Developmental changes in the human GH receptor and its signal transduction pathways

in Journal of Endocrinology
View More View Less
  • 1 Division of Experimental Medicine, Department of Pediatrics, McGill University, Montreal, Quebec, Canada

We previously reported the presence of functional human GH receptors (hGHRs) in the human fetal hepatocyte (FH) as early as the first trimester. Interestingly, fetal serum levels of hGH are in the acromegalic range, yet certain hGH-dependent factors are expressed at very low levels (IGF-I, IGF-binding protein-3), suggesting that fetal liver has limited responsiveness to hGH. To determine whether this is due to the fetal tissue levels of hGHR or factors in the hGH/hGHR axis that might influence hGHR function, we compared hGHR isoforms and downstream signaling proteins in FH versus human adult liver (HAL). Immunoprecipitation/immunoblotting (IB) analyses found similar precursor and mature hGHR forms while RT-PCR assays of truncated (T) hGHR1279, dominant negative for the full-length (FL) receptor, showed similar T/FL mRNA ratios in FH and HAL. IB demonstrated that Janus kinase (JAK) 2, signal transducers and activators of transcription (STAT(1, 3, 5A/B)), and suppressors of cytokine signaling (SOCS(1, 2, 3, cytokine-inducible SH2-containing protein (CIS))) proteins were detectable in all FH and HAL tested (12 weeks of fetal age to 60 years); the levels were similar (STAT5B) or lower (JAK2/STAT1/STAT3/STAT5A: 38–53%, SOCS/CIS: 58–76%) in FH compared with HAL. Our studies to date demonstrate that, during hepatocyte development, hGHR levels are lower in the fetal cells but the hGHR isoforms, including the relative amount of truncated versus FL, remain unchanged. The JAK2/STAT/SOCS signaling molecules are present in the FH as early as the first trimester. However, they are generally at <50% level in postnatal liver. These data suggest that low expression of both hGHR and major hGHR signaling components may explain the limited responsiveness of the fetal cells to the high circulating levels of hGH.

Abstract

We previously reported the presence of functional human GH receptors (hGHRs) in the human fetal hepatocyte (FH) as early as the first trimester. Interestingly, fetal serum levels of hGH are in the acromegalic range, yet certain hGH-dependent factors are expressed at very low levels (IGF-I, IGF-binding protein-3), suggesting that fetal liver has limited responsiveness to hGH. To determine whether this is due to the fetal tissue levels of hGHR or factors in the hGH/hGHR axis that might influence hGHR function, we compared hGHR isoforms and downstream signaling proteins in FH versus human adult liver (HAL). Immunoprecipitation/immunoblotting (IB) analyses found similar precursor and mature hGHR forms while RT-PCR assays of truncated (T) hGHR1279, dominant negative for the full-length (FL) receptor, showed similar T/FL mRNA ratios in FH and HAL. IB demonstrated that Janus kinase (JAK) 2, signal transducers and activators of transcription (STAT(1, 3, 5A/B)), and suppressors of cytokine signaling (SOCS(1, 2, 3, cytokine-inducible SH2-containing protein (CIS))) proteins were detectable in all FH and HAL tested (12 weeks of fetal age to 60 years); the levels were similar (STAT5B) or lower (JAK2/STAT1/STAT3/STAT5A: 38–53%, SOCS/CIS: 58–76%) in FH compared with HAL. Our studies to date demonstrate that, during hepatocyte development, hGHR levels are lower in the fetal cells but the hGHR isoforms, including the relative amount of truncated versus FL, remain unchanged. The JAK2/STAT/SOCS signaling molecules are present in the FH as early as the first trimester. However, they are generally at <50% level in postnatal liver. These data suggest that low expression of both hGHR and major hGHR signaling components may explain the limited responsiveness of the fetal cells to the high circulating levels of hGH.

Introduction

The human growth hormone receptor (hGHR), a member of class I cytokine receptor superfamily, is a 620 amino acid peptide containing an extracellular hormone-binding region, a single transmembrane segment, and a cytoplasmic signaling domain. It is encoded by a single gene spanning more than 300 kb on chromosome 5p13.1-12 (Leung et al. 1987, Barton et al. 1989, Wei et al. 2006). Three hGHR isoforms have been reported to date: full-length (FL), exon 3 deleted (d3), and truncated (T). Transcripts for all three isoforms have been detected in human tissues from early in fetal life (Esposito et al. 1994, Zogopoulos et al. 1996b, Goodyer et al. 2001a).

d3 hGHR mRNAs are expressed in an individual-specific manner, likely due to a retrovirus-mediated recombination mechanism at the genomic level; they translate into a hGHR that binds specific hGH-related hormones with similar affinities as the FL receptor (Bass et al. 1991, Urbanek et al. 1992, Sobrier et al. 1993, Wickelgren et al. 1995, Zogopoulos et al. 1996b, Clackson et al. 1998, Pantel et al. 2000). Whether the d3 hGHR has a different functional ability relative to the FL form remains controversial (Dos Santos et al. 2004, Audi et al. 2006, Binder et al. 2006, Blum et al. 2006, Carrascosa et al. 2006, Hujeirat et al. 2006, Jorge et al. 2006, Pilotta et al. 2006, Buzi et al. 2007, Kenth et al. 2007, Lettre et al. 2007, Mericq et al. 2007, Raz et al. 2008).

Two truncated hGHRs, 1–277 and 1–279, are produced at low levels in all human tissues examined, including fetal liver and placenta, with variable truncated to FL receptor (T/FL) ratios among the tissues (Dastot et al. 1996, Ballesteros et al. 2000, Fisker et al. 2001, 2004, Goodyer et al. 2001a). These truncated receptors are capable of binding hGH with high affinity, but they do not activate intracellular signal transduction pathways and, in fact, act as dominant-negative receptors (Ross et al. 1997, Ayling et al. 1999, Iida et al. 1999).

GH binds with high affinity to two molecules of hGHR, forming a stable heterotrimer complex (De Vos et al. 1992, Frank 2002, Gent et al. 2003, Waters et al. 2006, Brooks et al. 2008). Because the GHR is characterized by the absence of intrinsic catalytic activity within its intracellular domain, signal transduction through this receptor is mediated primarily by Janus kinase (JAK) 2, a cytoplasmic tyrosine kinase that associates with Box 1 of the GHR cytoplasmic region (Argetsinger et al. 1993, Carter-Su et al. 1996). Rapid and transient activation of JAK2 results in phosphorylation of JAK2, GHR, and a number of intracellular signaling and adaptor proteins (Fig. 1; Argetsinger et al. 1993, Carter-Su et al. 1996, Herrington & Carter-Su 2001, Piwien-Pilipuk et al. 2002, Lanning & Carter-Su 2006, Brooks et al. 2008). The major GHR signaling factors in hepatocytes are the signal transducers and activators of transcription (STATs): STAT1, STAT3, STAT5A, and STAT5B associate with specific tyrosine phosphorylated sites on the activated GHR through their SH2 domains (Levy & Darnell 2002, Lanning & Carter-Su 2006, Rosenfeld et al. 2007). Once phosphorylated by JAK2, the STAT proteins form homo- or heterodimers that migrate to the nucleus and bind to specific sites on the promoters of GH-responsive genes (Herrington et al. 2000, Piwien-Pilipuk et al. 2002, Lanning & Carter-Su 2006, Brooks et al. 2008).

Figure 1
Figure 1

Schematic representation of the hGH/hGHR signaling molecules examined in the present study.

Citation: Journal of Endocrinology 198, 1; 10.1677/JOE-07-0648

The family of cytokine-inducible proteins, termed suppressors of cytokine signaling (SOCS), is important in the negative regulation of GHR signaling in the hepatocyte (Krebs & Hilton 2001, Flores-Morales et al. 2006). SOCS1, SOCS2, SOCS3, and cytokine-inducible SH2-containing protein (CIS) expression are induced by GH in the rat liver with different kinetics (Adams et al. 1998, Ram & Waxman 1999, Tollet-Egnell et al. 1999). SOCS1 appears to inhibit JAK2 kinase activity directly, by binding to its kinase activation loop (Naka et al. 1997). SOCS2, SOCS3, and CIS, on the other hand, indirectly inhibit GHR signaling, by binding to the GHR and interfering with JAK2 activation and STAT5 binding (Hansen et al. 1999, Ram & Waxman 1999, Uyttendaele et al. 2007). Mice deficient in SOCS2 display gigantism (Metcalf et al. 2000), while those transgenic for CIS are growth retarded (Matsumoto et al. 1999), suggesting that SOCS2 and CIS may play especially important roles in the termination of GHR signaling. In addition, the SOCS proteins have been shown to be a new class of ubiquitin ligases that may regulate proteosomal degradation of the GHR and JAK2 (Flores-Morales et al. 2006, Lanning & Carter-Su 2006).

Pivotal roles for hGH and its receptor have been well established in the postnatal period (Veldhuis et al. 2005). However, their functions during human gestation remain unclear. Only a subset of newborns with hGH deficiency or hGHR dysfunction have decreased birth length, suggesting that, while fetal hGH can affect fetal growth, the presence of other fetal growth factors (e.g., insulin, placental lactogen, prolactin) ensures relatively normal intrauterine growth (Gluckman et al. 1992, Wit & van Unen 1992, Woods et al. 1997, Jensen et al. 2005, Mehta et al. 2005, Osafo et al. 2005).

Interestingly, fetal plasma levels of immunoreactive hGH are significantly elevated relative to the normal adult, up to 150 ng/ml at mid-gestation (Kaplan et al. 1976). This is likely due to the greater sensitivity of the human fetal pituitary somatotrope to its hypothalamic stimulatory factor, GH-releasing hormone (GHRH), than its inhibitory factor, somatostatin, throughout in utero development (Delitala et al. 1978, Goodyer et al. 1993a,b). Similar hGH levels postnatally cause highly elevated serum levels of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3), resulting in gigantism in the prepubertal child and acromegaly in the adult (Kato et al. 2002). However, circulating levels of IGF-I and IGFBP-3 in the fetus are low, reaching significant levels only late in the third trimester (Butler & Le Roith 2001, Gohlke et al. 2004), suggesting a blunted responsiveness of fetal tissues to hGH during earlier stages in development, at either the hGH receptor or the signaling level.

hGHR mRNA and protein have been identified in a wide variety of human tissues from the first trimester of fetal life (Hill et al. 1992, Simard et al. 1996, Zogopoulos et al. 1996a,b). There is a decrease in lung, kidney, and intestinal levels of hGHR postnatally and a significant increase in liver when early to mid-gestation fetal versus adult tissues are compared (Goodyer et al. 2001a). We and others have previously demonstrated that the hGHR present in fetal hepatocytes (FH) specifically binds hGH, and that these cells have biological responses (proliferation, IGF production, glucose uptake) following exposure to hGH, although the effects are lower compared with adult hepatocytes (Strain et al. 1987, Hill et al. 1988, 1989, Goodyer et al. 2001a).

In the present study, we have tested the hypothesis that, in addition to lower tissue levels of hGHR, other factors, including the hGHR protein forms (FL versus truncated) and the JAK/STAT and SOCS/CIS families of downstream signal transduction molecules, may be responsible for the limited responsiveness of FHs to the high circulating levels of hGH.

Materials and Methods

Materials

A rabbit antibody raised against the intracellular domain of the hGHR (AL47) was a generous gift from Dr Stuart Frank (University of Alabama, Birmingham, AL, USA) (Zhang et al. 2001). Antibodies directed against hGH signaling proteins were obtained from the following commercial sources: JAK2 from Imgenex (IMG-3007; San Diego, CA, USA); SOCS1 (ab3691-100) and SOCS2 (ab3692-100) from Abcam (Cambridge, MA, USA); SOCS3 (sc-9023) and CIS (sc-1529) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); and STAT1 (UBI 06-501), STAT3 (UBI 06-596), STAT5A (UBI 06-553), and STAT5B (UBI 06-554) from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Rabbit and mouse IgGs were obtained from Santa Cruz. A mouse monoclonal antibody for calnexin (C45520), the immunoblot loading control, was purchased from Transduction Laboratories (Lexington, KY, USA). The HRP-labeled secondary antibodies used were anti-rabbit (NEF812) and anti-mouse (NEF822) from Perkin–Elmer Life Sciences Inc. (Boston, MA, USA) and anti-goat (sc-2020) from Santa Cruz Biotechnology.

Tissue collections and cell lines

Human fetal tissues were obtained at the time of therapeutic abortion (10.75–19 weeks of fetal age (FA)); FA was determined by foot length (Munsick 1984). Postnatal liver specimens, perfused to remove all blood cells, were collected within 4–10 h after removal of organs for transplantation (15–60 years, donors were all males). Ethics approval for the study was obtained from local institutional research ethics boards, in accordance with the Canadian Council on Health Sciences Research Involving Human Subjects guidelines. Written informed consent was obtained in each case. Four primate cell lines were analyzed: CV1 (African green monkey kidney fibroblasts, ATCC), human embryonic kidney epithelial cells (HEK293 epithelial cells, ATCC), and two human postnatal hepatoma cell lines, HepG2 (ATCC) and Huh7 (kindly provided by Dr Ken K Ho, Garvan Institute of Medical Research, Sydney, New South Wales, Australia). CV1, HEK293, and HepG2 cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin G, and 1.6 mg/ml gentamicin sulfate. Huh7 cells were cultured in Earle's MEM (Invitrogen) supplemented with 10% FBS, 7.5 mM HEPES, 100 IU/ml penicillin G, and 1.6 mg/ml gentamicin sulfate. All cells were maintained at 37 °C with 5% CO2 in a humid environment.

FH preparations

Fetal hepatic tissues were minced and dispersed by collagenase, followed by gravity separation, yielding >95% pure hepatocytes, as described previously (Zogopoulos et al. 1996a). The isolated hepatocytes were plated onto collagen-coated Petri dishes in William's E medium (Invitrogen) with 10% FBS, 100 IU/ml penicillin G, 1.6 mg/ml gentamicin sulfate, and 39.2 μg/ml dexamethasone. Two hours after plating, the hepatocytes were rinsed thoroughly (three to five times) with 1× PBS to remove all remaining traces of hematopoietic cells, replaced with fresh, complete William's E medium with 39.2 μg/ml dexamethasone, and incubated overnight. After 24 h, hepatocytes were washed with 1× PBS and collected by scraping. The hematopoietic cells were pelleted, flash frozen, and stored at −80 °C until extracted for RNA.

Protein and RNA extractions

Homogenized tissue fragments or cell pellets were solubilized in ice-cold lysis buffer (50 mM Tris (pH 7.5), 0.1% Triton X-100, 150 mM NaCl, and 2 mM EDTA) containing protease and phosphatase inhibitors (complete cocktail tablets (Roche Diagnostics)) for 15 min on ice. To remove insoluble material, lysates were centrifuged at 13 000 g for 10 min at 4 °C. The supernatants were collected and protein concentrations measured with the Bradford kit (Bio-Rad Laboratories, Inc.), using BSA as a standard. Alternatively, tissue fragments and the cell pellets were extracted for RNA using the TRIzol method (Invitrogen).

RT-PCR assay for FL versus truncated hGHR

For each RT reaction, hepatic total RNA (5 μg) was reverse transcribed for 1 h at 42 °C in the presence of 200 U Superscript II (Invitrogen) and a specific exon 9 reverse primer (9AS) (5′-TAATCTTTGGAACTGGAACT-3′) designed to recognize both the FL and truncated (1–279) hGHR mRNAs (Goodyer et al. 2001a). RT products (3 μl) were amplified for 35 cycles with 1.25 U Taq DNA polymerase (Invitrogen), hGHR exon 7 sense (7S: 5′-ATAAGGAATATGAAGTGCGTGTGAG-3′), and hGHR exon 9 antisense primers. The cycles consisted of one cycle at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min, ending with a final elongation at 72 °C for 10 min. In each assay, an aliquot of H2O was amplified in parallel as a negative PCR control. PCR products were separated on 10% polyacrylamide gels and stained with SybrGold (Molecular Probes, Eugene, OR, USA). Densitometric analyses were carried out using the Bio-Rad GelDoc system.

Immunoprecipitation and immunoblotting

Liver lysate protein (0.5–1 mg) were incubated by rotation in lysis buffer described above with anti-hGHR (αAL47, 1:250) overnight at 4 °C, followed by the addition of 50 μl protein G-agarose beads (Amersham Biosciences, Baie, D'urfé, QC, Canada) for 1 h at 4 °C. Antibody complexes were washed three times with lysis buffer followed by a single wash with 50 mM Tris (pH 8.0). hGHR immunoprecipitates or 50–100 μg whole cell extracts were boiled for 5 min in Laemmli loading buffer (50 mM Tris (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 100 mM DTT) and proteins were resolved by SDS-PAGE (8–15%) and transferred (Mini Trans-blot Cell; Bio-Rad Laboratories, Inc.) to polyvinylidene fluoride Immobilon-P transfer membranes (Millipore Corporation, Mississauga, ON, Canada).

The membranes were blocked overnight at 4 °C with 5% non-fat dry milk in PBST buffer, and then incubated with the appropriate primary antibody (hGHR AL47 (1:2000), JAK2 (1:700), STAT1 (1:1000), STAT3 (1:1000), STAT5A (1:1000), STAT5B (1:1000), SOCS1 (1:1000), SOCS2 (1:1000), SOCS3 (1:200), CIS (1:100), or calnexin (1:1000)) for 3 h at room temperature. After washing three times with PBST, the membranes were incubated with an anti-mouse (1:2000), anti-rabbit (1:1000), or anti-goat (1:1000) IgG conjugated to horseradish peroxidase for 1 h at room temperature. Protein bands were visualized by an enhanced chemiluminescence detection system (Perkin–Elmer Life Sciences Inc).

To probe the same membrane for another protein, the membranes were incubated for 30 min at 50 °C with stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris (pH 6.8)). Calnexin was used to normalize for protein loading; pilot western blots showed a linear increase in calnexin protein levels over 20–80 μg of both fetal and postnatal hepatic protein (Supplementary Figure 1, see Supplementary data in the online of version of the Journal of Endocrinology at http://joe.endocrinology-journals.org/content/vol198/issue1/). Densitometric analyses were carried out using the Bio-Rad GelDoc system.

Statistical analyses

Data are presented as Mean±s.e.m. Differences between two groups were analyzed using Student's t-test (two tailed). Differences among several groups were analyzed using ANOVA followed by Bonferroni's multiple group comparison test.

Results

Analysis of hGHR molecular forms in human FHs versus postnatal liver

We previously analyzed hGHR mRNAs from fetal and postnatal hepatocytes and found transcripts for FL, truncated (1–279), and exon 3 forms at both developmental stages (Zogopoulos et al. 1996b, Goodyer et al. 2001a). In the present study, FH (n=2, 13.8, and 15 weeks FA) and postnatal liver (n=2, 15, and 47 years) samples were analyzed for hGHR protein by immunoprecipitation (IP) followed by immunoblotting (IB), using the same antibody (AL47) (Fig. 2). Two major forms of the hGHR, precursor (∼70 kDa) and mature (∼120 kDa), were detected in all cases. Since the antibody used for the IP/IB analyses was directed against the intracellular domain of the hGHR, we were only able to identify the FL, as opposed to the truncated, forms (Zhang et al. 2001). These data demonstrate that the FL hGHR forms are similar at the two developmental stages.

Figure 2
Figure 2

Immunoblot (IB) analyses of immunoprecipitated (IP) hGHR in human adult liver (HAL) and fetal hepatocytes (FH) using the AL47 antibody raised against the intracellular domain of the GHR (Zhang et al. 2001). Molecular markers are indicated at the left side with arrowheads and arrows indicate the two major (∼70 and ∼120 kDa) IP/IB hGHR bands. IgG, rabbit IgG.

Citation: Journal of Endocrinology 198, 1; 10.1677/JOE-07-0648

Truncated (1–279) hGHR mRNA in human FHs versus postnatal liver

To analyze whether increases in the major 1–279 dominant-negative truncated (T) form of the hGHR (Ross 1999) might play a role in the lower fetal biological responses to hGH, we determined the relative ratios of mRNAs for the FL and T forms. We used a specific RT-PCR assay that recognizes the FL and T hGHR mRNAs as distinct PCR products in each sample (Fig. 3A, C and E) (Goodyer et al. 2001a). Pilot studies with PCR cycle numbers ranging from 29 to 39 showed a parallel amplification of the two bands in both fetal and postnatal hepatic samples (Fig. 3B).

Figure 3
Figure 3

Analysis of full-length (FL) and truncated (T) (1–279) hGHR mRNA. (A) A specific primer in exon 9 (9AS) was designed to recognize both the FL and T forms of hGHR mRNA for RT–PCR assays. (B) Pilot RT-PCR assays showed that there were no significant differences in T versus FL GHR amplicon amplification over a wide range of PCR cycle number, in both fetal and postnatal hepatic samples (n=3 each). (C and D) Comparison of FL and T hGHR mRNAs in intact fetal liver (n=12, 12.7–19.5 weeks), postnatal liver (n=3, 37–60 years), fetal hepatocytes (n=7, 11.5–17.5 weeks), and fetal liver hematopoietic cells (n=5, 12.7–19.5 weeks) by RT-PCR. (C) Representative polyacrylamide gel of PCR products detects FL hGHR mRNA at 252 bp and T hGHR mRNA at 226 bp. (D) Comparative data for intact postnatal liver, intact fetal liver, fetal hepatocytes, and fetal hematopoietic cells, expressed as a ratio of T/FL transcripts (Mean±s.e.m). Statistical differences are calculated relative to fetal hepatocytes: ***P<0.001. (E and F) Comparison of FL and T hGHR mRNAs in various fetal tissues. (E) Representative polyacrylamide gel of PCR products. pBS-SK+/Hpa II marker is indicated at 242 bp. CNS, central nervous system. (F) T hGHR mRNA is detected in a wide variety of fetal tissues. Each tissue type was tested using three different samples and the final data are expressed as a ratio of T/FL transcripts (Mean±s.e.m). Statistical differences are calculated relative to intact fetal liver: *P<0.05, **P<0.01. (G and H) Comparison of FL and T hGHR mRNAs in various primate cell lines. (G) Representative polyacrylamide gel of PCR products. pBS-SK+/Hpa II marker is indicated at 242 bp. (H) Graphic analysis of T/FL hGHR mRNA in three cell lines and human adult liver (HAL) (HepG2 band densities were too low to be accurately analyzed). Each sample was tested using three to five different RNA pools (Mean±s.e.m). Statistical differences are calculated relative to HAL: **P<0.01, ***P<0.001.

Citation: Journal of Endocrinology 198, 1; 10.1677/JOE-07-0648

In a previous study, we determined T/FL hGHR mRNA ratios in intact fetal liver (0.18±0.03 (Mean±s.e.m.), n=8, 14–18 weeks) versus intact postnatal liver (0.09±0.02, n=3, 21–54 years) (Goodyer et al. 2001a). While the FL transcript always predominated, there was a trend for fetal liver to have a twofold higher ratio. However, fetal liver is the primary hematopoietic organ during this stage of development and blood cells have a number of cytokine receptors, including hGHR (Welniak et al. 2000). Therefore, for the present study, we dissociated the intact fetal liver into its two major cell components: hepatocytes and hematopoietic cells. This isolation permitted a more accurate comparison of fetal with postnatal liver, since the postnatal liver tissues used for these studies had been perfused for transplantation and, therefore, did not contain blood cells.

When intact postnatal liver, intact fetal liver, isolated FHs, and isolated fetal hematopoietic cells were compared, the highest T/FL hGHR mRNA ratios were detected in fetal liver (0.20±0.02, n=12, 12.7–19.5 weeks) and fetal hematopoietic cells (0.24±0.04, n=5, 12.7–19.5 weeks). FHs (0.07±0.01, n=7, 11.5–17.5 weeks) showed low ratios, similar to postnatal liver (0.09±0.01, n=3, 37–60 years) (Fig. 3C and D). Our findings of the same T/FL ratios in fetal and postnatal hepatocytes suggests that hGHR functional differences are not likely due to the dominant-negative form of the hGHR.

Truncated (1–279) hGHR mRNA in fetal tissues

To place these T/FL ratios in a biological context, we also assessed the relative abundance of T and FL hGHR mRNAs in other fetal tissues. Like the fetal liver, all nine other tissues predominantly expressed the FL hGHR transcript (Fig. 3E and F). The dominant-negative T hGHR mRNA was also detected in all tissues, but T/FL hGHR mRNA ratios varied considerably, from a mean of 0.002 in skin to means of 0.53 and 0.65 in kidney cortex and medulla respectively (Fig. 3E and F).

Characterization of T/FL hGHR mRNA isoforms in human and primate cell lines

Four different human and primate cell lines (CV1, HEK293, HepG2, and Huh7) that are routinely used as cell models to examine hGHR binding and signaling pathways were also analyzed for the different hGHR isoforms. The Huh7 hepatoma and CV1 cell lines demonstrated relatively high T/FL ratios (Huh7: 1.48+0.26, CV1: 1.27±0.19), while the HEK293 cell ratios were quite low (0.19+0.05) (Fig. 3G and H). Unfortunately, the HepG2 band densities were too low to be analyzed accurately.

Expression of JAK/STAT proteins in human fetal hepatocytes and intact liver versus postnatal liver

JAK2 and STAT1, STAT3, STAT5A, and STAT5B are major members of the hGH/hGHR intracellular signaling cascade (Lanning & Carter-Su 2006). To determine whether they are expressed in lower amounts in FHs, thereby decreasing responsiveness to hGH, we analyzed their levels in human intact fetal livers (n=3, 10.75–18 weeks), FHs (n=3, 12–18 weeks), and postnatal livers (n=3, 15–60 years). These five proteins were found in all of the samples tested demonstrating that they are expressed as early as the third month of fetal life (Fig. 4A and B). All but STAT5B were lower in FHs compared with postnatal liver; this difference was significant for STAT1 (↓42%, P<0.05) (Fig. 4A). Although a similar trend was seen for JAK2 (↓38%), STAT3 (↓38%), and STAT5A (↓53%), they did not reach statistical significance, likely due to the small sample size. Unfortunately, hepatocyte preparations require substantial amounts of fetal liver tissue and these are in limited supply.

Figure 4
Figure 4

Analysis of JAK/STAT proteins. Representative immunoblots of JAK/STAT and calnexin expression in (A) fetal hepatocytes and (B) fetal liver. Relative abundance of JAK2 (∼120 kDa), STAT1 (∼91 kDa), STAT3 (∼92 kDa), STAT5A (∼95 kDa), and STAT5B (∼92 kDa) proteins in (A) fetal hepatocytes (n=3, 12–18 weeks) or (B) fetal liver (n=3, 10.75–18 weeks) (Mean±s.e.m). Data are normalized to calnexin (∼90 kDa) and compared with postnatal liver (HAL: n=3, 15–60 years). Statistical differences are expressed relative to HAL: *P<0.05, **P<0.03, ***P<0.01.

Citation: Journal of Endocrinology 198, 1; 10.1677/JOE-07-0648

Interestingly, we observed quite different levels of the JAK/STATs when we analyzed intact human fetal versus postnatal liver samples (Fig. 4B). While STAT1 and STAT3 were still lower than human adult liver (HAL) (↓29%), JAK2, STAT5A, and STAT5B were markedly increased (↑100%, P<0.06; ↑680%, P<0.01; and ↑130%, P<0.03 respectively). These results, along with the T/FL hGHR data, indicate that the presence of hematopoietic cells in the intact fetal liver can markedly influence tissue biological expression patterns. In addition, the data suggest that the JAK2/STAT5A and STAT5B pathways may be very important for fetal hematopoietic cell function. Because of the limited number of hepatocyte preparations, we were, however, unable to carry out parallel studies in isolated hematopoietic cells.

Expression of SOCS proteins in human fetal hepatocytes and intact liver versus postnatal liver

SOCS proteins inhibit hGHR signaling in hGH target cells, including the hepatocytes (Flores-Morales et al. 2006). To test whether increased levels might be responsible for a lower response to hGH in fetal tissues, we compared the levels of SOCS/CIS proteins in human fetal livers (n=3, 10.75–18 weeks), FHs (n=3, 12–19 weeks), and postnatal livers (n=3, 15–60 years). The four SOCS proteins were detected in all of these samples (Fig. 5A and B). There were markedly lower levels of the inhibitory proteins in the FHs relative to the postnatal livers: SOCS1 (↓61%), SOCS2 (↓76%), SOCS3 (↓61%), and CIS (↓58%), although these changes did not achieve statistical significance, again likely due to limited sample numbers (Fig. 5A). Thus, human FHs do contain the four SOCS family members from as early as 12 weeks of FA, but they are present at less than half the levels observed in postnatal livers.

Figure 5
Figure 5

Analysis of SOCS proteins. Representative immunoblots of SOCS/CIS and calnexin expression in (A) fetal hepatocytes and (B) fetal liver. Relative abundance of SOCS1 (∼24 kDa), SOCS2 (∼23 kDa), SOCS3 (∼30 kDa), and CIS (∼32 kDa) proteins in (A) fetal hepatocytes (n=3, 12–19 weeks) or (B) fetal liver (n=3, 10.75–18 weeks) (Mean±s.e.m). Data are normalized to calnexin (∼90 kDa) and compared with postnatal liver (HAL: n=3, 15–60 years). Statistical differences are expressed relative to HAL: **P<0.04.

Citation: Journal of Endocrinology 198, 1; 10.1677/JOE-07-0648

When we analyzed the SOCS levels in intact human fetal livers, we observed that CIS was significantly decreased compared with the postnatal livers (CIS: ↓67%, P<0.04), while there were consistent decreases in the other family members: SOCS1 (↓31%), SOCS2 (↓41%), and SOCS3 (↓35%) (Fig. 5B). In contrast to the JAK/STAT results, these data are similar to the findings obtained for the FHs, suggesting that the SOCS/CIS family members are present at similar levels in both the fetal hematopoietic and hepatic cells.

Discussion

Although pivotal roles for GH, its receptor and their downstream signal transduction pathways have been well established in postnatal tissues, their functions during gestation remain unclear (Hill 1992, Symonds et al. 2001, Waters & Kaye 2002). Surprisingly, fetal serum levels of GH are tenfold higher than those in the postnatal period in both primates and subprimates (Gluckman et al. 1981). We have previously demonstrated that this may be because human fetal somatotropes have a much higher sensitivity to GHRH than somatostatin (Goodyer et al. 1993a,b). In corroboration, clinical studies have shown that newborns have an exaggerated response to GHRH and a delayed and blunted response to somatostatin (Delitala et al. 1978, Shimano et al. 1985). Postnatally, these elevated hGH levels would cause high serum levels of IGF-I and IGFBP-3, resulting in gigantism in a prepubertal child and acromegaly in an adult (Kato et al. 2002). However, in the fetus, serum IGF-I and IGFBP-3 levels are very low or undetectable until late in the third trimester (Butler & Le Roith 2001, Gohlke et al. 2004). These data suggest that fetal tissues have a relatively low responsiveness to hGH.

In subprimates, the onset of significant GHR mRNA expression occurs late in gestation (Adams et al. 1990, Walker et al. 1992, Ymer & Herington 1992). In the human fetus, ubiquitous transcription of the hGHR gene has been documented from as early as the first trimester; postnatal changes in hGHR mRNA expression are tissue specific, with decreases in the lung, kidney, and intestine but a sixfold increase in liver after birth (Zogopoulos et al. 1996a,b, Goodyer et al. 2001b). Binding (Goodyer et al. 2001a), immunohistochemical (Hill 1992, Hill et al. 1992, Simard et al. 1996), and IB experiments (present study) have identified functional hGHR protein in multiple human tissues from as early as 9–11 weeks of fetal life. In the present study, we asked whether there are factors other than the levels of hGHR, which may be responsible for the decreased responsiveness to hGH in fetal tissues. We focused on several hGHR isoforms and their downstream signaling pathway molecules, comparing their levels in FHs versus postnatal liver.

Based on its amino acid sequence, the nascent hGHR is predicted to be ∼70 kDa in size while, when resolved on SDS-PAGE gels under reducing conditions, the mature hGHR migrates at 100–140 kDa (Hocquette et al. 1989, 1990, Alele et al. 1998, Zhang et al. 2001, Cowan et al. 2005). These differences are due primarily to the presence of five asparagine-linked glycosylation sites within the extracellular domain, a variable number of which are glycosylated in the mature hGHR (Harding et al. 1994). In addition, hGHRs can be modified by ubiquitination, a step that can target the receptor for internalization and degradation (Govers et al. 1997, Strous et al. 1997, van Kerkhof et al. 2002). cDNA sequencing (Zogopoulos et al. 1996a) and now IP and IB experiments indicate no major size differences between the fetal and postnatal hepatic hGHR: the mRNA sequences are identical and both the immature and mature forms of the fetal hGHR in hepatocytes have very similar SDS-PAGE migration patterns as the postnatal hGHR.

Ross et al. (1997) previously demonstrated that cells expressing a T/FL GHR cDNA ratio of 1:10 exhibited a 10–30% decrease in FL GHR activity, while a 1:1 T/FL ratio resulted in a 40–80% inhibition and a 10:1 ratio completely blocked activity of the FL GHR. When we examined the T/FL hGHR mRNA ratios in FHs and postnatal liver, we found that they were similar and quite low (∼1:12), making it unlikely that the truncated form plays a role in the functional hGHR differences observed at these two developmental stages. However, we observed a relatively high T/FL hGHR mRNA ratio in hepatic hematopoietic cells (1:4), fetal kidney tissues (∼1:2), and two primate cell lines (∼1.4:1), suggesting that the dominant-negative isoforms of hGHR may play a functionally significant role in these cells and tissues, decreasing the ability of hGH to activate its intracellular signaling and, thus, its biological effectiveness. This is especially interesting in the case of the kidney, given the links between hGH and kidney function (Rabkin & Schaefer 2004, Reddy et al. 2007). It would be interesting, for example, to compare T/FL hGHR mRNA ratios in kidney tissue biopsies under normal versus diabetic conditions, to determine whether the T/FL ratio shifts from a high to low level with progression of the diabetic state, leading to increased responsiveness to hGH and diabetic nephropathy.

The only previous study of JAKs and STATs in human liver was undertaken by Hellgren et al. (1999): they determined by western blots, immunohistochemistry, and IP that JAK1, JAK2, JAK3, and TYK2 are all present in adult hepatocytes, and that the hGHR specifically associates with JAK1, JAK2, and TYK2. Our analysis of the hGHR downstream signaling factors demonstrates that JAK2 and STAT1, STAT3, STAT5A, and STAT5B are present in FHs from the 12th week of fetal life. With the exception of STAT5B, FHs contain ∼40–50% lower levels of the JAK/STAT proteins compared with postnatal liver. Whether this actually creates a functional difference in hGH signaling is impossible to know with our present data. To determine this, future experiments should examine the relative amounts of hGHR/JAK/STAT molecules that are phosphorylated or the relative responses of target genes following hGH treatment of fetal versus postnatal hepatocytes.

There have been two fetal versus postnatal liver analyses of JAK/STATs in the rat (Shoba et al. 1999, Phornphutkul et al. 2000). Contrary to our data (Goodyer et al. 2001a), Phornphutkul et al. (2000) found no difference in GH binding levels between late gestation (d17, d19) fetal and postnatal rat livers. In vitro or in vivo GH treatments caused a similar phosphorylation of GHR, JAK2, STAT1, and/or STAT5 in the fetal and postnatal hepatocytes. Finally, GH was able to enhance CAT expression of two GHRE reporter vectors transfected into FHs, leading Phornphutkul et al. (2000) to conclude that hepatic GH signaling mechanisms are functional in the late gestation rat. Similar to our data (Goodyer et al. 2001a), Shoba et al. (1999) found a marked increase in GHR mRNA and protein in fetal (d17) compared with postnatal (4 months) rat livers. Over the same time frame, surprisingly, JAK2, STAT1, and STAT3 protein levels dropped while STAT5 levels remained unchanged. These last findings are quite different from our present hepatocyte and intact human liver data and may represent major species differences.

One interesting note is that we observed significantly elevated levels of STAT5A and STAT5B expression in the human intact fetal liver when compared with the FHs, suggesting that this is due to the hematopoietic cell population. Several published reports support this conclusion. STAT5A/STAT5B-deficient mice are severely anemic due to significant decreases in their hepatic erythroid precursor cell population (Socolovsky et al. 1999), demonstrating the importance of these two STATs for murine fetal liver hematopoiesis. The recent findings, by Rosenfeld et al., of immune deficiencies in two patients with STAT5B mutations indicate an important role for STAT5B in the human immune system as well (Rosenfeld et al. 2005, Hwa et al. 2005).

The SOCS proteins are well recognized to act as inhibitors of GHR signaling (Flores-Morales et al. 2006). Although we had hypothesized that there might be increased levels in the FHs, our IB studies of FH lysates showed ∼60–75% lower levels of all of the SOCS family members compared with postnatal liver. In the only other human study, Dey et al. (1998) examined a CLONTECH panel of fetal and adult human tissues: they also showed a lower level of SOCS2 mRNA levels in the fetal when compared with the postnatal liver. In the only animal study to date, Gentili et al. (2006) have reported that there is a significant increase in SOCS3 mRNA expression in the ovine fetal liver during late gestation (d125–d145), and that this expression is regulated by circulating levels of PRL but STAT5 does not appear to be involved.

One obvious explanation for the lower expression of the SOCS proteins in the human fetal liver is the lower amounts of hGHR, JAK2, and most of the STATs in the fetal cells. It is well known that transcription of the SOCS genes is upregulated by hGH and other cytokines through the STATs: SOCS1 has STAT1, STAT3, and STAT6 binding sites while SOCS2, SOCS3, and CIS promoters have multiple STAT5 consensus response elements (Matsumoto et al. 1997, Adams et al. 1998, Davey et al. 1999, He et al. 2003, Hebenstreit et al. 2003, Gatto et al. 2004). However, an interesting caveat is that SOCS2 has dose-dependent effects on GHR signaling: at low concentrations it inhibits GH-induced STAT5-dependent gene transcription while restoration of GH signaling is observed at higher concentrations, demonstrating that SOCS2 functions by regulating cell sensitivity to GH (Favre et al. 1999, Greenhalgh et al. 2002). This interesting finding suggests that the relatively low levels of SOCS2 in the human FHs may, in fact, be acting to inhibit hGHR signaling and, thus, may contribute to the lower response to hGH.

Once induced, the SOCS3/CIS families are major inhibitors of GHR signaling (Adams et al. 1998, Hansen et al. 1999, Ram & Waxman 1999, Ronn et al. 2002, Flores-Morales et al. 2006), not only by blocking STAT phosphorylation but also by targeting the GHR for ubiquitination and subsequent degradation by the proteosome (Kamura et al. 1998, Zhang et al. 1999, Ram & Waxman 2000, Kile & Alexander 2001, Wormald & Hilton 2004, Flores-Morales et al. 2006). Whether the SOCS–ubiquitin–proteosome pathway is active in the FH remains unknown.

Several other aspects have yet to be investigated in any species. These include whether in the fetal liver: 1) the GH/GHR complex is working through alternative intracellular signaling pathways to regulate fetal-specific biological outcomes (e.g., the MAPK and PI3-kinase cascades, Ca2+ channels), 2) there are desensitization processes (e.g., more rapid dephosphorylation of the activated receptor, endocytosis, proteolysis) that influence GH responsiveness in general, and 3) gender has an effect on the levels of the signaling proteins, given that the fetal testes is producing significant amounts of testosterone at 10–20 weeks of fetal life (Tapanainen et al. 1981, Frank 2001, Herrington & Carter-Su 2001, Zhu et al. 2001, Choi et al. 2006).

In conclusion, our studies to date demonstrate that there are lower levels of hGHR in fetal versus postnatal hepatocytes but that the hGHR isoforms are unchanged, including the relative amounts of the truncated dominant-negative (hGHR1279) form. The JAK2/STAT/SOCS signaling molecules are present in the human hepatocyte as early as the first trimester. However, they are generally at <50% the level in postnatal perfused liver, suggesting that decreased hGHR signaling ability may help to explain the relatively low cell responsiveness to the very high fetal serum hGH levels.

Declaration of Interest

The authors have no conflict of interest that would prejudice the research described in this article. C G G is a member of the Research Institute of the McGill University Health Centre, which is supported in part by the Fonds de Recherches en Santé du Québec.

Funding

This study was funded by the Canadian Institutes of Health Research (to C G G). G K and J A M M were supported by studentships from the McGill University Health Centre – Montreal Children's Hospital Research Institute.

Acknowledgements

The authors would like to thank Dr S Frank for the AL47 antibody and both Drs Stuart Frank and Indra Gupta for their critical reviews of the manuscript. In addition, they would like to express their gratitude to the OR surgical and nursing staff at Hôpital Maisonneuve-Rosemont, Hôpital Sainte-Justine, and the Royal Victoria Hospital for their help in obtaining the human tissue samples.

References

  • Adams TE, Baker L, Fiddes RJ & Brandon MR 1990 The sheep growth hormone receptor: molecular cloning and ontogeny of mRNA expression in the liver. Molecular and Cellular Endocrinology 73 135145.

    • Search Google Scholar
    • Export Citation
  • Adams TE, Hansen JA, Starr R, Nicola NA, Hilton DJ & Billestrup N 1998 Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling. Journal of Biological Chemistry 273 12851287.

    • Search Google Scholar
    • Export Citation
  • Alele J, Jiang J, Goldsmith JF, Yang X, Maheshwari HG, Black RA, Baumann G & Frank SJ 1998 Blockade of growth hormone receptor shedding by a metalloprotease inhibitor. Endocrinology 139 19271935.

    • Search Google Scholar
    • Export Citation
  • 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 237244.

    • Search Google Scholar
    • Export Citation
  • Audi L, Esteban C, Carrascosa A, Espadero R, Perez-Arroyo A, Arjona R, Clemente M, Wollmann H, Fryklund L & Parodi LA 2006 Exon 3-deleted/full-length growth hormone receptor polymorphism genotype frequencies in Spanish short small-for-gestational-age (SGA) children and adolescents (n=247) and in an adult control population (n=289) show increased fl/fl in short SGA. Journal of Clinical Endocrinology and Metabolism 91 50385043.

    • Search Google Scholar
    • Export Citation
  • Ayling RM, Ross RJ, Towner P, Von Laue S, Finidori J, Moutoussamy S, Buchanan CR, Clayton PE & Norman MR 1999 New growth hormone receptor exon 9 mutation causes genetic short stature. Acta Paediatrica 88 168172.

    • Search Google Scholar
    • Export Citation
  • Ballesteros M, Leung KC, Ross RJ, Iismaa TP & Ho KK 2000 Distribution and abundance of messenger ribonucleic acid for growth hormone receptor isoforms in human tissues. Journal of Clinical Endocrinology and Metabolism 85 28652871.

    • Search Google Scholar
    • Export Citation
  • Barton DE, Foellmer BE, Wood WI & Francke U 1989 Chromosome mapping of the growth hormone receptor gene in man and mouse. Cytogenetics and Cell Genetics 50 137141.

    • Search Google Scholar
    • Export Citation
  • Bass SH, Mulkerrin MG & Wells JA 1991 A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. PNAS 88 44984502.

    • Search Google Scholar
    • Export Citation
  • Binder G, Baur F, Schweizer R & Ranke MB 2006 The d3-growth hormone (GH) receptor polymorphism is associated with increased responsiveness to GH in Turner syndrome and short small-for-gestational-age children. Journal of Clinical Endocrinology and Metabolism 91 659664.

    • Search Google Scholar
    • Export Citation
  • Blum WF, Machinis K, Shavrikova EP, Keller A, Stobbe H, Pfaeffle RW & Amselem S 2006 The growth response to growth hormone (GH) treatment in children with isolated GH deficiency is independent of the presence of the exon 3-minus isoform of the GH receptor. Journal of Clinical Endocrinology and Metabolism 91 41714174.

    • Search Google Scholar
    • Export Citation
  • Brooks AJ, Wooh JW, Tunny KA & Waters MJ 2008 Growth hormone receptor; mechanism of action. International Journal of Biochemistry and Cell Biology (In Press).

    • Search Google Scholar
    • Export Citation
  • Butler AA & Le Roith D 2001 Control of growth by the somatropic axis: growth hormone and the insulin-like growth factors have related and independent roles. Annual Review of Physiology 63 141164.

    • Search Google Scholar
    • Export Citation
  • Buzi F, Mella P, Pilotta A, Prandi E, Lanfranchi F & Carapella T 2007 Growth hormone receptor polymorphisms. Endocrine Reviews 11 2835.

  • Carrascosa A, Esteban C, Espadero R, Fernandez-Cancio M, Andaluz P, Clemente M, Audi L, Wollman H, Fryklund L, Parodi L & the Spanish Study Group 2006 The d3-growth hormone receptor polymorphism does not influence the effect of GH treatment (66 μg/k/day) or the spontaneous growth in short non-GH-deficient small-for-gestational-age children: results from a two-year controlled prospective study in 170 Spanish patients. Journal of Clinical Endocrinology and Metabolism 91 32813286.

    • Search Google Scholar
    • Export Citation
  • Carter-Su C, Schwartz J & Smit LS 1996 Molecular mechanism of growth hormone action. Annual Review of Physiology 58 187207.

  • Choi JH, Kim HS, Kim SH, Yang YR, Bae YS, Chang JS, Moo KH, Ryu SH & Suh PG 2006 Phospholipase Cgamma1 negatively regulates growth hormone signalling by forming a ternary complex with Jak2 and protein tyrosine phosphatase-1B. Nature Cell Biology 8 13891397.

    • Search Google Scholar
    • Export Citation
  • Clackson T, Ultsch MH, Wells JA & De Vos AM 1998 Structural and functional analysis of the 1:1 growth hormone: receptor complex reveals the molecular basis for receptor affinity. Journal of Molecular Biology 277 11111128.

    • Search Google Scholar
    • Export Citation
  • Cowan JW, Wang X, Guan R, He K, Jiang J, Baumann G, Black RA, Wolfe MS & Frank SJ 2005 Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage. Journal of Biological Chemistry 280 1933119342.

    • Search Google Scholar
    • Export Citation
  • Dastot F, Sobrier ML, Duquesnoy P, Duriez B, Goossens M & Amselem S 1996 Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein. PNAS 93 1072310728.

    • Search Google Scholar
    • Export Citation
  • Davey HW, McLachlan MJ, Wilkins RJ, Hilton DJ & Adams TE 1999 STAT5b mediates the GH-induced expression of SOCS-2 and SOCS-3 mRNA in the liver. Molecular and Cellular Endocrinology 158 111116.

    • Search Google Scholar
    • Export Citation
  • Delitala G, Meloni T, Masala A, Alagna S, Devilla L & Costa R 1978 Action of somatostatin, levodopa and pyridoxine on growth hormone (GH) secretion in newborn infants. Biomedicine 29 1315.

    • Search Google Scholar
    • Export Citation
  • Dey BR, Spence SL, Nissley P & Furlanetto RW 1998 Interaction of human suppressor of cytokine signaling (SOCS)-2 with the insulin-like growth factor-I receptor. Journal of Biological Chemistry 273 2409524101.

    • Search Google Scholar
    • Export Citation
  • Dos Santos C, Essioux L, Teinturier C, Tauber M, Goffin V & Bougneres P 2004 A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone. Nature Genetics 36 720724.

    • Search Google Scholar
    • Export Citation
  • Esposito N, Paterlini P, Kelly PA, Postel-Vinay MC & Finidori J 1994 Expression of two isoforms of the human growth hormone receptor in normal liver and hepatocarcinoma. Molecular and Cellular Endocrinology 103 1320.

    • Search Google Scholar
    • Export Citation
  • Favre H, Benhamou A, Finidori J, Kelly PA & Edery M 1999 Dual effects of suppressor of cytokine signaling (SOCS-2) on growth hormone signal transduction. FEBS Letters 453 6366.

    • Search Google Scholar
    • Export Citation
  • Fisker S, Kristensen K, Rosenfalck AM, Pedersen SB, Ebdrup L, Richelsen B, Hilsted J, Christiansen JS & Jorgensen JO 2001 Gene expression of a truncated and the full-length growth hormone (GH) receptor in subcutaneous fat and skeletal muscle in GH-deficient adults: impact of GH treatment. Journal of Clinical Endocrinology and Metabolism 86 792796.

    • Search Google Scholar
    • Export Citation
  • Fisker S, Hansen B, Fuglsang J, Kristensen K, Ovesen P, Orskov H & Jorgensen JO 2004 Gene expression of the GH receptor in subcutaneous and intraabdominal fat in healthy females: relationship to GH-binding protein. European Journal of Endocrinology 150 773777.

    • Search Google Scholar
    • Export Citation
  • Flores-Morales A, Greenhalgh CJ, Norstedt G & Rico-Bautista E 2006 Negative regulation of growth hormone receptor signaling. Molecular Endocrinology 20 241253.

    • Search Google Scholar
    • Export Citation
  • Frank SJ 2001 Growth hormone signalling and its regulation: preventing too much of a good thing. Growth Hormone and IGF Research 11 201212.

    • Search Google Scholar
    • Export Citation
  • Frank SJ 2002 Receptor dimerization in GH and erythropoietin action – it takes two to tango, but how? Endocrinology 143 210.

  • Gatto L, Berlato C, Poli V, Tininini S, Kinjyo I, Yoshimura A, Cassatella MA & Bazzoni F 2004 Analysis of SOCS-3 promoter responses to interferon gamma. Journal of Biological Chemistry 279 1374613754.

    • Search Google Scholar
    • Export Citation
  • Gent J, Van Den EM, van Kerkhof P & Strous GJ 2003 Dimerization and signal transduction of the growth hormone receptor. Molecular Endocrinology 17 967975.

    • Search Google Scholar
    • Export Citation
  • Gentili S, Waters MJ & McMillen IC 2006 Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 290 R1044R1051.

    • Search Google Scholar
    • Export Citation
  • Gluckman PD, Grumbach MM & Kaplan SL 1981 The neuroendocrine regulation and function of growth hormone and prolactin in the mammalian fetus. Endocrine Reviews 2 363395.

    • Search Google Scholar
    • Export Citation
  • Gluckman PD, Gunn AJ, Wray A, Cutfield 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. The International Board of the Kabi Pharmacia International Growth Study. Journal of Pediatrics 121 920923.

    • Search Google Scholar
    • Export Citation
  • Gohlke BC, Fahnenstich H, Dame C & Albers N 2004 Longitudinal data for intrauterine levels of fetal IGF-I and IGF-II. Hormone Research 61 200204.

  • Goodyer CG, Branchaud CL & Lefbvre Y 1993a Effects of growth hormone (GH)-releasing factor and somatostatin on GH secretion from early to midgestation human fetal pituitaries. Journal of Clinical Endocrinology and Metabolism 76 12591264.

    • Search Google Scholar
    • Export Citation
  • Goodyer CG, Branchaud CL & Lefebvre Y 1993b In vitro modulation of growth hormone (GH) secretion from early to midgestation human fetal pituitaries by GH-releasing factor and somatostatin: role of Gs-adenylate cyclase-Gi complex and Ca2+ channels. Journal of Clinical Endocrinology and Metabolism 76 12651270.

    • Search Google Scholar
    • Export Citation
  • Goodyer CG, Figueiredo R, Krackovitch S, DeSouza Li L, Manalo J & Zogopoulos G 2001a Characterisation of the growth hormone receptor in human dermal fibroblasts and liver during development. American Journal of Physiology. Endocrinology and Metabolism 281 12131220.

    • Search Google Scholar
    • Export Citation
  • Goodyer CG, Zogopoulos G, Schwartzbauer G, Zheng H, Hendy GN & Menon RK 2001b Organisation and evolution of the human growth hormone receptor gene 5′ flanking region. Endocrinology 142 19231934.

    • Search Google Scholar
    • Export Citation
  • Govers R, van Kerkhof P, Schwartz AL & Strous GJ 1997 Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor. EMBO Journal 16 48514858.

    • Search Google Scholar
    • Export Citation
  • Greenhalgh CJ, Metcalf D, Thaus AL, Corbin JE, Uren R, Morgan PO, Fabri LJ, Zhang JG, Martin HM & Willson TA 2002 Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling. Journal of Biological Chemistry 277 4018140184.

    • Search Google Scholar
    • Export Citation
  • Hansen JA, Lindberg K, Hilton DJ, Nielsen JH & Billestrup N 1999 Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins. Molecular Endocrinology 13 18321843.

    • Search Google Scholar
    • Export Citation
  • Harding PA, Wang XZ, Kelder B, Souza S, Okada S & Kopchick JJ 1994 In vitro mutagenesis of growth hormone receptor Asn-linked glycosylation sites. Molecular and Cellular Endocrinology 106 171180.

    • Search Google Scholar
    • Export Citation
  • He B, You L, Uematsu K, Zang K, Xu Z, Lee AY, Costello JF, McCormick F & Jablons DM 2003 SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. PNAS 100 1413314138.

    • Search Google Scholar
    • Export Citation
  • Hebenstreit D, Luft P, Schmiedlechner A, Regl G, Frischauf AM, Aberger F, Duschl A & Horejs-Hoeck J 2003 IL-4 and IL-13 induce SOCS-1 gene expression in A549 cells by three functional STAT6-binding motifs located upstream of the transcription initiation site. Journal of Immunology 171 59015907.

    • Search Google Scholar
    • Export Citation
  • Hellgren G, Jansson JO, Carlsson LM & Carlsson B 1999 The growth hormone receptor associates with Jak1, Jak2 and Tyk2 in human liver. Growth Hormone and IGF Research 9 212218.

    • Search Google Scholar
    • Export Citation
  • Herrington J & Carter-Su C 2001 Signaling pathways activated by the growth hormone receptor. Trends in Endocrinology and Metabolism 12 252257.

    • Search Google Scholar
    • Export Citation
  • Herrington J, Smit LS, Schwartz J & Carter-Su C 2000 The role of STAT proteins in growth hormone signaling. Oncogene 19 25852597.

  • Hill DJ What is the role of growth hormone and related peptides in implantation and the development of the embryo and fetus Hormone Research 38 Suppl 1 1992 2834.

    • Search Google Scholar
    • Export Citation
  • Hill DJ, Freemark M, Strain AJ, Handwerger S & Milner RD 1988 Placental lactogen and growth hormone receptors in human fetal tissues: relationship to fetal plasma human placental lactogen concentrations and fetal growth. Journal of Clinical Endocrinology and Metabolism 66 12831290.

    • Search Google Scholar
    • Export Citation
  • Hill DJ, Camacho-Hubner C, Rashid P, Strain AJ & Clemmons DR 1989 Insulin-like growth factor (IGF)-binding protein release by human fetal fibroblasts: dependency on cell density and IGF peptides. Journal of Endocrinology 122 8798.

    • Search Google Scholar
    • Export Citation
  • Hill DJ, Riley SC, Bassett NS & Waters MJ 1992 Localization of the growth hormone receptor, identified by immunocytochemistry, in second trimester human fetal tissues and in placenta throughout gestation. Journal of Clinical Endocrinology and Metabolism 75 646650.

    • Search Google Scholar
    • Export Citation
  • Hocquette JF, Postel-Vinay MC, Kayser C, de Hemptinne B & Amar-Costesec A 1989 The human liver growth hormone receptor. Endocrinology 125 21672174.

  • Hocquette JF, Postel-Vinay MC, Djiane J, Tar A & Kelly PA 1990 Human liver growth hormone receptor and plasma binding protein: characterization and partial purification. Endocrinology 127 16651672.

    • Search Google Scholar
    • Export Citation
  • Hujeirat Y, Hess O, Shalev S & Tenenbaum-Rakover Y 2006 Growth hormone receptor sequence changes do not play a role in determining height in children with idiopathic short stature. Hormone Research 65 210216.

    • Search Google Scholar
    • Export Citation
  • Hwa V, Little B, Adiyaman P, Kofoed EM, Pratt KL, Ocal G, Berberoglu M & Rosenfeld RG 2005 Severe growth hormone insensitivity resulting from total absence of signal transducer and activator of transcription 5b. Journal of Clinical Endocrinology and Metabolism 90 42604266.

    • Search Google Scholar
    • Export Citation
  • Iida K, Takahashi Y, Kaji H, Takahashi MO, Okimura Y, Nose O, Abe H & Chihara K 1999 Functional characterization of truncated growth hormone (GH) receptor-(1–277) causing partial GH insensitivity syndrome with high GH-binding protein. Journal of Clinical Endocrinology and Metabolism 84 10111016.

    • Search Google Scholar
    • Export Citation
  • Jensen RB, Jeppesen KA, Vielwerth S, Michaelsen KF, Main KM, Skakkebaek NE & Juul A 2005 Insulin-like growth factor I (IGF-I) and IGF-binding protein 3 as diagnostic markers of growth hormone deficiency in infancy. Hormone Research 63 1521.

    • Search Google Scholar
    • Export Citation
  • Jorge AA, Marchisotti FG, Montenegro LR, Carvalho LR, Mendonca BB & Arnhold IJ 2006 Growth hormone (GH) pharmacogenetics: influence of GH receptor exon 3 retention or deletion on first-year growth response and final height in patients with severe GH deficiency. Journal of Clinical Endocrinology and Metabolism 91 10761080.

    • Search Google Scholar
    • Export Citation
  • Kamura T, Sato S, Haque D, Liu L, Kaelin WG Jr, Conaway RC & Conaway JW 1998 The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes and Development 12 38723881.

    • Search Google Scholar
    • Export Citation
  • Kaplan SL, Grumbach MM & Aubert ML 1976 The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Recent Progress in Hormone Research 32 161243.

    • Search Google Scholar
    • Export Citation
  • Kato Y, Murakami Y, Sohmiya M & Nishiki M 2002 Regulation of human growth hormone secretion and its disorders. Internal Medicine 41 713.

  • Kenth G, Shao Z, Cole DE & Goodyer CG 2007 Relationship of the human growth hormone receptor Exon 3 genotype with final adult height and bone mineral density. Journal of Clinical Endocrinology and Metabolism 92 725728.

    • Search Google Scholar
    • Export Citation
  • van Kerkhof P, Smeets M & Strous GJ 2002 The ubiquitin-proteasome pathway regulates the availability of the GH receptor. Endocrinology 143 12431252.

    • Search Google Scholar
    • Export Citation
  • Kile BT & Alexander WS 2001 The suppressors of cytokine signalling (SOCS). Cellular and Molecular Life Sciences 58 16271635.

  • Krebs DL & Hilton DJ 2001 SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19 378387.

  • Lanning NJ & Carter-Su C 2006 Recent advances in growth hormone signaling. Reviews in Endocrine and Metabolic Disorders 7 225235.

  • Lettre G, Butler JL, Ardlie KG & Hirschhorn JN 2007 Common genetic variation in eight genes of the GH/IGF1 axis does not contribute to adult height variation. Human Genetics 122 129139.

    • Search Google Scholar
    • Export Citation
  • Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ & Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330 537543.

    • Search Google Scholar
    • Export Citation
  • Levy DE & Darnell JE Jr 2002 Stats: transcriptional control and biological impact. Nature Reviews. Molecular Cell Biology 3 651662.

  • Matsumoto A, Masuhara M, Mitsui K, Yokouchi M, Ohtsubo M, Misawa H, Miyajima A & Yoshimura A 1997 CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation. Blood 89 31483154.

    • Search Google Scholar
    • Export Citation
  • Matsumoto A, Seki Y, Kubo M, Ohtsuka S, Suzuki A, Hayashi I, Tsuji K, Nakahata T, Okabe M & Yamada S 1999 Suppression of STAT5 functions in liver, mammary glands, and T cells in cytokine-inducible SH2-containing protein 1 transgenic mice. Molecular and Cellular Biology 19 63966407.

    • Search Google Scholar
    • Export Citation
  • Mehta A, Hindmarsh PC, Stanhope RG, Turton JP, Cole TJ, Preece MA & Dattani MT 2005 The role of growth hormone in determining birth size and early postnatal growth, using congenital growth hormone deficiency (GHD) as a model. Clinical Endocrinology 63 223231.

    • Search Google Scholar
    • Export Citation
  • Mericq V, Roman R, Iniguez G, Angel B, Salazar T, Avila A, Perez-Bravo F & Cassorla F 2007 Relationship between nocturnal growth hormone concentrations, serum IGF-I/IGFBP-3 levels, insulin sensitivity and GH receptor allelic variant in small for gestational age children. Hormone Research 68 132138.

    • Search Google Scholar
    • Export Citation
  • Metcalf D, Greenhalgh CJ, Viney E, Willson TA, Starr R, Nicola NA, Hilton DJ & Alexander WS 2000 Gigantism in mice lacking suppressor of cytokine signalling-2. Nature 405 10691073.

    • Search Google Scholar
    • Export Citation
  • Munsick RA 1984 Human fetal extremity lengths in the interval from 9 to 21 menstrual weeks of pregnancy. American Journal of Obstetrics and Gynecology 149 883887.

    • Search Google Scholar
    • Export Citation
  • Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, Nishimoto N, Kajita T, Taga T & Yoshizaki K 1997 Structure and function of a new STAT-induced STAT inhibitor. Nature 387 924929.

    • Search Google Scholar
    • Export Citation
  • Osafo J, Wei Y, Kenth G & Goodyer CG 2005 Growth hormone during development. Reviews in Endocrine and Metabolic Disorders 6 173182.

  • Pantel J, Machinis K, Sobrier ML, Duquesnoy P, Goossens M & Amselem S 2000 Species-specific alternative splice mimicry at the growth hormone receptor locus revealed by the lineage of retroelements during primate evolution. Journal of Biological Chemistry 275 1866418669.

    • Search Google Scholar
    • Export Citation
  • Phornphutkul C, Frick GP, Goodman HM, Berry SA & Gruppuso PA 2000 Hepatic growth hormone signaling in the late gestation fetal rat. Endocrinology 141 35273533.

    • Search Google Scholar
    • Export Citation
  • Pilotta A, Mella P, Filisetti M, Felappi B, Prandi E, Parrinello G, Notarangelo LD & Buzi F 2006 Common polymorphisms of the growth hormone (GH) receptor do not correlate with the growth response to exogenous recombinant human GH in GH-deficient children. Journal of Clinical Endocrinology and Metabolism 91 11781180.

    • Search Google Scholar
    • Export Citation
  • Piwien-Pilipuk G, Huo JS & Schwartz J 2002 Growth hormone signal transduction. Journal of Pediatric Endocrinology and Metabolism 15 771786.

  • Rabkin R & Schaefer F 2004 New concepts: growth hormone, insulin-like growth factor-I and the kidney. Growth Hormone and IGF Research 14 270276.

    • Search Google Scholar
    • Export Citation
  • Ram PA & Waxman DJ 1999 SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. Journal of Biological Chemistry 274 3555335561.

    • Search Google Scholar
    • Export Citation
  • Ram PA & Waxman DJ 2000 Role of the cytokine-inducible SH2 protein CIS in desensitization of STAT5b signaling by continuous growth hormone. Journal of Biological Chemistry 275 3948739496.

    • Search Google Scholar
    • Export Citation
  • Raz B, Janner M, Petkovic V, Lochmatter D, Eble A, Dattani MT, Hindmarsh PC, Fluck CE & Mullis PE 2008 Influence of growth hormone receptor d3 and full-length isoforms on growth hormone response and final height in patients with severe growth hormone deficiency. Journal of Clinical Endocrinology and Metabolism 93 974980.

    • Search Google Scholar
    • Export Citation
  • Reddy GR, Pushpanathan MJ, Ransom RF, Holzman LB, Brosius FC III, Diakonova M, Mathieson P, Saleem MA, List EO & Kopchick JJ 2007 Identification of the glomerular podocyte as a target for growth hormone action. Endocrinology 148 20452055.

    • Search Google Scholar
    • Export Citation
  • Ronn SG, Hansen JA, Lindberg K, Karlsen AE & Billestrup N 2002 The effect of suppressor of cytokine signaling 3 on GH signaling in beta-cells. Molecular Endocrinology 16 21242134.

    • Search Google Scholar
    • Export Citation
  • Rosenfeld RG, Kofoed E, Buckway C, Little B, Woods KA, Tsubaki J, Pratt KA, Bezrodnik L, Jasper H & Tepper A 2005 Identification of the first patient with a confirmed mutation of the JAK-STAT system. Pediatric Nephrology 20 303305.

    • Search Google Scholar
    • Export Citation
  • Rosenfeld RG, Belgorosky A, Camacho-Hubner C, Savage MO, Wit JM & Hwa V 2007 Defects in growth hormone receptor signaling. Trends in Endocrinology and Metabolism 18 134141.

    • Search Google Scholar
    • Export Citation
  • Ross RJM 1999 Truncated growth hormone receptor isoforms. Acta Paediatrica 428 164166.

  • Ross RJ, Esposito N, Shen XY, Von Laue S, Chew SL, Dobson PR, Postel-Vinay MC & Finidori J 1997 A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Molecular Endocrinology 11 265273.

    • Search Google Scholar
    • Export Citation
  • Shimano S, Suzuki S, Nagashima K, Yagi H, Sakaguchi M & Kuroume T 1985 Growth hormone responses to growth hormone releasing factor in neonates. Biology of the Neonate 47 367370.

    • Search Google Scholar
    • Export Citation
  • Shoba L, An MR, Frank SJ & Lowe WL Jr 1999 Developmental regulation of insulin-like growth factor-I and growth hormone receptor gene expression. Molecular and Cellular Endocrinology 152 125136.

    • Search Google Scholar
    • Export Citation
  • Simard M, Manthos H, Giaid A, Lefebvre Y & Goodyer CG 1996 Ontogeny of growth hormone receptors in human tissues: an immunohistochemical study. Journal of Clinical Endocrinology and Metabolism 81 30973102.

    • Search Google Scholar
    • Export Citation
  • Sobrier ML, Duquesnoy P, Duriez B, Amselem S & Goossens M 1993 Expression and binding properties of two isoforms of the human growth hormone receptor. FEBS Letters 319 1620.

    • Search Google Scholar
    • Export Citation
  • Socolovsky M, Fallon AE, Wang S, Brugnara C & Lodish HF 1999 Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98 181191.

    • Search Google Scholar
    • Export Citation
  • Strain AJ, Hill DJ, Swenne I & Milner RD 1987 Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin- C. Journal of Cellular Physiology 132 3340.

    • Search Google Scholar
    • Export Citation
  • Strous GJ, van Kerkhof P, Govers R, Rotwein P & Schwartz AL 1997 Growth hormone-induced signal tranduction depends on an intact ubiquitin system. Journal of Biological Chemistry 272 4043.

    • Search Google Scholar
    • Export Citation
  • Symonds ME, Mostyn A & Stephenson T 2001 Cytokines and cytokine receptors in fetal growth and development. Biochemical Society Transactions 29 3337.

    • Search Google Scholar
    • Export Citation
  • Tapanainen J, Kellokumpu-Lehtinen P, Pelliniemi L & Huhtaniemi I 1981 Age-related changes in endogenous steroids of human fetal testis during early and midpregnancy. Journal of Clinical Endocrinology and Metabolism 52 98102.

    • Search Google Scholar
    • Export Citation
  • Tollet-Egnell P, Flores-Morales A, Stavreus-Evers A, Sahlin L & Norstedt G 1999 Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat. Endocrinology 140 36933704.

    • Search Google Scholar
    • Export Citation
  • Urbanek M, MacLeod JN, Cooke NE & Liebhaber SA 1992 Expression of a human growth hormone (hGH) receptor isoform is predicted by tissue-specific alternative splicing of exon 3 of the hGH receptor gene transcript. Molecular Endocrinology 6 279287.

    • Search Google Scholar
    • Export Citation
  • Uyttendaele I, Lemmens I, Verhee A, De Smet AS, Vandekerckhove J, Lavens D, Peelman F & Tavernier J 2007 MAPPIT analysis of STAT5, CIS and SOCS2 interactions with the growth hormone receptor. Molecular Endocrinology 21 28212831.

    • Search Google Scholar
    • Export Citation
  • Veldhuis JD, Roemmich JN, Richmond EJ, Rogol AD, Lovejoy JC, Sheffield-Moore M, Mauras N & Bowers CY 2005 Endocrine control of body composition in infancy, childhood, and puberty. Endocrine Reviews 26 114146.

    • Search Google Scholar
    • Export Citation
  • De Vos AM, Ultsch M & Kossiakoff AA 1992 Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255 306312.

    • Search Google Scholar
    • Export Citation
  • Walker JL, Moats-Staats BM, Stiles AD & Underwood LE 1992 Tissue-specific developmental regulation of the messenger ribonucleic acids encoding the growth hormone receptor and the growth hormone binding protein in rat fetal and postnatal tissues. Pediatric Research 31 335339.

    • Search Google Scholar
    • Export Citation
  • Waters MJ & Kaye PL 2002 The role of growth hormone in fetal development. Growth Hormone and IGF Research 12 137146.

  • Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA & Brown RJ 2006 New insights into growth hormone action. Journal of Molecular Endocrinology 36 17.

  • Wei Y, Rhani Z & Goodyer CG 2006 Characterization of growth hormone receptor messenger ribonucleic acid variants in human adipocytes. Journal of Clinical Endocrinology and Metabolism 91 19011908.

    • Search Google Scholar
    • Export Citation
  • Welniak LA, Tian ZG, Sun R, Keller JR, Richards S, Ruscetti FW & Murphy WJ 2000 Effects of growth hormone and prolactin on hematopoiesis. Leukemia & Lymphoma 38 435445.

    • Search Google Scholar
    • Export Citation
  • Wickelgren RB, Landin KL, Ohlsson C & Carlsson LM 1995 Expression of exon 3-retaining and exon 3-excluding isoforms of the human growth hormone-receptor is regulated in an interindividual, rather than a tissue-specific, manner. Journal of Clinical Endocrinology and Metabolism 80 21542157.

    • Search Google Scholar
    • Export Citation
  • Wit JM & van Unen H 1992 Growth of infants with neonatal growth hormone deficiency. Archives of Disease in Childhood 67 920924.

  • Woods KA, Dastot F, Preece MA, Clark AJ, Postel-Vinay MC, Chatelain PG, Ranke MB, Rosenfeld RG, Amselem S & Savage MO 1997 Phenotype: genotype relationships in growth hormone insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism 82 35293535.

    • Search Google Scholar
    • Export Citation
  • Wormald S & Hilton DJ 2004 Inhibitors of cytokine signal transduction. Journal of Biological Chemistry 279 821824.

  • Ymer SI & Herington AC 1992 Developmental expression of the growth hormone receptor gene in rabbit tissues. Molecular and Cellular Endocrinology 83 3949.

    • Search Google Scholar
    • Export Citation
  • Zhang JG, Farley A, Nicholson SE, Willson TA, Zugaro LM, Simpson RJ, Moritz RL, Cary D, Richardson R & Hausmann G 1999 The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. PNAS 96 20712076.

    • Search Google Scholar
    • Export Citation
  • Zhang Y, Guan R, Jiang J, Kopchick JJ, Black RA, Baumann G & Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. Journal of Biological Chemistry 276 2456524573.

    • Search Google Scholar
    • Export Citation
  • Zhu T, Goh EL, Graichen R, Ling L & Lobie PE 2001 Signal transduction via the growth hormone receptor. Cellular Signalling 13 599616.

  • Zogopoulos G, Albrecht S, Pietsch T, Alpert L, von Schweinitz D, Lefebvre Y & Goodyer CG 1996a Fetal- and tumor-specific regulation of growth hormone receptor mRNA expression in human liver. Cancer Research 56 29492953.

    • Search Google Scholar
    • Export Citation
  • Zogopoulos G, Figueiredo R, Jenab A, Ali Z, Lefebvre Y & Goodyer CG 1996b Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development. Journal of Clinical Endocrinology and Metabolism 81 775782.

    • Search Google Scholar
    • Export Citation
*

(G Kenth and J A M Mergelas contributed equally to this work)

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

      Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 307 47 1
PDF Downloads 187 46 0
  • View in gallery

    Schematic representation of the hGH/hGHR signaling molecules examined in the present study.

  • View in gallery

    Immunoblot (IB) analyses of immunoprecipitated (IP) hGHR in human adult liver (HAL) and fetal hepatocytes (FH) using the AL47 antibody raised against the intracellular domain of the GHR (Zhang et al. 2001). Molecular markers are indicated at the left side with arrowheads and arrows indicate the two major (∼70 and ∼120 kDa) IP/IB hGHR bands. IgG, rabbit IgG.

  • View in gallery

    Analysis of full-length (FL) and truncated (T) (1–279) hGHR mRNA. (A) A specific primer in exon 9 (9AS) was designed to recognize both the FL and T forms of hGHR mRNA for RT–PCR assays. (B) Pilot RT-PCR assays showed that there were no significant differences in T versus FL GHR amplicon amplification over a wide range of PCR cycle number, in both fetal and postnatal hepatic samples (n=3 each). (C and D) Comparison of FL and T hGHR mRNAs in intact fetal liver (n=12, 12.7–19.5 weeks), postnatal liver (n=3, 37–60 years), fetal hepatocytes (n=7, 11.5–17.5 weeks), and fetal liver hematopoietic cells (n=5, 12.7–19.5 weeks) by RT-PCR. (C) Representative polyacrylamide gel of PCR products detects FL hGHR mRNA at 252 bp and T hGHR mRNA at 226 bp. (D) Comparative data for intact postnatal liver, intact fetal liver, fetal hepatocytes, and fetal hematopoietic cells, expressed as a ratio of T/FL transcripts (Mean±s.e.m). Statistical differences are calculated relative to fetal hepatocytes: ***P<0.001. (E and F) Comparison of FL and T hGHR mRNAs in various fetal tissues. (E) Representative polyacrylamide gel of PCR products. pBS-SK+/Hpa II marker is indicated at 242 bp. CNS, central nervous system. (F) T hGHR mRNA is detected in a wide variety of fetal tissues. Each tissue type was tested using three different samples and the final data are expressed as a ratio of T/FL transcripts (Mean±s.e.m). Statistical differences are calculated relative to intact fetal liver: *P<0.05, **P<0.01. (G and H) Comparison of FL and T hGHR mRNAs in various primate cell lines. (G) Representative polyacrylamide gel of PCR products. pBS-SK+/Hpa II marker is indicated at 242 bp. (H) Graphic analysis of T/FL hGHR mRNA in three cell lines and human adult liver (HAL) (HepG2 band densities were too low to be accurately analyzed). Each sample was tested using three to five different RNA pools (Mean±s.e.m). Statistical differences are calculated relative to HAL: **P<0.01, ***P<0.001.

  • View in gallery

    Analysis of JAK/STAT proteins. Representative immunoblots of JAK/STAT and calnexin expression in (A) fetal hepatocytes and (B) fetal liver. Relative abundance of JAK2 (∼120 kDa), STAT1 (∼91 kDa), STAT3 (∼92 kDa), STAT5A (∼95 kDa), and STAT5B (∼92 kDa) proteins in (A) fetal hepatocytes (n=3, 12–18 weeks) or (B) fetal liver (n=3, 10.75–18 weeks) (Mean±s.e.m). Data are normalized to calnexin (∼90 kDa) and compared with postnatal liver (HAL: n=3, 15–60 years). Statistical differences are expressed relative to HAL: *P<0.05, **P<0.03, ***P<0.01.

  • View in gallery

    Analysis of SOCS proteins. Representative immunoblots of SOCS/CIS and calnexin expression in (A) fetal hepatocytes and (B) fetal liver. Relative abundance of SOCS1 (∼24 kDa), SOCS2 (∼23 kDa), SOCS3 (∼30 kDa), and CIS (∼32 kDa) proteins in (A) fetal hepatocytes (n=3, 12–19 weeks) or (B) fetal liver (n=3, 10.75–18 weeks) (Mean±s.e.m). Data are normalized to calnexin (∼90 kDa) and compared with postnatal liver (HAL: n=3, 15–60 years). Statistical differences are expressed relative to HAL: **P<0.04.