Computational and functional analysis of growth hormone-regulated genes

Using computational and functional approaches, mechanisms by which growth hormone (GH) regulates gene expression to mediate changes in growth and metabolism were analyzed. Microarray analysis of 3T3-F442A adipocytes treated with GH for critical time periods identified 561 GH-responsive probe sets which clustered into sequential waves of gene expression. Computational methods identified biological functions significantly over-represented among the GH-regulated genes, which include regulators of transcription at early times (0.5--4 h), and lipid and cholesterol biosynthesis, carbohydrate metabolism, and mediators of immune responses at later times (48 h). Two genes sharing similar time-dependent patterns of expression in response to GH encode Activating Transcription Factor 3 (ATF3) and Growth Arrest and DNA Damage-Inducible 45 gamma (GADD45). They share a promoter sequence which contains a CCAAT/Enhancer Binding Protein (C/EBP) site previously observed in the Gadd45gamma promoter. C/EBPbeta was found to bind to the corresponding C/EBP site predicted in Atf3. C/EBPbeta enhanced Atf3 promoter activation, which was disrupted by mutation of the C/EBP site. GH also activates the Atf3 promoter, and this response was disrupted by mutation of the C/EBP site, suggesting that the C/EBP site is involved GH-regulated Aft3 promoter activity. In contrast to the sustained increase in Atf3 expression induced by GH in adipocytes, 3T3-F442A pre-adipocytes were stimulated only transiently by GH. The early stimulation of Atf3 expression by GH was found to be dependent on an intact MAPK signaling pathway. The delayed increase in Atf3 expression in adipocytes appears to be mediated by a distinct mechanism. These studies identify novel GH targets in adipocytes, and provide new insights into mechanisms for GH-stimulated gene expression. These findings open new avenues for understanding mechanisms for GH action in normal and pathological conditions, including insulin resistance.