Growth Hormone Induction of Hepatic Serine Protease Inhibitor 2.1 Transcription Is Mediated by a Stat5-related Factor Binding Synergistically to Two γ-Activated Sites

Abstract

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...

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

“…Stat5, first purified as the prolactin (PRL)-responsive mammary gland factor from sheep mammary glands (20), has since been purified and cloned from mouse liver and IM-9 lymphocytes (19,21). We purified a GH-inducible nuclear factor complex, GHINF, from rat liver, in which the major DNA binding component was Stat5 (15). We also found that the GHINF complex contained additional unknown proteins.…”

“…Electrophoretic Mobility Shift Assays (EMSAs)-Preparation of crude rat hepatic nuclear extracts, affinity purification of GHINF, and EMSA have all been reported previously (15). Briefly, in an EMSA reaction, 5 g of hepatic nuclear extracts or ϳ2 ng of GHINF were incubated with 20 fmol of the radiolabeled probe of interest in a buffer containing 20 mM HEPES (pH 7.6), 10% glycerol, 2 mM MgCl 2 , 5 mM CaCl 2 , 0.1 mg/ml bovine serum albumin, 4% Ficoll 400, 1 mM spermidine, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 g of poly(dI⅐dC).…”

“…Plasmid Constructions and Functional Assays-The construction of Spi 2.1 (Ϫ275/ϩ85) into the HindIII/PstI sites of the parent CAT reporter plasmid pCAT(An), designated here as Spi-A-CAT, and that of mutations in place of the wild type GHRE sequence in Spi-A-CAT were described previously (15). Based on our observation that the insertion of the Xba site at Ϫ116/Ϫ111 does not disrupt the GH response, we constructed long PCR primers extending from this site to 15 base pairs (bp) 5Ј of the putative YY1 recognition site.…”

“…Five l of the resultant supernatant were then loaded onto a 7.5% SDS-polyacrylamide gel. Immunoblotting was performed as reported previously (15).…”

“…We delineated a GH response element (GHRE) in the promoter of Spi 2.1 that includes two ␥-activated sites (GAS), both of which are necessary for a GH response in functional assays (15). In the hypophysectomized rat, as well as in several cell lines, GH activates Stat1, -3, and/or -5 (7)(8)(9)(10)(15)(16)(17)(18)(19). Stat5, first purified as the prolactin (PRL)-responsive mammary gland factor from sheep mammary glands (20), has since been purified and cloned from mouse liver and IM-9 lymphocytes (19,21).…”