WNT5A/Ror2/PKC Signaling Pathways Through Downregulate The Expression Of CyclinD1 Inhibition Of Vascular Smooth Muscle Cell Proliferation

Objective: Investigate the effect of WNT5 A on vascular smooth muscle cells(VSMCs) proliferation and the underly mechanism.Methods: VSMCs cell lines were stable over-expressed with WNT5 A,transfected with si RNA-WNT5 A or treated with recombinant WNT5 A.The effect of WNT5 A on proliferation of VSMCs were detected by MTT,Plate clone and Edu.Flow cytometry was used to detect the effects of WNT5 A on cell cycle. The co-location and binding of WNT5 A and Ror2 were verified by immunoprecipitation and co-precipitation. MTT, Plate clone,Edu and Flow cytometry were applied to detect the effects of si RNA-Ror2 on VSMCs proliferation and cell cycle.Western-blot was used to detect the expression of WNT5A/Ror2/PKC/P53/Cyclin D1 proteins.1) WNT5 A decreases obviously in serum cultured VSMCs in a time dependent manner.Overexpression of WNT5 A and Exogenous recombinant WNT5 A inhibits the proliferation of VSMCs.si RNA-WNT5 A promotes the proliferation of VSMCs.Results:2) WNT5 A induces G0/G1 cell-cycle arrest by promoting the expression of P53 and decreasing the expression of Cyclin D1.3) WNT5 A up-regulates the expression of the receptor Ror2.There is spatial co-localization between WNT5 A and Ror2 indicates they are able to form a combination.Knockdown of Ror2 by si RNA reverts the inhibition of proliferation by WNT5 A, alleviates G0/G1 cell-cycle arrest,increases Cyclin D1 and decreases P53 expression.4) WNT5 A increases phosphorylated PKC level, which is decreased by si RNA knockdown of Ror2. With PKC agonist PMA treated VSMCs,WNT5 A and P53 were significantly increased, and Cyclin D1 was decreased. While in PKC inhibitor GF10923 X treated VSMCs, WNT5 A and P53 were decreased, Cyclin D1 was increased.Conclusions:1) WNT5 A inhibits the proliferation of VSMCs.2) WNT5 A inhibits VSMCs proliferation through induction G0/G1cell-cycle arrest, which might be related to upregulated cyclin D1 and downregulated P53.3) Ror2 conducts the inhibitory effect of WNT5 A on the proliferation of VSMCs.4)WNT5A/Ror2 induction G0/G1 cell-cycle arrest to inhibit VSMCs proliferation by regulating P53 and cyclin D1 may be PKC signaling activation dependent, and there are feedbacks between WNT5 A and PKC.