Profiles of Growth Hormone (GH)-regulated Genes Reveal Time-dependent Responses and Identify a Mechanism for Regulation of Activating Transcription Factor 3 By GH

Jeffrey S Huo,Richard C Mceachin,Tracy Xiao Cui,Nisha K Duggal,Tsonwin Hai,David J States,Jessica Schwartz

doi:10.1074/jbc.m508492200

Abstract

In examination of mechanisms regulating metabolic responses to growth hormone (GH), microarray analysis identified 561 probe sets showing time-dependent patterns of expression in GH-treated 3T3-F442A adipocytes. Biological functions significantly over-represented among GH-regulated genes include regulators of transcription at early times, and lipid biosynthesis, cholesterol biosynthesis, and mediators of immune responses at later times (48 h). One novel GH-induced gene encodes activating transcription factor 3 (ATF3). Atf3 mRNA expression and promoter activity were stimulated by GH. Genes for ATF3 and growth arrest and DNA damage-inducible gene 45 gamma (GADD45gamma) showed similar time-dependent patterns of responses to GH, suggesting similar regulatory mechanisms. A conserved sequence in the promoters of the Atf3 and Gadd45gamma genes contains a CCAAT/enhancer-binding protein (C/EBP) site previously observed in the Gadd45gamma promoter, suggesting a novel corresponding C/EBP site in the Atf3 promoter. C/EBPbeta was found to bind to the predicted Atf3 C/EBP site, and C/EBPbeta enhanced the activation of the wild-type Atf3 promoter. Mutation of the predicted Atf3 C/EBP site disrupted Atf3 promoter activation not only by C/EBPbeta but also by GH. These findings suggest that GH regulates transcription of Atf3 through a mechanism utilizing factors, such as C/EBPbeta, which bind to a novel C/EBP site.

Highlights

The molecular mechanisms controlling growth hormone (GH)-regulated gene expression have been studied in several genes, including Fos (c-fos), which encodes a transcription factor implicated in growth regulation [4]; Spi2.1, which encodes serine protease inhibitor 2.1 [5]; and Igf1, which encodes insulin-like growth factor 1, which mediates many of the growth-promoting effects of GH [6, 7]
To identify GH-regulated genes potentially involved in insulin resistance, cells were treated with GH for 48 h when 3T3-F442A adipocytes show impaired responses to insulin
When GH-regulated genes sharing temporal patterns of gene expression were sorted into groups by biological function, the role and timing of biological processes regulated by GH in adipocytes were suggested

Summary

EXPERIMENTAL PROCEDURES

Materials—3T3-F442A cells were provided by Dr H. Fetal calf serum for use in differentiation of 3T3-F442A cells into adipocytes was purchased from Atlanta Biologicals. University of Michigan), in which the CMV-C/EBP␤ vector was constructed, was used to normalize total amounts of transfected DNA. Transcriptional Activation—CHO-GHR cells (1 ϫ 105 cells/35 mm well) were pre-incubated for 4 h with Dulbecco’s modified Eagle’s medium supplemented with 8% calf serum, and transiently transfected as described previously [23] with WT or mC/EBP ATF3-Luc plasmids (500 ng/well), and plasmids for C/EBP␤ (10 ng/well) or fulllength rat GHR (500 ng/well) as indicated. For GH treatment experiments, cells were transfected for 18 h, followed by 4 h in Ham’s F-12 medium with 8% fetal calf serum, 18 h in Ham’s F-12 medium containing 1% bovine serum albumin instead of serum, to which GH was added for 8 more hours. Statistical significance was assessed using a Student’s t test

RESULTS

No of genes

DISCUSSION