Recruitment of cAMP-response Element-binding Protein and Histone Deacetylase Has Opposite Effects on Glucocorticoid Receptor Gene Transcription

Abstract

Glucocorticoids control the synthesis of the glucocorticoid receptor (GR) in various tissues through a negative feedback regulation of the mRNA. In this study, we have identified feedback regulatory domains in the human GR gene promoter and examined the roles of GR, the cAMP-response element-binding protein (CREB), and HDAC-6 in association with promoter elements of the human GR gene. Using breast cancer T47D and HeLa-GR cells, we identify specific negative glucocorticoid-response elements in the GR gene. The feedback regulatory domains were also involved in interactions with CREB. GR-bound negative glucocorticoid-response elements recruited HDAC-6, and this was dependent on treatment with dexamethasone. Both CREB and HDAC-6 formed complexes with GRdexamethasone. The HDAC-6 LXXLL motif between amino acids 313 and 418 made direct contact with the GR AF-1 domain. Interestingly enough, although the level of GR decreased in CREB knockdown cells, it was elevated in HDAC-6 knockdown cells. Our results suggest that CREB-P is dephosphorylated and that HDAC-6 is recruited by the GR, and they play opposite roles in the negative feedback regulation of the GR gene.

Final steps in the feedback regulation of human glucocorticoid receptor gene and role of nuclear protein phosphatase 2A

“…For in vitro CREB-P de-phosphporylation analyses, CREB-32 P was purified and the assay performed as described previously. Briefly, 3,000 cpm of 32 P-CREB was incubated with increasing amount of GST-hGR [7] in the presence of 10ng of purified PPP2R3C expressed in bacteria either in the absence or in the presence of 100nM DEX. The complexes were reconstituted with 10µg of whole cell extract from exponentially growing T47D cells grown in the absence of ligands.…”

“…The pM cloning vector is used to generate fusions of GRwt and its N-terminal serine mutants S113A, S141A, S203A, S211A and S226A respectively in the GAL4 DNA-BD vector with primers (IDT DNA, USA) designed for GR fusion ( Table 2). The generation of hGR mutants S113A, S141A, S203A, S211A and S226A were described before [7]. Similarly, pVP16 is used to construct fusions of PPP2R3C, PPP2R3C1-103 and PPP2R3C1-253 deletion mutants were amplified using primers ( Table 2), digested with EcoRV+Xba1 and cloned into pVP16 vectors at Sma1+Xba1 vector, to an AD derived from the VP16 protein of herpes simplex virus.…”

“…The de-phosphorylation assay was performed using CREB-32 P and GST GR captured PP2A as previously described [7]. IgGs against actin (SC-7210); CREB (SC-58); CREB-1 (Ser-133) (SC-7978) were from Santa Cruz Biotechnologies.…”

“…The ligand activated GR affects the phosphorylation status of CREB in cAMP stimulated corticotropinreleasing hormone-reporter expression in AtT-20 cells [9]. A recent report shows that dephosphorylation of CREB-P is dependent on dexamethasone (DEX) bound GR [7] and involves a nuclear protein phosphatase [10]. PP2A, one of the four major serine/ threonine phosphatases, is implicated in the negative control of cell growth and division, cell cycle progression, DNA replication, transcription and translation [11].…”

“…The functional response of the regulation of GR gene transcription is located -2846 bp upstream of the Cap site of the GR gene promoter [6]. More precisely, a feedback regulatory element (FBRE) is located between -1532 bp and -971 bp, which contains two nGREs and two CREs [7]. Activation of the GR is a multi-step process that requires among others, displacement of proteins, recruitment of co-regulators and phosphorylation of specific residues such as serines ( Figure 1A), all dictated by tissue specificity.…”