Abstract
Growth hormone (GH) regulates transcription factors associated with c-fos, including C/EBPbeta. Two forms of C/EBPbeta, liver-activating protein (LAP) and liver inhibitory protein (LIP), are dephosphorylated in GH-treated 3T3-F442A fibroblasts. GH-induced dephosphorylation of LAP and LIP is reduced when cells are preincubated with phosphatidylinositol 3'-kinase (PI3K) inhibitors. GH activates Akt and inhibits glycogen synthase kinase-3 (GSK-3). Lithium, a GSK-3 inhibitor, increases GH-dependent dephosphorylation of LAP and LIP. Both are in vitro substrates of GSK-3, suggesting that GSK-3 inactivation contributes to GH-promoted dephosphorylation of C/EBPbeta. Alkaline phosphatase increases binding of LAP homodimers and decreases binding of LIP homodimers to c-fos, suggesting that dephosphorylation of C/EBPbeta modifies their ability to bind DNA. Both alkaline phosphatase- and GH-mediated dephosphorylation comparably increase binding of endogenous LAP in 3T3-F442A cells. In cells overexpressing LAP and GSK-3, LAP binding decreases, suggesting that GSK-3-mediated phosphorylation interferes with LAP binding. Expression of constitutively active GSK-3 reduced GH-stimulated c-fos promoter activity. These studies indicate that PI3K/Akt/GSK-3 mediates signaling between GH receptor and the nucleus, promoting dephosphorylation of C/EBPbeta. Dephosphorylation increases binding of LAP complexes to the c-fos promoter and may contribute to the participation of C/EBPbeta in GH-stimulated c-fos expression.
Highlights
The CCAAT/Enhancer-Binding Proteins (C/EBPs) have recently been shown to participate in Growth hormone (GH)-regulated transcription of c-fos [14]
Treatment with the MEK inhibitor PD098059 (40 M, 30 min) prior to GH did not interfere with the GH-promoted dephosphorylation of liver inhibitory protein (LIP) and liver-activating protein (LAP), indicating that the MEK-ERK pathway is unlikely to be a major contributor to this response to GH
It has been known for some time that GH can initiate phosphatidylinositol 3-kinase (PI3K) signaling by stimulating tyrosine phosphorylation of insulin receptor substrate family proteins, and the association of insulin receptor substrates 1 and 2 with PI3K (44 – 47)
Summary
Materials—3T3-F442A fibroblasts were provided by Dr H. Polyclonal antibodies against an oligopeptide corresponding to amino acids 16 –26 of GSK-3␣ phosphorylated on Ser-21 (anti-P-GSK-3) and to a peptide corresponding to amino acids 203–219 of GSK-3 (anti-GSK-3) were purchased from Upstate Biotechnology Inc. Immunoblotting—Cell lysis and immunoblotting for C/EBP were performed as previously described [14] and for Akt and GSK-3 as follows: confluent 3T3-F442A cells on 100-mm plates were washed with phosphate-buffered saline with vanadate (10 mM Tris (pH 7.4), 150 mM sodium phosphate, 1 mM sodium vanadate) and scraped in 0.3 ml of L-RIPA lysis buffer (50 mM Hepes, pH 7.0, 250 mM NaCl, 0.5% Triton X-100) containing 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml each of aprotinin and leupeptin. Gene Expression Assay—CHO-GHR (1 ϫ 105 cells/35-mm well) were transiently transfected by calcium phosphate coprecipitation [28] with wt-fos-Luc (0.4 g) and the RSV -galactosidase plasmid (0.1 g), with or without a plasmid encoding a constitutively active GSK-3 (GSK-3 S9A 0.8 g), in the presence or absence of CMV-LAP DNA (1 ng) or corresponding amounts of pcDNA3.1 vector per 35-mm well. Analysis of variance with factorial Scheffe F test was used to analyze data as indicated
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