RESUMO
Transforming growth factor (TGF)-ß signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-ß signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-ß signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-ß signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-ß type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-ß-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-ß. Consistently, TGF-ß-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-ß signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-ß signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-ß signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-ß signaling in a coordinated manner when cells are stimulated with high levels of TGF-ß.
Assuntos
Proteínas de Membrana/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Motivos de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Western Blotting , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The TGF-ß and Wnt pathways are involved in cell fate and tumorigenicity. A recent report indicated that a TGF-ß target gene, TMEPAI (transmembrane prostate androgen-induced RNA), is possibly also a downstream target of Wnt signaling. Although TMEPAI was believed to be involved in tumorigenicity because of its blockage of TGF-ß signaling, how TGF-ß and Wnt signals affect the activation of the TMEPAI gene is not well understood. Herein, we show that the TMEPAI promoter is regulated synergistically by TGF-ß/Smad and Wnt/ß-catenin/T cell factor (TCF) 7L2. The critical cis-element for dual signals, termed TGF-ß-responsive TCF7L2-binding element (TTE), is located in intron 1 of the TMEPAI gene. TCF7L2, but not Smad proteins, bound to TTE, whereas the disruption of TTE by mutagenesis remarkably counteracted both TGF-ß and TCF7L2 responses. The introduction of mutations in critical Smad-binding elements blocked the activation of the TMEPAI promoter by TCF7L2. Furthermore, our DNA-protein interaction experiments revealed the indirect binding of TCF7L2 to Smad-binding elements via Smad3 upon TGF-ß stimulation as well as its TGF-ß-dependent association with TTE. We demonstrate that the Wnt/ß-catenin/TCF7L2 pathway is preferentially able to alter the transcriptional regulation of the TGF-ß-target gene, TMEPAI.
