Abstract
Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release, which is intermittent in male rats and nearly continuous in females. To investigate the influence of these GH secretory patterns on intracellular hepatocyte signaling, we compared the pattern of liver nuclear protein tyrosine phosphorylation in male and female rats. An M(r) approximately 93,000 polypeptide, p93, was found to be tyrosine phosphorylated to a high level in male but not female rats. GH, but not prolactin, rapidly stimulated p93 tyrosine phosphorylation in hypophysectomized rats. Intermittent plasma GH pulses triggered repeated p93 phosphorylation, while continuous GH exposure led to desensitization and a dramatic decline in liver nuclear p93. p93 was cross-reactive with two monoclonal antibodies raised to mammary Stat 5, whose tyrosine phosphorylation is stimulated by prolactin. Intermittent GH pulsation translocated liver Stat 5/p93 protein from the cytosol to the nucleus and also activated its DNA binding activity, as demonstrated using a mammary Stat 5-binding DNA element derived from the beta-casein gene. p93 is thus a liver-expressed, Stat 5-related DNA binding protein that undergoes tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation and is proposed to be an intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene expression.
Highlights
Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release, which is intermittent in male rats and nearly continuous in females
Intermittent plasma GH pulses triggered repeated p93 phosphorylation, while continuous GH exposure led to desensitization and a dramatic decline in liver nuclear p93. p93 was cross-reactive with two monoclonal antibodies raised to mammary Stat 5, whose tyrosine phosphorylation is stimulated by prolactin
Intermittent GH pulsation translocated liver Stat 5/p93 protein from the cytosol to the nucleus and activated its DNA binding activity, as demonstrated using a mammary Stat 5-binding DNA element derived from the p-casein gene. p93 is a liver-expressed, Stat 5-related DNA binding protein that undergoes tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation and is proposed to be an intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene expression
Summary
Animal Treatments-Adult male and female Fischer 344 rats (8-10 weeks of age) were untreated or were hypophysectomized by the supplier (Taconic, Inc., Germantown, NY), with follow-up care provided as previously described [43]. In other experiments, hypophysectomized rats were treated with human GH (NIDDK-hGH-B-1, BIO) or with rat prolactin (hormonally pure grade, NIDDK-rPRL-B-8SIAFP) at 12.5 ",g/100 g body weight, intraperitoneally, and the animals were killed 45 min later. Gel Mobility Sh ift A naly sis-Double-stranded oligon ucleoti de probes, 32P_end lab eled on one st ra nd usin g T4 kinase, wer e in cubated for 30 min at room temp erature with 5 Mg of liver nu clea r ext ract protein dissolved in 5 MI of NE D buffer an d 10 Ml of 10 rnxt Tris-H Cl buffer, pH 7.5, containing 10 fmol of DNA prob e, 2 Mg of polytdl -dC), 4% glycerol, 1 mxt MgCl2, 0.5 mxt EDTA, 0.5 m xt dit hiothreitol, 50 mxt NaCl (15 MI, tot al volume ). Unlabeled doubl e-stranded oligon ucleot ides used as com petitors for the gel shift experi ments wer e obta ined from Santa Cr uz Biotechn ology, Inc. a nd included SIE mutant oligon ucleoti de (muta t ion of TTCCCG to CCACCG; sc-2536), a nd GASIISRE conse nsus oligonucleo tide (sc-2537)
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