Orphan nuclear receptor TR2, a mediator of preadipocyte proliferation, is differentially regulated by RA through exchange of coactivator PCAF with corepressor RIP140 on a platform molecule GRIP1

Orphan nuclear receptor TR2 is a preadipocyte proliferator. Knockdown of TR2 in 3T3-L1 preadipocytes reduced their proliferation efficiency, whereas specific elevation of TR2 in these cells facilitated their proliferation. All-trans retinoic acid (RA) stimulates cellular proliferation in 3T3-L1 preadipocytes by activating TR2 through an IR0-type RA response element, which further activates c-Myc expression. In post-differentiated adipocytes, RA becomes a repressive signal for TR2 and rapidly down-regulates its expression. The biphasic effect of RA on TR2 expression in 3T3-L1 is mediated by differential RA-dependent coregulator recruitment to the receptor/Glucocorticoid Receptor-Interacting Protein 1 (GRIP1) complex that binds IR0 on the TR2 promoter. RA induces the recruitment of histone acetyl transferase-containing/GRIP1/p300/CBP-associated factor (PCAF) complex to the TR2 promoter in undifferentiated cells, whereas it triggers recruitment of histone deacetylase-containing/GRIP1/receptor-interacting protein 140 (RIP140) complex in differentiated cells. GRIP1 directly interacts with RIP140 through its carboxyl terminal AD2 domain. GRIP1 interacts with PCAF and RIP140 directly and differentially, functioning as a platform molecule to mediate differential RA-induced coregulator recruitment to TR2 promoter target. This results in a biphasic effect of RA on the expression of TR2 in undifferentiated and differentiated cells, which is required for RA-stimulated preadipocyte proliferation.     

Limiting effects of RIP140 in estrogen signaling: potential mediation of anti-estrogenic effects of retinoic acid

The receptor interacting protein 140 (RIP140) belongs to a unique subclass of nuclear receptor coregulators with the ability to bind and repress the action of a number of agonist-bound hormone receptors. We have previously demonstrated that all-trans-retinoic acid (RA) induction of RIP140 constitutes a rate-limiting step in the regulation of retinoid receptor signaling. Here we demonstrate that RIP140 is also a limiting regulator of estrogen receptor signaling. Overexpression of RIP140 dose dependently inhibits estrogen-dependent reporter activity in human breast cancer cells. Furthermore, small interfering RNA to RIP140 enhances estrogen-dependent signaling. Our previous studies indicate that RIP140 is a direct target of RA. We report here that RA can abrogate estrogen-mediated cell cycle re-entry. In addition, RA treatment of estrogen-dependent breast cancer cells opposes estrogen receptor-dependent reporter activity, implying that a proportion of RA effects are anti-estrogenic. We provide evidence for a role for RIP140 in mediating anti-estrogenic effects of RA. RIP140 small interfering RNA blocks RA-mediated repression of estrogen receptor activity and provides a growth advantage to estrogen-dependent cells. Together these data implicate a regulatory role for RIP140 in mediating anti-estrogenic effects of RA in estrogen-dependent breast cancer cells and suggest that acute regulation of coregulator expression may be a general mechanism to integrate diverse hormone signals.     

Ligand-dependent formation of retinoid receptors, receptor-interacting protein 140 (RIP140), and histone deacetylase complex is mediated by a novel receptor-interacting motif of RIP140

Receptor-interacting protein 140 (RIP140) interacts with retinoic acid receptor and retinoid X receptor in a ligand-dependent manner and suppresses retinoic acid (RA) induction of its target genes. The receptor-interacting motif is mapped to a C-terminal peptide sequence (LTKTNPILYYMLQK) of RIP140. The functional role of this motif in mediating the suppressive effects of RIP140 on RA induction is demonstrated in mutation studies. RA induces coimmunoprecipitation of histone deacetylase 3 with retinoic acid receptor/retinoid X receptor in the presence of wild type RIP140, but not in the presence of the C-terminal motif-deleted RIP140. A decrease in histone acetylation on the promoter region that carries a RA response element is associated with the expression of wild type RIP140, but not with expression of the mutant RIP140, in a dose-dependent manner. These data provide a molecular explanation for RIP140 acting as a novel ligand-dependent, negative modulator of RA-regulated gene expression.     

Protein kinase C activity during sphinganine potentiation of retinoic acid-induced differentiation in a human leukemia cell line (HL-60).

The differentiation of HL-60 promyelocytic cells toward mature granulocytic cells induced by retinoic acid (RA) was accompanied by a decrease in protein kinase C (PKC) activity. The enhancement of RA-induced differentiation and the potentiation of the decrease of PKC activity by sphinganine (SP) seemed to correlate with each other. Kinetically, PKC activity during RA-induced differentiation without SP decreased to its lowest (75% of the control) after 48h; about 50% of the reduction was observed at 24h. In the presence of SP, PKC activity decreased more rapidly to its lowest (60% of the control) within 24h of incubation of RA. SP, added 24h before or concomitantly with the addition of RA, could potentiate the RA-induced differentiation and the reduction of PKC activity. Our results indicate that the effect of SP and the role of PKC during RA-induced differentiation may be critical at the early stages of induction of differentiation (within 24h of RA exposure).     

Retinoic Acid Mediates Regulation of Network Formation by COUP-TFII and VE-Cadherin Expression by TGFβ Receptor Kinase in Breast Cancer Cells

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFβ signaling pathway were induced by RA, and specific inhibition of the TGFβ type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFβ pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA.     

Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21(CIPI/Waf1) and Bax proteins, as well as activation of p34(cdc2) kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27(Kip1) expression. Olomoucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-mediated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cdc2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.     

Characteristics of cyclic AMP enhancement of retinoic acid induction of increased transglutaminase activity in HL60 cells

When the human myeloid leukemia cell line (HL60) is induced to differentiate with retinoic acid (RA), there is a concentration-dependent increase in transglutaminase (TGase) activity which peaks on day 5. While dibutyryl 3',5'-cyclic adenosine monophosphate (db-cAMP) alone produced only a slight increase in TGase activity in HL60 cells, the concomitant addition of db-cAMP (100 microM) with RA (10(-12)-10(-4) M) potentiates RA induction of TGase activity. Maximal increases in TGase activity (2- to 10-fold) were observed with 10(-4)-10(-7) M RA and when db-cAMP was present from 24 to 48 h after the addition of RA. The cyclic nucleotide enhancement was dose-dependent from 10 to 100 microM of cAMP. Less marked increases were observed with 8-bromo-cAMP and with the phosphodiesterase inhibitor theophylline. Although the simultaneous addition of PGE1 or PGE2 (10(-8)-10(-6) M) produced no enhancement of RA-induced TGase activity, adding PGE1 or PGE2 24 or 48 h following RA treatments produced an enhancement of TGase activity. The phosphodiesterase inhibitor potentiated the increases produced by db-cAMP and the prostaglandins. Dibutyryl cAMP enhanced the ability of RA to induce the cells to reduce nitroblue tetrazolium (NBT), a functional measure of differentiation, at lower concentrations of RA and with shorter treatment durations. cAMP potentiates RA-induced TGase activity in HL60 cells and the combination appears to be associated with enhanced RA-induced differentiation.     

Suppression of receptor interacting protein 140 repressive activity by protein arginine methylation

Receptor interacting protein 140 (RIP140), a ligand-dependent corepressor for nuclear receptors, can be modified by arginine methylation. Three methylated arginine residues, at Arg-240, Arg-650, and Arg-948, were identified by mass spectrometric analysis. Site-directed mutagenesis studies demonstrated the functionality of these arginine residues. The biological activity of RIP140 was suppressed by protein arginine methyltransferase 1 (PRMT1) due to RIP140 methylation, which reduced the recruitment of histone deacetylases to RIP140 and facilitated its nuclear export by enhancing interaction with exportin 1. A constitutive negative (Arg/Ala) mutant of RIP140 was resistant to the effect of PRMT1, and a constitutive positive (Arg/Phe) mutation mimicked the effect of arginine methylation. The biological activities of the wild type and the mutant proteins were examined in RIP140-null MEF cells. This study uncovered a novel means to inactivate, or suppress, RIP140, and demonstrated protein arginine methylation as a critical type of modification for corepressor.     

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.     

An intronic Ikaros-binding element mediates retinoic acid suppression of the kappa opioid receptor gene, accompanied by histone deacetylation on the promoters

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in mouse embryonal carcinoma P19 stem cells and suppressed by retinoic acid (RA) in cells undergoing neuronal differentiation. A negative regulatory element is located within intron 1 of the KOR gene, which contains an Ikaros (Ik)-binding site (GGGAAgGGGAT). This sequence is an Ik-1 respondive, functionally negative element as demonstrated in the context of both natural KOR and heterologous promoters. The two underlined G residues of the second half-site are critical for Ik-1 binding and Ik-mediated repression of the KOR gene. RA induces Ik-1 expression within 1 day of treatment and suppresses KOR expression between 2 and 3 days. Overexpression of Ik-1 in P19 suppresses endogenous KOR gene expression, accompanied by increased binding of Ik-1 to the Ik-binding site and chromatin histone deacetylation on KOR promoters. It is proposed that in an RA-induced P19 differentiation model, RA elevates Ik-1 expression, which recruits histone deacetylase to intron 1 of the KOR gene and silences KOR gene promoters.     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.