads, resolved by PAGE, transferred to PVDF, and immunoblotted with antibodies directed toward phosphotyrosine (upper panel). The immunoblot was then stripped and reprobed with JAK2 antibody (lower panel). The position of the 130-kDa JAK2 protein is indicated to the right of the autoradiograms.

 
The signal immediately downstream from JAK activation, STAT tyrosine phosphorylation, was studied next. Fetal and adult hepatocytes were kept in culture for 48 h, then stimulated with EGF or rGH for 20 min. Lysates were subjected to immunoprecipitation with antibodies directed toward STAT1, STAT3, and STAT5. The immunoprecipitates were analyzed by sequential Western blotting for STAT phosphorylation and STAT content. Results (Fig. 5Go, left panel) showed that GH stimulated the tyrosine phosphorylation of STAT1, STAT3, and STAT5 in cultured adult hepatocytes. STAT1 and STAT5 were phosphorylated in response to GH in cultured fetal hepatocytes. STAT3 did not show GH-stimulated phosphorylation. These results were confirmed in three independent experiments. As EGF has been shown to stimulate the hepatic tyrosine phosphorylation and activation of STAT1, STAT3, and STAT5 in the adult mouse liver (16) and adult rat hepatocytes (28), we chose to use EGF as a comparison. EGF stimulated tyrosine phosphorylation of STAT1 and STAT5, but only in adult hepatocytes. No response was observed in fetal hepatocytes. Of note, STAT1, STAT3, and STAT5 contents, determined by Western immunoblotting, were similar in fetal and adult hepatocytes. An additional experiment (Fig. 5Go, right panel) was performed to directly compare STAT5 content and phosphorylation in fetal and adult hepatocytes. For this experiment, the two were prepared and analyzed in parallel. The results showed that fetal hepatocytes responded to GH stimulation with a higher level of STAT5 phosphorylation than adult hepatocytes. An additional difference, a decrease in total STAT5 content after adult, but not fetal, hepatocyte stimulation with GH, was consistently observed. This indicates that the STAT5 protein turnover rate after GH activation may differ in fetal vs. adult hepatocytes.



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  		Figure 5. Left panel, Tyrosine phosphorylation of STAT1, -3, and -5 in cultured fetal and adult hepatocytes in response to rGH and EGF. Cells were maintained in culture for 48 h, then lysed after no additions (C; control), or exposure to rGH or EGF for 20 min. Cells were lysed in extraction buffer containing 1% Triton X-100. Cell lysates were immunoprecipitated with antibodies directed toward STAT1, STAT3, or STAT5. The immunoprecipitates were resolved by PAGE and transferred to PVDF. The resulting Western blots were probed with phospho-STAT1 (for STAT1 immunoprecipitates), phospho-STAT3 (for STAT3 immunoprecipitates), or phosphotyrosine antibody (for STAT5 immunoprecipitates; upper panel for each group). Blots were then stripped and reprobed with anti-STAT antibodies (lower panel for each group). Numbers to the right of the autoradiograms indicate molecular mass in kilodaltons. Note that these immunoblots were exposed for varying periods of time to optimize the signals obtained. Therefore, direct fetal/adult comparisons are not possible. Right panel, Tyrosine phosphorylation of STAT5 in cultured fetal and adult hepatocytes in response to rGH. Duplicate cell lysates were prepared as above and analyzed for phospho- and total STAT5. Numbers to the right of the autoradiograms indicate molecular mass in kilodaltons.

 
As we were able to demonstrate that the GH signaling pathways are intact in fetal hepatocytes in vitro, we were interested in evaluating the functional response to GH in hepatocytes. Fetal and adult hepatocytes were transiently transfected with two GH-responsive plasmids [Spi2.1 (-275/+85)-CAT and 8xGHRE-TKCAT] (29) to study the functional response to GH. Transfection with empty vector, pbl-CAT, was used as a control. Both response elements exhibited GH sensitivity (Fig. 6Go). We interpreted our results as indicating that transcriptional regulation downstream from the GH receptor is intact in cultured fetal rat hepatocytes. Because fetal and adult hepatocytes require different transfection conditions, we did not attempt to perform direct comparisons for GH-mediated transcriptional activation.



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  		Figure 6. Functional response to rGH in fetal hepatocytes transfected with Spi2.1-CAT and 8xGHRE-TKCAT. Fetal hepatocytes were transfected with either of two GH response elements linked to CAT, Spi2.1-CAT, or 8xGHRE-TKCAT. Control cells, transfected with pbl-CAT, were also studied. Cell lysates were prepared either before ({square}) or after ({blacksquare}) exposure to rGH for 24 h. Lysates were analyzed for CAT activity. Results are expressed as the mean ± 1 SD for three wells per condition.

 
As we were able to demonstrate intact fetal hepatocyte GH signaling in vitro, we returned to the in vivo situation. E19 fetal and adult male rats received rGH by ip injection. This led to similar, but barely detectable, levels of JAK2 phosphorylation in E19 fetal and adult liver, as detected by Western immunoblotting (not shown). However, the same liver homogenates immunoprecipitated with STAT5 antibody showed a clear phosphotyrosine signal in response to GH in both fetuses and adults (Fig. 7Go, left panel). STAT5 content was similar in the two. In an additional experiment (Fig. 7Go, right panel) we compared STAT5 tyrosine phosphorylation in response to EGF vs. GH injection in E19 fetal and adult rats. Again, similar GH responses were seen in the two. Although an EGF response could be detected in adult rats, it was minimal compared with the GH response. No EGF response could be detected in the E19 fetuses.



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  		Figure 7. In vivo GH response in fetal and adult rats. Animals were injected ip with rGH or hEGF between 0930–1030 h. Livers were obtained 20 or 15 min after injection for GH and EGF, respectively. Samples were homogenized in extraction buffer containing 1% Triton X-100. Homogenates were immunoprecipitated with STAT5 antibody beads, resolved by PAGE, and transferred to PVDF. Western blots were probed with phosphotyrosine antibody (above), stripped, and reprobed with STAT5 antibody (below). Molecular mass is designated by the numbers to the right of the figure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although GHr mRNA and GHBP mRNA are low or undetectable in E19 fetal liver (6, 7), GH-inducible genes are expressed late in gestation (4), and their expression correlates with the plasma GH surge that occurs in late fetal life (10, 11). Taken together, these data indicate that GH may be physiologically active before term. Thus, we undertook a direct evaluation of GH signal transduction in late gestation rat hepatocytes and in the intact late gestation fetal rat. Our results indicate that levels of GHr binding are similar in E19 and adult liver homogenates, in contradistinction to GHr mRNA levels.

The gradual decline in hepatic GHr binding that we observed between E17 and term (E21) coincides with the decline in liver GHr mRNA expression in the rat reported by Berry et al. (4). Circulating fetal cortisol levels increase as term approaches in the rat (30). It has recently been shown in sheep that when this cortisol surge is abolished by fetal adrenalectomy, GHr mRNA expression is significantly attenuated (31). From these studies, one might anticipate a cortisol-mediated increase in GHr expression and content as fetuses approach term. Our observations that in vivo GHr binding declines and GHr binding (as determined through in vitro cross-linking analyses and signaling studies) increases with time in cultured hepatocytes may indicate that GHr expression is actively inhibited in vivo at the time of the preterm cortisol surge. The presence of glucocorticoid in our primary hepatocyte cultures may mediate this in vitro induction of GHr binding and activity. Alternatively, the increase in GHr binding and activity after hepatocyte culture may reflect the recovery of GH receptors that are lost during the process of hepatocyte isolation.

We went on to examine proximal GH signal transduction events. In fetal hepatocytes, GHr underwent tyrosine phosphorylation in response to GH, a presumed consequence of the activation of JAK-2, which was also observed. Interestingly, fetal hepatocytes appeared to be more responsive to GH relative to adult hepatocytes when these proximal signaling events were compared. Downstream signaling, as indicated by the stimulation of STAT protein phosphorylation, was also intact in fetal hepatocytes. A direct comparison of STAT5 activation in fetal and adult hepatocytes showed greater responsiveness in the fetal cells. Activation of the transcription of two GH-responsive constructs was also intact in fetal hepatocytes. A direct fetal hepatocyte/adult hepatocyte comparison was not possible for this outcome variable. The two cell types behaved very differently under similar transfection conditions, perhaps due to the high rate at which fetal hepatocytes proliferate in the absence of growth factors or serum (21). Our data, therefore, only permitted the conclusion that transcriptional activation in response to GH was intact in cultured fetal hepatocytes. Of note, the greater response to GH in cultured fetal hepatocytes compared with adult hepatocytes was not reflected in apparent in vivo differences. Not only were GH binding results similar, but STAT5 phosphorylation in response to in vivo GH administration was similar for the two developmental ages. Thus, it would appear that the culture of hepatocytes results in attenuation of GH signaling in adult cells, amplification of GH signaling in fetal cells, or both.

The physiological relevance of our in vitro findings was, to a degree, dependent on the demonstration of intact GH signaling in the fetal rat in situ. We were reluctant to employ hypophysectomy, a approach commonly used to attenuate basal GH signaling in adult rats, because this procedure would produce marked physiological perturbations in the late gestation fetuses. Instead, the strategy for adult rats involved performing studies between 0930–1030 h, when baseline GH secretion is at a nadir (22). This succeeded, in that GH signaling events were not activated in vehicle- injected control rats. Fortunately, baseline hepatic GH signaling was also negligible in unperturbed or vehicle-injected E19 fetuses. In fact, we were anticipating a significant basal level of JAK-STAT signal transduction due to the hemopoietic compartment in E19 fetal liver (32) and the key role of this pathway in hemopoiesis (33). However, we did not find basal signaling that could be attributed to a contribution from this compartment. Thus, we were able to confirm that ip administration of GH potently induces STAT5 phosphorylation in E19 fetuses, similar to the induction observed by us in intact adult rats and by others (12, 17) studying hypophysectomized adult rats.

Whether fetal and adult hepatic GH signaling pathways are the same is still open to question. Our in vitro hepatocyte studies showed subtle differences. In adult hepatocytes STAT1, STAT3, and STAT5 were tyrosine phosphorylated in response to GH, whereas in fetal hepatocytes only STAT 1 and STAT5 responded. Considering that the transcriptional actions of this system require complex events involving multiple signaling proteins and subtle temporal discrepancies in their activation (34, 35), functional differences due to our observed differences in signaling seem likely. Notwithstanding such a likelihood, the finding that GH signaling in cultured fetal hepatocytes was intact to the level of transcriptional activation of two reporter genes supports the presence of intact GH signaling in the late gestation rat fetus.

One of our reasons for undertaking these studies was our interest in mitogenic signaling during liver development. Transcription factors of the STAT family have been implicated in the regulation of cell proliferation (33). Although EGF has been shown to activate hepatic STAT signaling in adult mice (16), we previously observed significant differences in EGF-regulated, hepatic mitogenic signaling comparing fetal and adult rats (24, 36). In fact, we observed a difference between EGF activation of STAT phosphorylation in the present fetal/adult comparisons. EGF at a physiological dose did not stimulate phosphorylation of STAT1, STAT3, or STAT5 in fetal hepatocytes, whereas adult hepatocytes showed a potent STAT5 response and a small, but reproducible, STAT1 response. In vivo, we observed a small, but detectable, STAT5 phosphorylation response to the ip administration of EGF to adult rats. A similar response was not observed in E19 fetuses.

In summary, our findings support the conclusion that hepatic GH signal transduction is intact in late gestation rat fetuses. GHr binding and the ability to activate downstream signaling appear to be similar in E19 fetal and adult hepatocytes. It seems logical to conclude that at the time of the GH surge in late fetal life, developing hepatocytes are responsive to GH. Nonetheless, conclusions regarding the role and mechanisms of GH signaling in late fetal life are difficult to draw from the available data. GH deficiency in humans is generally associated with normal intrauterine growth (37, 38). On the other hand, there have been case reports of growth failure in utero and early during postnatal development in patients with neonatal GH deficiency (39, 40). In addition, it is widely recognized that newborns with GH deficiency often present with hypoglycemia (41), a component of which may relate to altered hepatic metabolic function. In animal studies, early inhibition of growth is noted after neonatal hypophysectomy (42), and treatment with anti-rGH serum reduced growth in day 2 rats (43). These data indicate that GH may have a physiological growth-regulating role during late perinatal life, a conclusion supported by our direct observations of intact GH signaling in the late gestation fetal rat.


   Acknowledgments
 
We thank Joan Boylan for her valuable advice and expertise, and Theresa Bienieki for her assistance in the performance of animal and cell culture studies.


   Footnotes
 
1 This work was supported by NIH Grants HD-24455 and HD-11343 (to P.A.G.), DK-19392 (to H.M.G.), and DK-32817 (to S.A.B.) and by a Fellowship Research Training Grant from Eli Lilly & Co. (to C.P.). Back

Received January 12, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Charlton HM, Clarke RG, Robinson IC, Goff AE, Cox BS, Bugnon C, Bloch BA 1988 Growth hormone-deficient dwarfism in the rat: a new mutation. J Endocrinol 119:51–58[Abstract]
  2. Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Cataldo L, Coschigamo K, Wagner TE, Baumann G, Kopchick JJ 1997 A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 94:13215–13220[Abstract/Free Full Text]
  3. D’Ercole J, Stiles AD, Underwood LE 1984 Tissue concentration of somatomedin C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanism of action. Proc Natl Acad Sci USA 81:935–939[Abstract/Free Full Text]
  4. Berry SA, Bergad PL, Bundy MV 1993 Expression of GH-responsive mRNA in perinatal rat liver. Am J Physiol 264:E973–E980
  5. Mathews LS, Hammer RE, Brinster RL, Palmiter RD 1988 Expression of insulin-like factor I in transgenic mice with elevated level of growth hormone is correlated with growth. Endocrinology 123:433–437[Abstract]
  6. Walker JL, Moats-Staats BM, Stiles AD, Underwood LE 1992 Tissue-specific developmental regulation of the mRNA encoding the growth hormone receptor and growth hormone binding protein in rat fetal and postnatal tissue. Pediatr Res 31:335–339[Medline]
  7. Tiong TS, Herington AC 1992 Ontogeny of mRNA for the rat GHR and serum binding protein. Mol Cell Endocrinol 83:133–141[CrossRef][Medline]
  8. Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905–9910[Abstract/Free Full Text]
  9. Ymer S, Stevenson JL, Herington AC 1989 Differences in the developmental patterns of somatotrphic and lactogenic receptors in rabbit liver cytosol. Endocrinology 125:516–523[Abstract]
  10. Rieutort M 1974 Pituitary content and plasma level of GH in fetal and weaning rats. J Endocrinol 60:261–268[Medline]
  11. Walker P, Dussault JH, Alvarado-Urbina G, Dupont A 1977 The development of the hypothalamic-pituitary axis in the neonatal rat: hypothalamic somatostatin and pituitary and serum GH concentrations. Endocrinology 101:782–787[Abstract]
  12. Han Y, Leaman DW, Watling D, Rogers NC, Groner B, Kerr IM, Wood WI, Stark GR 1996 Participation of JAK and STAT proteins in growth hormone-induced signaling. J Biol Chem 271:5947–5952[Abstract/Free Full Text]
  13. Argetsinger LS, Campbell GS, Yang X, Witthuhn BA, Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74:237–244[CrossRef][Medline]
  14. Argetsinger LS, Carter-Su C 1996 Mechanism of signaling by growth hormone receptor. Physiol Rev 76:1089–1107[Abstract/Free Full Text]
  15. Darnell JE, Kerr IM, Stark GR 1994 JAK-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421[Abstract/Free Full Text]
  16. Ruff-Jamison S, Chen K, Cohen S 1995 Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver. Proc Natl Acad Sci USA 92:4215–4218[Abstract/Free Full Text]
  17. Ram PA, Park S-H, Choi HK, Waxman DJ 1996 Growth hormone activation of STAT 1, STAT 3 and STAT5 in rat liver. J Biol Chem 270:13262–13270[Abstract/Free Full Text]
  18. Mayor F, Cuezva JM 1985 Hormonal and metabolic changes in the perinatal period. Biol Neonate 48:185–196[Medline]
  19. Greengard O 1969 Enzymic differentiation in mammalian liver. Science 163:891–895[Free Full Text]
  20. Frick GP, Tai L-R, Baumbach WR, Goodman HM 1998 Tissue distribution, turnover, and glycosylation of the long and short growth hormone receptor isoforms in rat tissues. Endocrinology 139:2824–2830[Abstract/Free Full Text]
  21. Curran TR, Bahner RI, Oh W, Gruppuso PA 1993 Mitogenic-independent DNA synthesis by fetal rat hepatocytes in primary culture. Exp Cell Res 209:53–57[CrossRef][Medline]
  22. Tannenbaum GS, Martin JB 1976 Evidence of an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562–570[Abstract]
  23. Seglen PO 1976 Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83[Medline]
  24. Boylan J, Gruppuso PA 1996 A comparative study of the hepatic mitogen-activated protein kinase and Jun-NH2-terminal kinase pathways in the late-gestation fetal rat. Cell Growth Differ 7:1261–1269[Abstract]
  25. Yoon J-B, Berry SA, Seelig S, Towle HC 1990 An inducible nuclear factor binds to a growth hormone-regulated gene. J Biol Chem 265:19947–19954[Abstract/Free Full Text]
  26. Tseng YT, Waschek JA, Padbury JF 1995 Functional analysis of the 5' flanking sequence in the ovine ß1-adrenergic receptor gene. Biochem Biophys Res Commun 215:606–612[CrossRef][Medline]
  27. Robetto EJ, Caamano CA, Fernandez HN, Dellacha JM 1989 Proteins associated with somatogenic and lactogenic receptors in microsomal membranes and intact rat hepatocytes. Biochim Biophys Acta 1013:223–230[Medline]
  28. Guren TK, Abrahamsen H, Thoresen GH, Babaie E, Berg T, Christoffersen T 1999 EGF-induced activation of STAT1, STAT3, and STAT5b is unrelated to the stimulation of DNA synthesis in cultured hepatocytes. Biochem Biophys Res Commun 258:565–71[CrossRef][Medline]
  29. Bergad PL, Shih H-M, Towle HC, Schwarzenberg SJ, Berry SA 1995 Growth hormone induction of hepatic serine protease inhibitor 2.1 transcription is mediated by a STAT5-related factor binding synergistically to two {gamma}-activated sites. J Biol Chem 270:24903–24910[Abstract/Free Full Text]
  30. Brooks AN, Hagan DM, Howe DC 1996 Neuroendocrine regulation of pituitary-adrenal function during fetal life. Eur J Endocrinol 135:153–165[Abstract]
  31. Li J, Gilmour GS, Saunders JC, Duancey MJ, Fowden AL 1999 Activation of the adult mode of ovine growth hormone receptor gene expression by cortisol during late fetal development. FASEB J 13:545–552[Abstract/Free Full Text]
  32. Greengard O, Federman M, Knox WE 1972 Cytomorphometry of developing rat liver and its application to enzymic differentiation. J Cell Biol 52:261–272[Abstract/Free Full Text]
  33. Darnell JE 1997 STATs and gene regulation. Science 277:1630–1635[Abstract/Free Full Text]
  34. Yi W, Kim S-O, Jiang J, Park S-H, Djaft AS, Waxman DJ, Frank SJ 1996 Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase. Mol Endocrinol 10:1425–1443[Abstract]
  35. Ram PA, Waxman DJ 1997 Interaction of growth hormone-activated STATs with SH2-containing phosphotyrosine phosphatase SHP-1 and nuclear JAK2 tyrosine kinase. J Biol Chem 272:17694–17702[Abstract/Free Full Text]
  36. Boylan JM, Gruppuso PA 1998 Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat. J Biol Chem 273:3784–3790[Abstract/Free Full Text]
  37. Lovinger RD, Kaplan SL, Grumbach MM 1975 Congenital hypopituitarism associated with neonatal hypoglycemia and microphallus: four cases secondary to hypothalamic hormone deficiency. J Pediatr 87:1171–81[CrossRef][Medline]
  38. Karlberg J, Albertson-Wikland K 1988 Infancy growth pattern related to growth hormone deficiency. Acta Pediatr Scand 77:385–391[Medline]
  39. Gluckman PD, Gunn AJ, Wray A, Cutfeild WS, Chatelain PG, Guilbaud O, Ambler GR, Wilton P, Albertsson-Wikland K 1992 Congenital idiopathic growth hormone deficiency associated with prenatal and early postnatal growth failure. J Pediatr 121:920–923[CrossRef][Medline]
  40. Wit JM, van Unen H 1992 Growth of infants with neonatal growth hormone deficiency. Arch Dis Child 67:920–924[Abstract]
  41. Cornblath M, Schwartz R 1991 Disorders of Carbohydrate Metabolism in Infancy, Ed 3. Blackwell, Boston
  42. Glasscock GF, Gelber SE, Lamson G, McGee-Tekula R, Rosenfeld RG 1990 Pituitary control of the neonatal rat: effect of neonatal hypophysectomy on somatic and organ growth, serum insulin-like growth factor-I and II levels and expression of IGF binding protein. Endocrinology 127:1792–1803[Abstract]
  43. Flint DJ, Gardner MJ 1989 Inhibition of neonatal rat growth and circulating concentrations of insulin like growth factor-I using an antiserum to rat growth hormone. J Endocrinol 122:79–86[Abstract]



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