RESUMO
RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-beta (TGF-beta) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-beta target gene PAI-1 after stimulation by TGF-beta when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-beta and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-beta and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-beta signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-beta signaling pathways.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , DNA Complementar/genética , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Receptores X do Fígado , Camundongos , Camundongos Knockout , Coativadores de Receptor Nuclear , Receptores Nucleares Órfãos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Serpina E2 , Serpinas/genética , Serpinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Células U937RESUMO
The suppressor of cytokine signalling (SOCS) protein family negatively regulates cytokine action. In this study, we investigated the effects of estrogen (E2) on SOCS-3 expression in T47D and MCF-7 human breast cancer cells. Real-time PCR analysis of E2-treated T47D cells revealed a ligand and time-dependent increase in of SOCS-3 mRNA levels. Cloning of a 1.7 kb fragment of the human SOCS-3 5' flanking sequence, and subsequent analysis of potential transcription factor-binding sites identified an incomplete ERE motif located -1493 to -1489 upstream of the start site. Transient transfection of the cloned fragment in MCF-7 cells showed that both E2 and genistein treatment caused an increase in reporter gene activity, which was inhibited by co-treatment with ICI 182,780. Chromatin immunoprecipitation analysis revealed an E2 and time-dependent recruitment of ERalpha to the E2 responsive region of the human SOCS-3 promoter. In summary, this study shows that ERalpha directly regulates human SOCS-3 promoter activity in human breast cancer cells, thus modulating cytokine activity.