14-3-3 Protein Is Involved In ERK1/2 Signaling Transduction Pathway Of Rat VSMCs Proliferation Induced By Apelin

AIM To investigate the effects of apelin-13 induced cell proliferation and activated ERK1/2 function via PKC and 14-3-3 protein in rat VSMCs. Further to find new signaling pathway of apelin-APJ.METHODS VSMCs were prepared from male Sprague-Dawley rats'thoracic aortas by the primary-explant method. Immunofluorescence was used to identify localization of apelin and APJ receptor in VSMCs. Expression of 14-3-3, pRaf-1, pERK1/2, ERK1/2, CyclinD1, CyclinE and Caveolin-1 were detected by Western Blotting. The blockade effect of GF109203X and Difopein were measured by MTT assay. Formation of high molecular weight polyprotein component including 14-3-3 and Raf-1 was detected by immunoprecipitation.RESULTS Western Blotting analysis showed that apelin-13 promoted the concentration- dependent and time-dependent expression of 14-3-3,pRaf-1 and pERK1/2 during 2μM and 4h. The potent PKC inhibitor GF109203X and 14-3-3 protein inhibitor Difopein decreased the expression of 14-3-3,pRaf-1,pERK1/2,CyclinD1 and CyclinE, and increased the expression of Caveolin-1 induced by apelin-13. Similarly, MTT assay showed that GF109203X and Difopein significantly inhibited the VSMCs proliferation stimulated by apelin-13. The immunoprecipitate results suggested that apelin-13 promoted the combination of 14-3-3 protein and Raf-1, furthermore GF109203X,Difopein andβ-CD significantly inhibited the combination of 14-3-3 and Raf-1 stimulated by apelin-13.CONCLUSION Apelin-13 promoted cell proliferation in rat VSMCs, and the effects maybe involved in apelin/APJ/PKC/Raf-1/14-3-3/ERK1/2/Caveolin-1 signal cascades.