RESUMO
Retinoids are derivatives of vitamin A and powerful inhibitors of cell proliferation and inflammation. Angiotensin II (Ang II) contributes to vascular lesions by promoting cell growth of vascular smooth muscle cells (VSMCs). Therefore, we examined whether retinoids interfere with the proproliferative actions of Ang II in VSMCs via AT(1) receptor-dependent or activator protein-1 (AP-1)-dependent mechanisms. VSMCs express retinoid receptor proteins, ie, retinoic acid receptor (RAR) alpha and retinoid X receptor (RXR) alpha. Long-term exposure to 1 micromol/L all-trans retinoic acid (RA) dose-dependently inhibited Ang II-induced cell proliferation (P<0.005) as well as DNA and protein synthesis (P<0.001). All-trans RA blocked Ang II stimulation of transforming growth factor-beta(1) mRNA (P<0.005). All-trans RA inhibition of vascular VSMC growth was mediated both via RAR- and RXR-dependent pathways, as shown by receptor-specific synthetic retinoids. Transfection experiments revealed that inhibition of AP-1-dependent gene transcription is one mechanism by which all-trans RA inhibits Ang II action. RARalpha cotransfection enhanced the anti-AP-1 effects of all-trans RA dose-dependently. AP-1 activity was similarly inhibited by cotransfection with either RARalpha or RXRalpha. Ang II-induced gene expression of c-fos was abrogated by all-trans RA treatment (P<0.005). In VSMCs, all-trans RA downregulated AT(1) receptor mRNA (P<0.01) and reduced B(max) (P<0.001). All-trans RA repressed Ang II-stimulated AT(1) receptor promoter activity. The all-trans RA inhibitory effect was abolished when the AP-1 consensus site on the AT(1) receptor promoter was deleted. Our findings demonstrate that retinoids are potent inhibitors of the actions of Ang II on VSMCs. The findings support the notion that retinoids may interfere with proliferative vascular disease.
Assuntos
Angiotensina II/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Retinoides/farmacologia , Alitretinoína , Animais , Benzoatos/farmacologia , Células COS , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/genética , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/fisiologia , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Tetra-Hidronaftalenos/farmacologia , Fator de Transcrição AP-1/genética , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1 , Tretinoína/farmacologiaRESUMO
Retinoic acid receptor gamma (RARgamma) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARgamma1 and serines 66 and 68 in RARgamma2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7.cyclin H. MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARgamma is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARgamma phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARgamma transcriptional activity in a response gene-dependent manner.
Assuntos
Quinases Ciclina-Dependentes , Proteínas Serina-Treonina Quinases/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , Animais , Células COS , Humanos , Camundongos , Fosforilação , Fator de Transcrição TFIIH , Ativação Transcricional , Quinase Ativadora de Quinase Dependente de Ciclina , Receptor gama de Ácido RetinoicoRESUMO
The nuclear receptor mouse retinoid X receptor alpha (mRXRalpha) was shown to be constitutively phosphorylated in its NH2-terminal A/B region, which contains potential phosphorylation sites for proline-directed Ser/Thr kinases. Mutants for each putative site were generated and overexpressed in transfected COS-1 cells. Constitutively phosphorylated residues identified by tryptic phosphopeptide mapping included serine 22 located in the A1 region that is specific to the RXRalpha1 isoform. Overexpression and UV activation of the stress-activated kinases, c-Jun NH2-terminal kinases 1 and 2 (JNK1 and JNK2), hyperphosphorylated RXRalpha, resulting in a marked decrease in its electrophoretic mobility. This inducible hyperphosphorylation involved three residues (serines 61 and 75 and threonine 87) in the B region of RXRalpha and one residue (serine 265) in the ligand binding domain (E region). Binding assays performed in vitro with purified recombinant proteins demonstrated that JNKs did not interact with RXRalpha but bound to its heterodimeric partners, retinoic acid receptors alpha and gamma (RARalpha and RARgamma). Hyperphosphorylation by JNKs did not affect the transactivation properties of either RXRalpha homodimers or RXRalpha/RARalpha heterodimers in transfected cultured cells.
