OS 02-07 WNT5A/ROR2 SUPPRESSES THE PROLIFERATION OF SMOOTH MUSCLE CELL VIA PKC SIGNALING PATHWAY

Abstract

Objective: Hypertension is closely associated with profound vascular remodeling, especially proliferation and hyperplasia of vascular smooth muscle cells (VSMCs). Aberrant Wnt5a signaling has been related to vascular diseases, but its role on the proliferation of VSMCs is unclear. The study evaluated the effect of Wnt5a on the proliferation of VSMCs and its possible mechanism. Design and Method: The capability of cell proliferation was detected by the assays of MTT, colony formation and EdU cell proliferation. Flow cytometry detections were applied to evaluate the cell cycle. The expressions of specific proteins were detected by western blotting. Immunofluorescence analysis was utilized to observe the localization of Wnt5a and Ror2. Results: The data show that serum induced proliferation of VSMC in a time-dependent manner, accompanied with a significant decrease of Wnt5a expression. Treatment with recombinant mouse Wnt5a or overexpression of Wnt5a leads to the proliferative inhibition of VSMCs and cell cycle arrest at G0/G1 phase, which related to the down-regulation of CyclinD1 and up-regulation of p53. Whereas knockdown of Wnt5a has an opposite effect. Both of Wnt5a and Ror2 are co-localized in the membrane surface. Additionally, knockdown of Ror2 decreases Wnt5a expression and the phenotype of cell proliferation induced by Wnt5a overexpression are rescued. Moreover, Wnt5a promotes Ror2 expression, which has been shown to specifically interact with the non-canonical Wnt signaling pathway. Therefore, protein kinase C (PKC) signaling was activated by Wnt5a in VSMCs, which was also accompanied with upregulation of p53 and reduction of CyclinD1. A positive feedback loop between Wnt5a and phospho-PKC to regulate CyclinD1 expression was also revealed. Furthermore, knockdown of p53 dramatically increases CyclinD1 expression. Conclusions: Taken together, Wnt5a exerts its beneficial effects on VSMCs proliferation and vascular remodeling. Wnt5a/Ror2/PKC signaling pathway is involved in suppression of VSMC proliferation by down-regulating and up-regulating the expression of CyclinD1 and p53 respectively.