Cooperative regulation of endogenous cAMP-response element binding protein and CCAAT/enhancer-binding protein beta in GH-stimulated c-fos expression.

GH activates the c-fos promoter by regulating multiple transcription factors. This study adds to our understanding of GH-regulated transcription by demonstrating that GH regulates the c-fos cAMP-response element (CRE) and its binding protein, CREB. Activation of the c-fos promoter by GH is impaired by expression of dominant-negative A-CREB. GH stimulates rapid and transient phosphorylation of CREB at Ser 133 (P-CREB), a critical site for transactivation by CREB, in 3T3-F442A preadipocytes. Mutation of this residue impairs GH-induced c-fos expression, suggesting that phosphorylation of CREB at Ser 133 contributes to GH-induced c-fos activation. The MEK inhibitor UO126 impaired the phosphorylation of CREB and that of C/EBPbeta, suggesting that ERKs mediate the phosphorylation of both proteins. UO126, but not the protein kinase A inhibitor H89, blocked GH-induced c-fos mRNA expression. A combination of CREB and C/EBPbeta enhanced c-fos promoter activation, and mutation of the CRE impaired the enhancement, as well as GH-stimulated c-fos activation. GH treatment increased the occupancy of both endogenous phospho-CREB and phospho-C/EBPbeta on the c-fos promoter. The increases were impaired by UO126. The active P-CREB and P-C/EBPbeta are induced by GH to occupy the same c-fos promoter DNA, suggesting that they may participate in a GH-regulated complex on c-fos. These findings suggest that coordinated phosphorylation of CREB and C/EBPbeta in response to GH is mediated by ERK1/2, and that the phosphorylated proteins are part of a regulatory complex that occupies c-fos in vivo to regulate c-fos transcription cooperatively in response to GH.

Multiple mechanisms of growth hormone-regulated gene transcription.

Diverse physiological actions of growth hormone (GH) are mediated by changes in gene transcription. Transcription can be regulated at several levels, including post-translational modification of transcription factors, and formation of multiprotein complexes involving transcription factors, co-regulators and additional nuclear proteins; these serve as targets for regulation by hormones and signaling pathways. Evidence that GH regulates transcription at multiple levels is exemplified by analysis of the proto-oncogene c-fos. Among the GH-regulated transcription factors on c-fos, C/EBPbeta appears to be key, since depletion of C/EBPbeta by RNA interference blocks the stimulation of c-fos by GH. The phosphorylation state of C/EBPbeta and its ability to activate transcription are regulated by GH through MAPK and PI3K/Akt-mediated signaling cascades. The acetylation of C/EBPbeta also contributes to its ability to activate c-fos transcription. These and other post-translational modifications of C/EBPbeta appear to be integrated for regulation of transcription by GH. The formation of nuclear proteins into complexes associated with DNA-bound transcription factors is also regulated by GH. Both C/EBPbeta and the co-activator p300 are recruited to c-fos in response to GH, altering c-fos promoter activation. In addition, GH rapidly induces spatio-temporal re-localization of C/EBPbeta within the nucleus. Thus, GH-regulated gene transcription mediated by C/EBPbeta reflects the integration of diverse mechanisms including post-translational modifications, modulation of protein complexes associated with DNA and re-localization of gene regulatory proteins. Similar integration involving other transcription factors, including Stats, appears to be a feature of regulation by GH of other gene targets.

Profiles of growth hormone (GH)-regulated genes reveal time-dependent responses and identify a mechanism for regulation of activating transcription factor 3 by GH.

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.

Endogenous CCAAT/enhancer binding protein beta and p300 are both regulated by growth hormone to mediate transcriptional activation.

The regulation of c-fos transcription by GH involves multiple factors, including CCAAT/enhancer binding protein (C/EBP) beta. Knockdown of C/EBPbeta by RNA interference prevents stimulation of endogenous c-fos mRNA by GH, indicating a key role for C/EBPbeta in GH-stimulated c-fos transcription. GH rapidly increases the occupancy of both endogenous C/EBPbeta and p300 on the c-fos promoter in 3T3-F442A preadipocytes as indicated by chromatin immunoprecipitation. The transient occupancy of p300 on c-fos and the presence of p300 in the anti-C/EBPbeta immunoprecipitate coincide with the transient increase in c-fos transcription with GH, suggesting that a nuclear complex containing both p300 and C/EBPbeta occupies the c-fos promoter in response to GH. Expression of p300 with C/EBPbeta markedly increases c-fos promoter activity when neither alone is effective, indicating that p300 coactivates C/EBPbeta-mediated c-fos promoter activation. Such coactivation can determine a baseline for c-fos activation by GH. Furthermore, the occupancy of phosphorylated murine C/EBPbeta (T188) on c-fos upon GH treatment is simultaneous with increased occupancy by p300, suggesting that phospho-C/EBPbeta recruits p300 in response to GH. Thus, endogenous C/EBPbeta and p300 on c-fos are dynamically regulated by GH to determine transcriptional activation. Phosphorylated C/EBPbeta and p300 appear to function as part of a regulated complex that mediates GH-stimulated transcription.