Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPbeta and C/EBPdelta. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPbeta. This results in a dramatic enhancement of C/EBPbeta-dependent transcription from the C/EBPalpha promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPbeta is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98-102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPbeta prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPbeta-directed transcription and to potentiate C/EBPbeta-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPbeta appeared to directly interfere with the interaction of HDAC1 with C/EBPbeta, suggesting that PCAF/GCN5-dependent acetylation of C/EBPbeta serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.
The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin ␣/ karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling. The glucocorticoid receptor (GR)5 is a nuclear hormone receptor of the steroid receptor subfamily whose activity is tightly regulated by glucocorticoids (1). GR is a modular protein that features a central DNA binding domain (DBD) and a C-terminal ligand binding domain (LBD). Two activation functions (AF-1 and AF-2) are located in the N terminus and the LBD of the receptor, respectively (2, 3). GR is directly and indirectly involved in both the activation and suppression of genes that are involved in cell differentiation, glucose uptake and homeostasis, inflammation, response to stress, neuronal function, lipid metabolism, and cancer (4, 5).Transcriptional regulation by GR is tightly controlled. The regulation of function by segregation in subcellular compartment has been proposed to act as an important regulatory checkpoint for a number of transcription factors, including steroid receptors (6, 7). Unlike other steroid receptors, such as estrogen receptor and progesterone receptor, that are nuclear in the absence of steroid, naive GR is largely cytoplasmic (8). In the cytoplasm GR is maintained in an inactive hsp90 cochaperone complex that includes hsp70, immunophilins, p23, and other factors (9). Upon ligand binding, GR dissociates from the chaperone complex, homodimerizes, and rapidly translocates to the nucleus to regulate target gene expression (10 -12). Nuclear import of GR is accomplished through two nuclear localization signals as follows: NL1, located in a hinge-like region of GR that separates the DBD from the LBD; and NL2, which is within the LBD (13). NL1 is a basic NLS that mediates the nuclear import of GR through interaction with importin ␣ and importin 7 (14 -16).NL2 is strictly steroid-dependent, and bot...
Glucocorticoids are synthesized locally in adipose tissue and contribute to metabolic disease through the facilitation of adipose tissue expansion. Here we report that exposure of human primary preadipocytes to glucocorticoids increases their sensitivity to insulin and enhances their subsequent response to stimuli that promote differentiation. This effect was observed in primary human preadipocytes but not in immortalized 3T3-L1 murine preadipocytes or in fully differentiated primary human adipocytes. Stimulation of insulin signaling was mediated through induction of insulin receptor (IR), IR substrate protein 1 (IRS1), IRS2, and the p85 regulatory subunit of phosphoinositide-3-3-kinase, which led to enhanced insulin-mediated activation of Akt. Although induction of IRS2 was direct, induction of IR and IRS1 by glucocorticoids occurred subsequent to primary induction of the forkhead family transcription factors FoxO1A and FoxO3A. These results reveal a new role for glucocorticoids in preparing preadipocytes for differentiation.
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