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 ϳ93 kDa and a minor band of ϳ70 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 ␥-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 ␤-casein gene. These studies indicate that GHINF is a Stat5-related factor binding synergistically to two GAS elements to activate Spi 2.1 transcription.Great strides have been made in the last year toward understanding the mechanisms of cytokine and growth factor signal transduction. These extracellular signaling proteins include growth hormone (GH), 1 prolactin, interleukins (IL), interferons, granulocyte-macrophage colony stimulating factor, and colony stimulating factor 1. The binding of these polypeptides to their specific surface receptors in target cells is followed by a cascade of events activating the Jak-STAT pathway. In this pathway, the Janus kinase (Jak) family of tyrosine kinases, known to be associated with these receptors, are activated and tyrosine-phosphorylated. These kinases, in turn, presumably activate a family of latent cytoplasmic proteins known as signal transducers and activators of transcription (STAT), through phosphorylation of tyrosine residues. The activated STAT proteins are then translocated to the nucleus where they, by themselves or in combination with otherwise weak DNA-binding proteins, bind to specific response elements on responsive genes and activate transcription (1). Six of these STAT proteins have been identified to date. Some of the STAT proteins are highly specific in their response to individual cytokines (e.g. Stat2 for interferon-␣), while others appear to be involved in multiple pathways (2). The STAT proteins recognize response elements that share homology with the ␥-interferon activation site (GAS) recognized by Stat1 (1).The involvement of Jak-STAT pathways in GH signal transduction has been evidenced recently. Jak2 has been shown to be associated with GH receptors following GH binding with phosphorylation of both Jak2 and the GH receptor and subsequent activation of signal transduction (3). Further, it has been observed that GH treatment appears to activate several STAT proteins resulting in their phosphorylation. This has been noted both in cultured cell systems (4 -7) and in liver (8, 9), a known targe...

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.

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, ϳ41-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.

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 GHINF⅐GHRE 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 ␥-activated sites, it contains a putative YY1 binding site between the two ␥-activated sites, overlapping them both. Mutation of this putative YY1 site results in a decrease of GHINF⅐GHRE 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.

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.