Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.

Transcriptional regulation by growth hormone (GH) represents the culmination of signal transduction pathways that are initiated by the cell surface GH receptor and are targeted to the nucleus. Recent studies have demonstrated that the activated GH receptor can stimulate Stat1, a cytoplasmic transcription factor that becomes tyrosine phosphorylated and translocates to the nucleus, where it can interact with specific DNA sequences to modulate gene expression. GH also has been found to induce protein binding to a portion of the rat serine protease inhibitor (Spi) 2.1 gene promoter that is required for GH-induced transcription of Spi 2.1. Using GH-deficient hypophysectomized rats as a model, we show that GH treatment rapidly and potently induces both nuclear Spi 2.1 mRNA expression in the liver and specific nuclear protein binding to a 45-bp segment of the Spi 2.1 gene promoter. A GH-inducible gel-shifted complex appears within 15 min of systemic hormone administration and can be inhibited by an antiphosphotyrosine monoclonal antibody but is not blocked by a polyclonal antiserum to Stat1, Stat3, or Stat4, even though the nucleotide sequence contains two gamma interferon-activated sequence-like elements that could interact with STAT proteins. By Southwestern (DNA-protein) blot analysis, approximately 41- and 35-kDa GH-inducible proteins were detected in hepatic nuclear extracts with the Spi 2.1 DNA probe. Thus, a GH-activated signaling pathway stimulates Spi 2.1 gene expression through a unique mechanism that does not appear to involve known members of the STAT family of transcription factors.

Binding of a growth hormone-inducible nuclear factor is mediated by tyrosine phosphorylation.

The nuclear mechanism by which GH acts to induce gene expression after binding to its receptor on the cell surface is not defined. We have characterized an element in the 5'-flanking region of the rat GH-responsive serine protease inhibitor (Spi) 2.1 gene responsible for its induction by GH. This element binds a hepatic nuclear protein(s) in a GH state-specific manner. Activation of binding by GH does not require de novo protein synthesis, suggesting that a reversible posttranslational process is required for binding to the element. To define the mechanism of this process, hepatic nuclear extracts were analyzed by electrophoretic mobility shift assays using a DNA fragment (-147 to -103) of the Spi 2.1 gene. Treatment of extracts with phosphatases resulted in a marked reduction of GH state-specific binding. Addition of phosphatase inhibitors antagonized the reduction in binding after phosphatase treatment. The specific nature of the phosphorylation event involved in binding was explored using phosphotyrosine antibodies and a protein tyrosine phosphatase. Treatment of nuclear extracts with either of these reagents ablated binding to the response element. Because the tyrosine-phosphorylated transcription factor protein p91 has recently been implicated in cytokine signal transduction mediated by JAK2, we sought evidence that p91 was part of the GH-responsive binding complex. Analysis of an enriched preparation of GH-inducible binding complexes by Western blots using anti-p91 demonstrated no immunoreactivity. We conclude that tyrosine phosphorylation of a nuclear factor is required for GH state-specific binding to this GH response element in vivo, but that p91 is not present in the binding complex.

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 gamma-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/T(N)A/G(GAA)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.

Inhibition of Na,K-ATPase by sodium selenite and reversal by glutathione.

Sodium selenite has been shown to inhibit Na,K-ATPase. Glutathione, at sufficient excess, is able to prevent or reverse the inhibition. Dithiothreitol can also reverse much of the inhibition, but KCN cannot. Selenomethionine does not inhibit Na,K-ATPase. The interactions of sodium selenite with Na,K-ATPase and glutathione may aid in understanding the early events in selenium cataractogenesis.

High performance liquid chromatography determination of cystathionase activity in human and rabbit lenses.

Cystathionase activity has been demonstrated in human and rabbit lenses, using L-homoserine as substrate. The amount of activity found was quite low, 0.05 and 0.08 nmol of alpha-ketobutyrate formed/h/mg protein in human and rabbit lenses, respectively. The human lens cystathionase was found to have a pH maximum of 8.5 and a Km of 23.8 mM for L-homoserine. A sensitive assay, involving the use of high performance liquid chromatography, was developed to measure the small amounts of alpha-ketobutyrate produced.

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.

Expression of growth hormone-responsive serpin mRNAs in perinatal rat liver.

Hormonal mechanisms controlling growth of the fetus are poorly understood, and generally growth hormone (GH) is not thought to influence perinatal growth. To examine the influence of GH in the expression of genes in perinatal rat liver, we measured RNA levels of several GH responsive and growth axis genes. Spi 2.1, Spi 2.2, Spi 2.3, insulin-like growth factors (IGF) I and II, and GH receptor mRNAs were measured in rat liver total RNA from gestational days 19, 20, 21, and postnatal day 2. Spi 2.1 and 2.3 genes were faintly expressed on day 20, 6% and 13 +/- 1% of adult levels on gestation day 21, and 6% and 31 +/- 6% of adult levels on day 2. Deoxyribonuclease I (DNase I)-hypersensitive sites in the 5' flanking region of the Spi 2.1 gene, which are concordant with GH response, were not present in DNA extracted from livers at gestation day 19 but were present at days 20, 21, and 2, suggesting the gene is transcriptionally competent after day 19 and that the areas of chromatin vulnerable to DNase I digestion are the same in pre- and postnatal life. Low levels of GH receptor mRNAs (approximately 10% of adult) were present on all measured days. IGF-I mRNA was below quantitatable levels in day 19 or 20 fetal samples and was only 2.7 +/- 0.1% of adult levels on day 21. Levels on day 2 were 9.6 +/- 1.9% of adult. IGF-II mRNA was essentially constant throughout this period, with a minimal increase at day 21 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Growth hormone induction of hepatic serine protease inhibitor 2.1 transcription is mediated by a Stat5-related factor binding synergistically to two gamma-activated sites.

A growth hormone (GH)-inducible nuclear factor (GHINF) from rat liver has been purified to near homogeneity. On SDS-polyacrylamide gel electrophoresis and UV-cross-linking, a major band of mass approximately 93 kDa and a minor band of approximately 70 kDa are detected in the purified fraction. DNase I footprinting using purified GHINF yields a protected region of -149/-115 on the rat serine protease inhibitor 2.1 (Spi 2.1) promoter encompassed within the growth hormone response element (GHRE). Mutational analysis demonstrated that GHINF binds synergistically to two gamma-interferon-activated sites (GAS) within the GHRE, with the 3' element being the pivotal binding domain. Functional assays show that both GAS elements are necessary for full GH response. GHINF has no immunoreactivity with either a C-terminal Stat1 antibody or an N-terminal Stat3 antibody, while cross-reacting with a C-terminal Stat5 monoclonal antibody. GHINF will bind to two GAS elements from the Stat5 binding region of the beta-casein gene. These studies indicate that GHINF is a Stat5-related factor binding synergistically to two GAS elements to activate Spi 2.1 transcription.

Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6.

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

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.