Growth Hormone Response Element Research Articles

Intrauterine growth restriction disrupts developmental epigenetics around distal growth hormone response elements on the rat hepatic IGF-1 gene.

Intrauterine growth restriction (IUGR) decreases serum IGF-1 levels. Postnatal IGF-1 expression is transcriptionally regulated by growth hormone (GH) through growth hormone response elements (GHREs). We hypothesized that IUGR disrupts the normal developmental maturation of hepatic IGF-1 intron 2 growth hormone response element (IN2GHRE) histone methylation of key lysines and DNA methylation. We also evaluated a 5' distal weak enhancer (IGF-1 5'-upstream region growth hormone response element; 5URGHRE) as a GHRE specificity control. IUGR was induced through a well-characterized model of bilateral uterine artery ligation of the pregnant rat. Offspring livers were tested at d 0 and 21. Chromatin immunoprecipitation and bisulfite sequencing quantified epigenetic characteristics. We found that distinct age-related developmental patterns of histone and DNA methylation characterize each GHRE. Development increased H3K4 trimethylation (me3) in both GHREs. However, H3K9me3 decreased with age at IN2GHRE and increased with age at 5URGHRE. IUGR altered the developmental pattern of H3K4me3 and K9me3 around the GHREs in a sex-specific manner at d 21. Developmental and IUGR-induced DNA methylation occurred in a GHRE-, CpG site-, and sex-specific manner. We conclude that IUGR disrupts developmental epigenetics around distal GHREs on the rat hepatic IGF-1 gene.

IGF1 Growth Hormone Response Element Undergoes Developmentally Regulated Demethylation Independent of STA5b

The insulin‐like growth factor 1 (IGF1)/growth hormone (GH) axis, a major factor in growth, development and metabolism, is significantly affected by intrauterine growth retardation. Some of these alterations may lead to permanent pathological programming of the IGF axis. Prenatally, IGF1 signaling is GH independent, while postnatally, it is partly or fully GH dependent. GH regulates IGF1 via binding of the intermediate transcription factor, STAT5b, to a GH response element (GHRE) in the hepatic IGF1 gene. During adolescence, the rodent hepatic GHRE converts from closed to open chromatin and remains open throughout life. However it is not known when epigenetic events first begin to occur that prepare the chromatin for opening. CpG methylation and demethylation is often associated with closed and open chromatin respectively. We therefore sought to follow changes in DNA methyaltion in the GHRE as a possible indicator of transition to open chromatin. The GHRE was subjected to Na‐bisulfite sequencing at various time points in mouse development in wild‐type and STA5b knockout mice. The GHRE was also fused to a CG‐free reporter to test the effect of methylation on enhancer activity. The GHRE undergoes 3 stages of gradual DNA demethylation declining from a high of 80% methylation in embryonic stem (ES) cells to <30% by 5 weeks of life and remains hypomethyated for 2 yrs. Demethylation of the mouse GHRE is independent of STAT5b and is required for GHRE function in vitro.

Human growth hormone receptor gene expression is regulated by Gfi-1/1b and GAGA cis-elements

Human growth hormone receptor (hGHR) gene regulation is complex: mRNAs are transcribed from multiple variant (V) 5′UTR exons, several ubiquitously while others only in the postnatal hepatocyte. The liver-specific V1 exon promoter contains Gfi-1/1b repressor sites adjacent to a GAGA box, a GH response element (GHRE) in several mammalian genes. GAGA boxes are also present in the ubiquitously expressing V3 exon promoter. Heterologous sites in bovine, ovine and murine GHR genes suggest conserved roles. GAGA factor stimulated V1 and V3 promoters while Gfi-1/1b repressed basal and GAF-stimulated V1 transcription. HGH treatment of HepG2 cells resulted in a new complex forming with V3 GAGA elements, suggesting a functional GHRE. Data suggest liver-specific V1 transcription is regulated by inhibitory Gfi-1/1b and stimulatory GAGA cis-elements and Gfi-1/1b may control the lack of V1 expression in fetal liver, hepatic tumours and non-hepatic tissues. In addition, hGH may regulate hGHR expression through V3 GAGA boxes.

In VivoImaging of Hepatic Growth Hormone Signaling

We developed a system to noninvasively and repeatedly image in vivo hepatic GH signaling. GH regulates postnatal growth and metabolism. It affects numerous tissues, but has major effects in liver. We used nude mice for adenoviral-mediated delivery of a signal transducer and activator of transcription 5-dependent GH response element, a luciferase reporter to detect GH signaling pathway activation. We detected by noninvasive bioluminescence imaging GH-induced hepatic GH signaling serially within intact mice. Statistically significant effects of GH dose and time dependence were detected in the liver luciferase signal that peaked 3 h after GH injection. Codelivery of GH receptor significantly enhanced GH response, an effect that was further augmented by fasting. Our imaging system allows detailed in vivo analysis of GH signaling and action and may be a paradigm for studies of additional signaling pathways in liver and other tissues.

Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.

Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression

Growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression through signal transducer and activator of transcription 5b (STAT5b) and STAT5a. The objective of this study was to identify the cis-regulatory DNA region involved in this process. By cotransfection analyses of shotgun DNA fragments of a bacterial artificial chromosome sequence containing the entire human IGF-I gene and a large 5'-flanking region, a approximately 700-bp DNA region approximately 75 kb 5' to the IGF-I gene was found to have the ability to enhance gene expression from both heterologous and homologous promoters in the presence of constitutively active STAT5a or STAT5b. This 700-bp DNA region contains two closely located consensus STAT5-binding sites, and its sequence appears to be evolutionarily conserved. Electrophoretic mobility shift assays verified the ability of the two putative STAT5-binding sites to bind to STAT5a and STAT5b. Cotransfection analyses confirmed that both STAT5-binding sites were necessary for the 700-bp DNA region to mediate STAT5a or STAT5b activation of gene transcription. Chromatin immunoprecipitation assays demonstrated that the chromosomal region containing these two STAT5-binding sites was bound by constitutively active STAT5b protein in HepG2 cells and that the binding was accompanied by increased expression of IGF-I mRNA. In reconstituted GH-responsive cells, this 700-bp DNA region was able to mediate GH-induced STAT5a or STAT5b activation of gene expression. These results together suggest that this STAT5-binding site-containing distal 5'-flanking region of IGF-I gene may be an enhancer mediating GH-induced STAT5 activation of IGF-I gene transcription.

Novel Gender-Related Regulation of CYP2C12 Gene Expression in Rats

The expression of CYP2C12 by GH occurs in female but not in male rat livers. Direct injection of the CYP2C12 promoter-luciferase gene into male rat livers showed that the CYP2C12 promoter was active in both male and female rats. Thus, to further examine one or more factors that regulate the gender-related expression of CYP2C12, male rats were treated with trichostatin A, a specific inhibitor of histone deacetylase capable of condensing the chromatin structure. Interestingly, the expression of CYP2C12 by GH was seen even in the livers of male rats, indicating that histone deacetylase contributes to the suppression of CYP2C12 expression in male rats. Deoxyribonuclease I hypersensitive assay using nuclei from the livers of male or female rats revealed that the chromatin structure of the CYP2C12 gene was gender specific: a hypersensitive site at a position -4.2 kb containing GH-responsive element that bound to signal transducer and activator of transcription 5 (STAT5), termed as HS (hypersensitive site) 1, was specific for female rat livers, whereas a hypersensitive site at a position -3 kb, designated as HSm (male-specific hypersensitive site), was characteristic of male rat livers. A -3425/-3275 region within HSm functioned as a negative regulatory region, when the region was inserted in front of simian virus 40 promoter. Gel shift assay demonstrated that both CCAAT/enhancer-binding protein alpha and beta bound to the -3425/-3275 region. Based on these results, we conclude that the gender-related expression of the CYP2C12 gene results from the inaccessibility of to STAT5 to the GH-responsive element by chromatin condensation seen in male rat livers, and from the presence of the male-specific HSm that acts as a silencer.

Transcriptional Regulation of the CYP2C12 Gene in Female Rats.

The purpose of this study was to clarify the mechanism (s) responsible for the growth hormone (GH)-induced expression of the CYP2C12 gene. To identify a functional GH-responsive element (GHRE) in vivo, we performed the direct injection of promoter-luciferase chimeric genes into female rat livers. The results showed that the luciferase activity was decreased to approximately 20% by the deletion of the sequence between nucleotides -4213 and -4161. Within this region, two copies of a possible GHRE were present. The sequence of the GHRE was overlapped with that of an interferon-y-activated sequence (GAS), known to be recognized by the signal transducer and activator of transcription (STAT) proteins. In fact, a super shift assay showed that STAT5 was capable of binding to the core sequence of the GHRE. Furthermore, a luciferase assay with reporter plasmids, which lacks HNF4 or HNF6-binding sites, revealed that the GH-stimulated expression of the CYP2C12 gene was regulated cooperatively by STAT5, HNF-4, HNF-6 and the factor(s) which binds to the elements, 2C12-I (-4095 to -4074) and 2C12-II (-4072 to -4045). The cooperative regulation by STAT5 and the liver-enriched transcription factors account for the GH-dependent and the liver-specific expression of the CYP2C12 gene in female rats.

Inhibition of growth hormone action in models of inflammation.

Growth hormone (GH) action is attenuated during the hepatic acute-phase response (APR). To understand this attenuation, we asked whether GH and cytokine-signaling pathways intersect during an APR. In hypophysectomized rats treated with lipopolysaccharide (LPS), accumulation of activated signal transducer and transcription activator 5 (Stat5) in hepatic nuclei in response to GH and its binding to a GH response element (GHRE) from the serine protease inhibitor (Spi) 2.1 promoter are diminished in a time-dependent manner. Similarly, accumulation of activated Stat3 in hepatic nuclei in response to LPS and its binding to a high-affinity sis-inducible element (SIE) are also diminished by the simultaneous administration of GH. In functional assays with primary hepatocytes, LPS-stimulated monocyte-conditioned medium (MoCM) inhibits the GH response of Stat5-dependent Spi 2.1 reporter activity but induces Stat3-dependent Spi 2.2 reporter activity, as in an APR. Similar results are obtained when hepatocytes are treated with either tumor necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1beta. TNF-alpha, IL-1beta, and IL-6 also inhibit GH-induced Spi 2.1 mRNA expression in hepatocytes. Thus inhibition of the GH signaling pathway during an APR results in reduced expression of GH-responsive genes.

Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.

GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.

Organization and regulation of the gene encoding the sheep acid-labile subunit of the 150-kilodalton insulin-like growth factor-binding protein complex.

In adult animals, most circulating insulin-like growth factor I (IGF-I) and IGF-II is sequestered in a 150-kDa complex composed of 1 molecule each of IGF, IGF-binding protein-3 or -5, and the acid-labile subunit (ALS). Capture of IGF in ALS-containing complexes increases their circulating half-lives and concentrations and suppresses their hypoglycemic potential. ALS has been studied almost exclusively in rodents and primates, and no information exists in the sheep despite its extensive use to study the circulating IGF system. To initiate studies in the sheep, we isolated the ovine ALS gene and characterized its spatial and developmental regulation. The ALS gene spans about 3.0 kb of chromosomal DNA and consists of 2 exons interrupted by a 977-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide; exon 2 encodes the remaining 27 amino acids of the signal peptide and the entire mature protein of 579 amino acids. Transcription initiation occurs at nucleotides -58 and -29 (ATG, + 1), 2 sites that are not preceded by TATA or initiator sequences. A DNA fragment extending from -727 to - 11 of the sheep ALS gene directed basal expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. GH increased promoter activity by 1.8-fold, consistent with conservation in the sheep promoter of the GH response element previously identified in the mouse gene. A survey of adult tissues by Northern analysis revealed the presence of a 2.2-kb transcript only in liver. Weak expression was first detected in liver on day 130 of fetal life, increased suddenly on day 7 of postnatal age, and changed little thereafter. The sheep is a useful model to understand the regulation and role of ALS, particularly around the time of birth, when final maturation of the circulating IGF system occurs.

Yin-yang 1 and Glucocorticoid Receptor Participate in the Stat5-mediated Growth Hormone Response of the Serine Protease Inhibitor 2.1 Gene

A growth hormone-inducible nuclear factor complex (GHINF), affinity-purified using the growth hormone response element (GHRE) from the promoter of rat serine protease inhibitor 2.1, was found to contain Stat5a and -5b, as well as additional components. The ubiquitous transcription factor yin-yang 1 (YY1) is present in GHINF. An antibody to YY1 inhibited the formation of the GHINF.GHRE complex in an electrophoretic mobility shift assay. Furthermore, Stat5 was co-immunoprecipitated from rat hepatic nuclear extracts with antibodies to YY1. An examination of the GHRE shows that, in addition to two gamma-activated sites, it contains a putative YY1 binding site between the two gamma-activated sites, overlapping them both. Mutation of this putative YY1 site results in a decrease of GHINF.GHRE complex formation in an electrophoretic mobility shift assay and a corresponding decrease in growth hormone (GH) response in functional assays. The glucocorticoid receptor was also present in GHINF, and Stat5 co-immunoprecipitates with glucocorticoid receptor in hepatic nuclear extracts from rats treated with GH. GH activation of serine protease inhibitor 2.1 requires the unique sequence of the GHRE encompassing the recognition sites of several transcription factors, and the interaction of these factors enhances the assembly of the transcription complex.

Growth hormone action in hypothyroid infant rats.

In neonatal rats, expression of serine protease inhibitors 2.1 and 2.3 mRNA peaks on d 2 of life and declines shortly thereafter, coinciding with levels of circulating GH. To evaluate the role of GH in this increase and to test the hypothesis that GH is active in perinatal life, we studied GH action in a model of GH deficiency. Maternal/neonatal hypothyroidism with consequent GH deficiency was induced by methimazole administration to pregnant dams. The resultant hypothyroid neonates were treated at d 2 or 7 of age with GH or saline for 1 h before exsanguination. In d-7 neonates, but not at d 2, GH administration resulted in significant serine protease inhibitors 2.1 and 2.3 mRNA induction. This treatment did not result in increased production of either GH receptor or IGF-I mRNA at either age. There was a slight GH-independent increase in GH receptor and IGF-I mRNA expression by d 7. Electromobility shift assays using hepatic nuclear extracts from these neonates and the GH response element from the serine protease inhibitor 2.1 promoter showed signal transducer and activator of transcription 5 (Stat5) binding in response to GH in extracts from d-7 rats only. Immunoblots of these extracts showed twice as much Stat5 in the nuclei of d-7 treated neonates compared with d-2 treated neonates. We conclude that there is apparent insensitivity to GH treatment in d-2 neonates that remits by d 7 and that this remission correlates with increased abundance of GH receptor and Stat5.

Cooperative Regulation of CYP2C12 Gene Expression by STAT5 and Liver-specific Factors in Female Rats

The purpose of this study was to clarify the mechanism(s) responsible for the growth hormone (GH)-induced expression of the CYP2C12 gene. To identify a functional GH-responsive element (GHRE) in vivo, we performed the direct injection of promoter-luciferase chimeric genes into female rat livers. The results showed that the luciferase activity was decreased to approximately 20% by the deletion of the sequence between nucleotides -4213 and -4161. Within this region, two copies of a possible GHRE were present. The sequence of the GHRE was overlapped with that of an interferon-gamma-activated sequence, known to be recognized by the signal transducer and activator of transcription (STAT) proteins. In fact, a supershift assay showed that STAT5 was capable of binding to the core sequence of the GHRE. Furthermore, a luciferase assay with reporter plasmids, Delta-4161/-3781, mutated hepatocyte nuclear factor-4 (HNF-4), and mutated HNF-6, revealed that the GH-stimulated expression of the CYP2C12 gene was regulated cooperatively by STAT5, HNF-4, HNF-6, and the factor(s) that binds to the elements, 2C12-I (-4095 to -4074) and 2C12-II (-4072 to -4045). The cooperative regulation by STAT5 and the liver-enriched transcription factors account for the GH-dependent and the liver-specific expression of the CYP2C12 gene in female rats.

Growth Hormone-Mediated Regulation of Insulin-Like Growth Factor I Promoter Activity in C6 Glioma Cells

The molecular mechanisms by which GH regulates insulin-like growth factor (IGF-I) gene expression remain obscure. One difficulty has been the lack of established GH-responsive cell lines that express the IGF-I gene. To develop such a cell line, we used rat C6 glioma cells which, as determined by RNase protection assay, express the IGF-I gene but not the GH receptor gene. To confer GH responsiveness, C6 cells were cotransfected with vectors that express the GH receptor (pRc/CMV WTrGHR) and Jak2 (pRc/CMV Jak2). GH responsiveness was demonstrated using luciferase reporter genes containing either the Sis-inducible element from the c-fos gene (pTK81-SIE-Luc) or 6 copies of the GH-responsive GAS-like element (GLE) from the rat spi2.1 gene (pSpi-GLE-Luc). The SIE is activated by binding of STAT1 and 3, whereas the GLE binds STAT5. In cells cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and either pTK81-SIE-Luc or pSpi GLE-Luc, treatment with 500 ng/ml GH for 24 h stimulated a 3.1- and 1.7-fold increase in luciferase activity, respectively. These data suggest that in C6 cells cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2, GH activates STAT1, 3, and 5. To determine whether GH-responsive IGF-I promoter activity could be demonstrated, C6 cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and an IGF-I-luciferase fusion gene that contained a fragment of the rat IGF-I gene that extended from −412 in the 5′-flanking region of exon 1 to the Met-22 in exon 3. GH stimulated a modest, but reproducible, 1.7-fold increase in luciferase activity in these cells, suggesting that a GH-responsive element is present in this region of the IGF-I gene. To better localize the GH-responsive element, cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2 plus one of several IGF-I-luciferase fusion genes containing either fragments of one of the two promoters in the IGF-I gene or a fragment of intron 2 that includes a GH-responsive DNase I hypersensitivity site. For all constructs, treatment with GH for 24 h did not stimulate a significant increase in luciferase activity, suggesting that GH-responsive sequences are not located in these specific regions of the IGF-I gene or that GH-directed transcription of the IGF-I gene is mediated via several different regions of the IGF-I gene and the effect of any one of these regions in isolation was not sufficiently robust to be detected in this model system. In summary, transient expression of the GH receptor and Jak2 in C6 cells creates a GH-responsive system that activates STAT1, 3, and 5. Moreover, a fragment of the IGF-I gene that contains exons 1 and 2, a fragment of exon 3, and introns 1 and 2 is GH responsive using this model system.

Growth hormone-mediated regulation of insulin-like growth factor I promoter activity in C6 glioma cells.

The molecular mechanisms by which GH regulates insulin-like growth factor (IGF-I) gene expression remain obscure. One difficulty has been the lack of established GH-responsive cell lines that express the IGF-I gene. To develop such a cell line, we used rat C6 glioma cells which, as determined by RNase protection assay, express the IGF-I gene but not the GH receptor gene. To confer GH responsiveness, C6 cells were cotransfected with vectors that express the GH receptor (pRc/CMV WTrGHR) and Jak2 (pRc/CMV Jak2). GH responsiveness was demonstrated using luciferase reporter genes containing either the Sis-inducible element from the c-fos gene (pTK81-SIE-Luc) or 6 copies of the GH-responsive GAS-like element (GLE) from the rat spi2.1 gene (pSpi-GLE-Luc). The SIE is activated by binding of STAT1 and 3, whereas the GLE binds STAT5. In cells cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and either pTK81-SIE-Luc or pSpi GLE-Luc, treatment with 500 ng/ml GH for 24 h stimulated a 3.1- and 1.7-fold increase in luciferase activity, respectively. These data suggest that in C6 cells cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2, GH activates STAT1, 3, and 5. To determine whether GH-responsive IGF-I promoter activity could be demonstrated, C6 cells were cotransfected with pRc/CMV WTrGHR, pRc/ CMV Jak2, and an IGF-I-luciferase fusion gene that contained a fragment of the rat IGF-I gene that extended from -412 in the 5'-flanking region of exon 1 to the Met-22 in exon 3. GH stimulated a modest, but reproducible, 1.7-fold increase in luciferase activity in these cells, suggesting that a GH-responsive element is present in this region of the IGF-I gene. To better localize the GH-responsive element, cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2 plus one of several IGF-I-luciferase fusion genes containing either fragments of one of the two promoters in the IGF-I gene or a fragment of intron 2 that includes a GH-responsive DNase I hypersensitivity site. For all constructs, treatment with GH for 24 h did not stimulate a significant increase in luciferase activity, suggesting that GH-responsive sequences are not located in these specific regions of the IGF-I gene or that GH-directed transcription of the IGF-I gene is mediated via several different regions of the IGF-I gene and the effect of any one of these regions in isolation was not sufficiently robust to be detected in this model system. In summary, transient expression of the GH receptor and Jak2 in C6 cells creates a GH-responsive system that activates STAT1, 3, and 5. Moreover, a fragment of the IGF-I gene that contains exons 1 and 2, a fragment of exon 3, and introns 1 and 2 is GH responsive using this model system.

Dual effects of suppressor of cytokine signaling (SOCS-2) on growth hormone signal transduction

A family of suppressors of cytokine signaling (SOCS) has recently been identified of which two members have been shown to block growth hormone (GH) signaling. Dose-response experiments were conducted in 293 cells and SOCS-1 and SOCS-3 were shown to inhibit the transcriptional activation of a GH-responsive element and suppressed Jak2 tyrosine kinase activity. SOCS-2 had two opposite effects: at low concentrations it inhibited GH-induced STAT5-dependent gene transcription, but restoration of GH signaling was observed at higher concentrations. In cotransfection studies, SOCS-2 was able to block the inhibitory effect of SOCS-1 but not that of SOCS-3 on GH signaling. These findings suggest that a major function for SOCS-2 is to restore the sensitivity to GH by overcoming the initial inhibitory effects of other endogenous SOCS molecules.

Definition of a high affinity growth hormone DNA response element

Rat liver contains a growth hormone inducible nuclear factor complex, GHINF, that binds to the growth hormone response element (GHRE) of the serine protease inhibitor (Spi) 2.1 gene. GHINF contains Stat5 and binds to paired γ-activated sites (GAS) within the GHRE, but poorly to either one alone. By analysis of the sequence of various GAS sites that bind the GHINF complex (based on the GHRE 3′ GAS motif), we demonstrate that a 13 nucleotide high affinity DNA recognition sequence (haGHRE) for GHINF complex binding is ANTTC C/ TN A/ GGAA A/ T A/ T. One copy of the haGHRE will replace the requirement for two GAS elements present in the wild type promoter in supporting a GH response in primary hepatocyte culture. Mutation of the native Spi 2.1 from a paired GAS site to a single haGHRE does not appreciably change its affinity for binding to the GHINF complex, nor does it alter its sensitivity to GH concentration.

Grb10 Identified as a Potential Regulator of Growth Hormone (GH) Signaling by Cloning of GH Receptor Target Proteins

The cloning of receptor targets procedure, used so far to identify proteins associated with tyrosine kinase receptors was modified to clone SH2 proteins able to bind to the growth hormone receptor (GHR). The cytoplasmic region of GHR, a member of the cytokine receptor superfamily does not contain tyrosine kinase activity. It was thus phosphorylated in bacteria by the Elk tyrosine kinase and radiolabeled to screen a mouse expression library. With this probe, we identified Shc and the p85 subunit of phosphatidylinositol 3-kinase as direct targets of the receptor. The other proteins identified, Csk, Shb, Grb4, and Grb10 are new potential transducers for cytokine receptors. We show in Huh-7 hepatoma cells that Grb10 and GHR associate under GH stimulation. Co-transfections in 293 cells further show that Grb10 interacts with both the GHR and Jak2. Functional tests demonstrate that Grb10 inhibits transcription of two reporter genes containing, respectively, the serum response element of c-fos and the GH response element 2 of the Spi2.1 gene, whereas it has no effect on a reporter gene containing only Stat5 binding elements. Our results suggest that Grb10 is a new target for a member of the cytokine receptor family that down-regulates some GH signaling pathways downstream of Jak2 and independently of Stat5.

Binding of STAT5a and STAT5b to a single element resembling a gamma-interferon-activated sequence mediates the growth hormone induction of the mouse acid-labile subunit promoter in liver cells.

After birth, the endocrine actions of insulin-like growth factor (IGF)-I and -II become increasingly important. In postnatal animals, most of circulating IGFs occur in 150-kDa complexes formed by association of an acid-labile subunit (ALS) with complexes of IGF and IGF-binding protein-3. ALS is synthesized almost exclusively in liver. GH stimulates the transcription of the ALS gene, resulting in increased hepatic mRNA and circulating ALS levels. To map the GH response element, a series of 5'-deletion fragments of the mouse ALS promoter (nt -2001 to -49, A(+1)TG) were inserted in the luciferase reporter plasmid pGL3 and transfected into the H4-II-E rat hepatoma cell line. GH stimulated the activity of promoter fragments with 5'-ends between nucleotide (nt) -2001 and nt -653 by 1.9- to 2.7-fold. This stimulation was abolished by deletion of the region located between nt -653 and nt -483. This region contains two sites, ALS-GAS1 and ALS-GAS2, that resemble the gamma-interferon activated sequence (GAS). Mutation of the ALS-GAS1 site, but not of the ALS-GAS2 site, eliminated the response to GH when assessed in the context of a GH-responsive promoter fragment, indicating that ALS-GAS1 was necessary for GH induction. Three tandem copies of ALS-GAS1 were sufficient to confer GH inducibility to the minimal promoter of the thymidine kinase gene. In electrophoretic mobility shift assays, ALS-GAS1 formed a specific, GH-dependent protein-DNA complex with nuclear extracts from H4-II-E cells. Using antibodies directed against members of the family of signal transducers and activators of transcription (STAT), this complex was shown to be composed of STAT5a and STAT5b. Identical results were obtained when transfections and mobility shift assays were performed in primary rat hepatocytes in which the endogenous ALS gene is expressed. Thus, the transcriptional activation of the mouse ALS gene by GH is mediated by the binding of STAT5 isoforms to a single GAS-like element.