ERK1/2 AND AKT ARE DOWN REGULATED BY VDR AGONISTS IN ENDOTHELIAL TUMOR CELLS EXPRESSING vGPCR

VERONICA GONZALEZ PARDO; ALEJANDRA SUARES

Boston

Congreso; 19TH WORKSHOP ON VITAMIN D; 2016

The Kaposi?s Sarcoma-associated Herpes virus G Protein-Coupled Receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi Sarcoma. At the molecular level, the angiogenic and paracrine transforming effect of vGPCR involves the activation of multiple mitogen activated protein kinases (MAPKs) and Akt. We have previously shown that VDR agonists, 1α,25(OH)2D3 and its less calcemic analog TX 527, have antiproliferative effects in endothelial cells transformed by vGPCR. In the present work, we studied the regulation of ERK1/2 and Akt by 1α,25(OH)2D3 and TX 527 and their role in the antiproliferative actions of VDR agonists in vGPCR cells. The results showed that incubation of vGPCRs cells with the MEK inhibitor PD 98059 (10 µM), or the PI3K inhibitor LY294002 (10 µM) decreased vGPCR cell number similarly to 1α,25(OH)2D3 alone (10 nM) after 72 h treatment. These changes were accompanied by a reduction of nuclear p-ERK1/2 and p-Akt. Time response studies assayed by western blot showed that 10 nM of 1α,25(OH)2D3 or TX 527 decreased p-ERK1/2 and p-Akt (24-48 h) protein expression. Moreover, studies with several concentrations of 1α,25(OH)2D3 (0.1- 100 nM) demonstrated that p-ERK and p-Akt decrease was dose-dependent. The magnitude and duration of MAPKs activity are linked to phosphatases actions which are able to dephosphorylate and inactivate MAPKs. ERK specific MAPK phosphatase-3 (MKP-3) is characterized as a highly specific phosphatase for attenuating ERK1/2 signaling. Time response studies analyzed by qRT-PCR and western blot exhibited that 1α,25(OH)2D3 or TX 527 increased mRNA and protein MKP-3 expression and this increase correlated with p-ERK1/2 inhibition. Since we have previously reported that VDR agonists induce apoptosis in vGPCRs, we investigated the role of ERK and Akt on the regulation of Bad, a pro-apoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL. Survival factors inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136. vGPCR cells treated with 1α,25(OH)2D3 (10 nM) or LY294002 or PD 98059 revealed that 1α,25(OH)2D3 increased Bad phosphorylation at Ser136 but not at Bad Ser112. On the contrary, the presence of both inhibitors did not altered Bad phosphorylation at both sites suggesting that p-Bad (Ser136)-regulated by 1α,25(OH)2D3 could be a survival mechanism to counteract ongoing apoptosis. Taken together, these results suggest that VDR agonists regulate ERK and Akt phosphorylation as part of their antiproliferative mechanism on vGPCR cells