MON-214 1α,25-DIHYDROXYVITAMIN D3 TARGETING OF NF-ΚB SUPPRESSES TNF-Α INDUCED ADHESION MOLECULE EXPRESSION IN HUMAN ENDOTHELIAL CELLS

Abstract

Vitamin D and its analogues have been documented to be associated with endothelial dysfunction in various diseases. However, the underlying mechanism remains unknown. Here, we conducted an in vitro study to evaluate the effect of 1α,25-dihydroxyvitamin D3, the active form of vitamin D3, on adhesion molecule expression in human endothelial cells. The possible mechanism involved in this process was also explored. Human umbilical vein cells (HUVECs) were cultured and treated according to the experiment requirement. Western Blot and RT-PCR were used to evaluate the expression of vascular cell adhesion molecular-1 (VCAM-1) and E-selectin. ChIP assay, immunofluorescence, Western Blot and co-immunoprecipitation were used to assess the effect of 1α,25-dihydroxyvitamin D3 on NF-κB signaling. 1α,25-dihydroxyvitamin D3 inhibited VCAM-1 and E-selectin mRNA and protein expression after TNF-α stimulation. ChIP assay showed that TNF-α increased the p65 binding to the promoter of VCAM-1 and E-selectin, which was suppressed by 1α,25-dihydroxyvitamin D3. 1α,25-dihydroxyvitamin D3 affected TNF-α induced IκBα phosphorylation and p65 NF-κB activation, leading to an inhibition of p65 nuclear translocation. These effects were reversed by a specific vitamin D receptor siRNA (VDR-siRNA). Co-immunoprecipitation revealed that 1α,25-dihydroxyvitamin D3 induced an increased binding of VDR to p65, which inhibited the ability of p65 binding to target gene promoters. 1,25-dihydroxyvitamin D3 suppresses TNF-α induced adhesion molecule expression in human endothelial cells by blocking the NF-κB pathway, and this was VDR dependent.