Differential role of two VDR coactivators, DRIP205 and SRC-3, in keratinocyte proliferation and differentiation

Cell programs such as proliferation and differentiation involve the selective activation and repression of gene expression. The vitamin D receptor (VDR), through 1,25(OH)(2)D(3), controls the proliferation and differentiation of keratinocytes. Previously, we have identified two VDR binding coactivator complexes. In proliferating keratinocytes VDR bound preferentially to the DRIP complex, whereas in differentiated keratinocytes the SRC complex was preferred. We proposed that different coactivators are required for sequential gene regulation in the transition from proliferation to differentiation. Here we examined the roles of DRIP205 and SRC-3 in this transition. Silencing of DRIP205 and VDR caused hyperproliferation of keratinocytes, demonstrated by increased XTT and BrdU incorporation. SRC-3 silencing, on the other hand, did not have an effect on proliferation. In contrast, SRC-3 as well as DRIP205 and VDR silencing blocked keratinocyte differentiation as shown by decreased expression of keratin 1 and filaggrin. These results are consistent with the differential localization of DRIP205 and SRC-3 in skin. These results indicate that DRIP205 is required for keratinocyte proliferation. Both DRIP205 and SRC-3 are required for the keratinocyte differentiation. These results support the concept that the selective use of coactivators by VDR underlies the selective regulation of gene expression in keratinocyte proliferation and differentiation.     

Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in adipogenesis. PPARgamma binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARgamma-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARgamma is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARgamma. Here we report that PPARgamma and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARgamma-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARgamma. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARgamma- and RXR-specific ligands may occur at the level of selective coactivator recruitment.     

Modulation of transcriptional sensitivity of mineralocorticoid and estrogen receptors

Recent reports describe the ability of factors to modulate the position of the dose–response curve of receptor–agonist complexes, and the amount of partial agonist activity of receptor–antagonist complexes, of androgen, glucocorticoid (GRs), and progesterone receptors (PRs). We now ask whether this modulation extends to the two remaining steroid receptors: mineralocorticoid (MRs) and estrogen receptors (ERs). These studies of MR were facilitated by our discovery that the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) is a new antimineralocorticoid with significant amounts of partial agonist activity. Elevated levels of MR, the co-activators TIF2 and SRC-1, and the co-repressor SMRT do modulate the dose–response curve and partial agonist activity of MR complexes. Interestingly, the precise responses are indistinguishable from those seen with GRs in the same cells. Thus, the unequal transactivation of common genes by MRs versus GRs probably cannot be explained by differential responses to changing cellular concentrations of homologous receptor, co-activators, or co-repressors. We also find that the dose–response curve of ER–estradiol complexes is left-shifted to lower steroid concentrations by higher amounts of exogenous ER. Therefore, the modulation of either the dose–response curve of agonists or the partial agonist activity of antisteroid, and in many cases the modulation of both properties, is a common phenomenon for all of the classical steroid receptors.