Growth hormone (GH) is the major regulator of postnatal growth. It acts upon its target cells to regulate their growth, differentiation and metabolism. GH is thought to act by altering gene expression in target cells, but few GH-regulated genes are known. In this thesis, to further understand the molecular mechanisms of growth hormone action I have sought to identify genes that are directly regulated by GH using in vivo and in vitro models of growth hormone action. Three complementary approaches were undertaken to identify GH-target genes and included: 1. screening of candidate transcription factors for GH-induced binding to their consensus elements 2. cDNA array analysis and 3. suppression subtractive hybridization (SSH) analysis. The function and mechanism of regulation of some of these identified genes were further investigated in a cell model, growth hormone-dependent adipogenesis of 3T3-F442A preadipocytes.
candidate transcription factors for GH-induced DNA binding revealed that the immediate early gene, Egr-1, displays rapid and transient GH-dependent binding to a high-affinity binding site. Furthermore, this was accompanied by a concomitant increase in Egr-1 mRNA. Truncations of the proximal 1kb of the Egr-1 promoter revealed that a 374bp region (-624 to -250) contributes around 80% of GH inducibility in 3T3-F442A cells and around 90% inducibility in CHO-Kl cells. This region contains three juxtaposed SRE/Ets site pairs known to be important for regulating Egr-1 transcription in response to exogenotis stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. EMSA studies incorporating these specific mutations showed that disruption of either Ets or SRE elements abrogated ternary complex formation at these composite sites. DNA binding of ternary complexes, but not binary complexes, was rapidly and transiently
increased by GH. EMSA supershifts indicated that these ternary complexes contained SRF and the Ets factors Elk-1 and Sap-1a. Co-expression of Sap-1a and Elk-1 resulted in a marked increase in GH induction of Egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. I conclude that GH stimulates transcription of Egr-1 primarily through activation of these Ets factors at multiple composite Ets/SRF sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximises this response.
cDNA array analysis was used to identify genes rapidly induced in the liver of GH-deficient dwarf rats following a single systemic injection of GH. Eight genes were found to have upregulated mRNA expression within 1-3 hours of GH administration, results that were confirmed by northern analysis. The identity of these genes indicates that GH influences a diversity of
cellular processes. Hepatic target genes include several associated with the acute phase response: fibrinogen B beta, IL-6 receptor (gpl30), Stat3 and IL-1 receptor (type 1). A role for GH in regulating cytokine and growth factor signalling is suggested by upregulation of mRNAs encoding the intracellular signalling molecule, p38MAPK (mitogen activated protein kinase). Two genes involved in DNA repair and cell cycle control, APEN (apurinic/apyridinimic endonuclease) and GADD45 (growth arrest and DNA damage 45) were upregulated. Other induced genes include a lactate transporter (MCT-1) and an extracellular matrix remodelling enzyme, MT1-MMP (membrane type 1 matrix metalloproteinase).
To identify genes involved in GH-initiated cell differentiation, I have studied the well-characterized model of GH-dependent adipogenesis of 3T3-F442A preadipocytes. Using the suppression subtractive hybridization technique, I have identified nine genes induced within 60 minutes of
GH treatment, and verified these by northern analysis. Seven were identifiable as Stat2, Stat3, Stra13, thrombospondin-1, oncostatin M receptor β chain, a DEAD box RNA helicase, and muscleblind, a developmental transcription factor. Bioinformatic approaches identified one of the two remaining unknown genes as a novel 436 residue serine/threonine kinase. As each of the identified genes have important developmental roles, they may be important in initiating GH-induced adipogenesis.
To establish if the transcription factors Stat3, Stra13, Stat5a are involved in GH-induced differentiation of 3T3-F442A preadipocytes these proteins together with constitutively active Stat3 and Stat5a were ectopically expressed in 3T3-F442A preadipocytes using a retroviral expression system. Overexpression of Stat3, Stat5 and Stral3 had no effect on differentiation as determined macroscopically by lipid accumulation. Overexpression of Stat3 and Stat5a constitutively active
mutant proteins demonstrated that Stat5a but not Stat3 was sufficient for GH-independent differentiation of 3T3-F442A preadipocytes. Constitutively active Stat5a was able to replace the requirement of GH in differentiation of 3T3-F442A preadipocytes in part by upregulation of the expression of key adipogenic transcription factors C/EBPβ, PPARγ and C/EBPα.