COUP-TFI and Tob1 regulate the interaction of BMP-4 and RA on POMC transcription

ROSMINO, JOSEFINA; NIETO, LEANDRO E.; ARZT, EDUARDO; FUERTES, MARIANA

Mar del Plata

Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2018

Sociedad Argentina de Investigación Clínica

Cushing?s disease pituitary corticotrophinomas have no definitive pharmacological treatment up-to-date. Studying the Proopiomelanocortin (POMC) gene expression as target, our group described in AtT-20 mouse corticotroph cell line model, the interaction between the signaling pathways of Retinoic Acid (RA) and Bone Morphogenetic Protein-4 (BMP-4) during transcriptional regulation in a putative response element for RA, in the proximal POMC promoter. Here we found that COUP-TFI (RA signaling blocker), disrupted the transcriptional complexes involving Smad proteins (BMP-4 signaling) and nuclear receptors for RA (RXRα, RXRγ and RARβ). We demonstrate that COUP-TFI blocked from 40-80% to 10% the inhibitory effect of 100nM RA on POMC-Luc reporter transcriptional activation and the potentiation of this inhibition in co-treatment with 100ng/ml BMP-4. COUP-TFI blocked the inhibitory effect of 100nM RA on POMC-Luc constructions with deletion or mutation of the BMPRE site, retaining the potentiated inhibition in co-treatment with 100ng/ml BMP-4. Tob1 negatively regulates BMP signaling. We observed by coimmunoprecipitation that Tob1 diminished the interaction between RARβ, RXRα and RXRγ with Smad4. Tob1 expression halted the inhibitory effect of 100ng/ml BMP-4 on POMC-Luc with deletion or mutation of the RARE site, without potentiation effect in co-treatment with 100nM RA. Moreover, the co-expression of COUP-TFI and Tob1 abolished the inhibitory effect of both 100nM RA and 100ng/ml BMP-4 treatments on complete POMC-Luc reporter, backing our observation in AtT-20 cells stably expressing a dominant negative of Smad4, where the effect of RA and BMP-4 is completely nullified. By EMSA with RARE sequence of POMC promoter as a probe and AtT-20 nuclear extracts, we observed a transcriptional complex binding to the RARE element which is displaced by α-Smad4 under RA and BMP-4 co-treatment. This supports our hypothesis about the involvement of BMP-4 pathway elements mediating the RA inhibitory action on POMC transcription.Supported by ANPCyT, CONICET, UBA and FOCEM (COF 03/11).