Lysophosphatidic Acid Induces Matricellular Protein CTGF Expression Via The PKCdelta-mediated ERK And JNK Pathway In Vascular Smooth Muscle Cells

Cardiovascular disease is a common and important disease threatening human health.Coronary heart disease is the most common one among all cardiovascular system diseases.Its main pathological manifestation is the formation of Atherosclerosis（AS）.During atherogenesis,Low density lipoprotein（LDL）accumulates in the intima of the artery and is Oxidized and modified to Oxidized Low density lipoprotein（ox-LDL）.Ox-LDL promotes Vascular smooth muscle cells（VSMC）proliferation and migration.Lysophospholipids acid（LPA）is one of the main active components in ox-LDL.Connective tissue growth factor（CTGF）is an important growth factor,which activates a series of genes related to AS and subsequent thrombosis pathogenesis,and promotes SMC proliferation,migration.Therefore,understanding the regulatory mechanism of CTGF expression in VSMC is essential to get full awareness of atherogenesis.Whether or not LPA regulates CTGF expression and how LPA regulates CTGF gene expression,especially the signal transduction of CTGF are currently unclear.The current study will address these questions.To investigate the effect of LPA on the expression of CTGF in vascular smooth muscle cells,the cells were stimulated with LPA,totalRNA was extracted with Trizol reagent for Northern blot detection,and cell lysates were collected for Western blot detection.The results showed that LPA significantly induced the expression of CTGF mRNA and protein in VSMC and and induced the expression of CTGF protein in a time and concentration dependent manner（P<0.05）.The peak value was reached at3 h after LPA stimulation,the expression of CTGF can be induced when the concentration of LPA is higher than 1μM.To further explore the role of Protein kinase C（PKC）and Mitogen-activated Protein kinases（MAPK）pathways in the induction of vascular smooth muscle cells by LPA.For this reason,used drug induction and inhibition methods.The result revealed,phosphorylation of PKC,ERK,and c-Jun N-terminal kinase（JNK）and p38was significantly and time dependently induced by LPA.The peak phosphorylation time of PKC,ERK and JNK was 2 min,and the activation time of p38 was 2 min to1 h.PKC,ERK and JNK specific inhibitors could block LPA-induced CTGF expression in a dose-dependent manner（P<0.01）,while p38 specific inhibitors had no significant effect on LPA-induced CTGF protein expression（P>0.05）.Furthermore,experiment showed that ERK-specific inhibitors inhibited JNK phosphorylation in a dose-dependent manner（P<0.01）.The above experimental results indicated that LPA induced the expression of matrix protein CTGF in smooth muscle cells required activation of PKC,ERK and JNK,but not activation of p38MAPK,and proved that ERK activation mediated JNK activation,which was necessary for LPA-induced CTGF expression.To investigate which PKC kinase involved in induces CTGF expression,it was demonstrated by PKC-specific small molecule interferingRNA（siRNA）assay,which reduced the activation of ERK and JNK,thereby blocking LPA-induced CTGF expression.To further explore the main receptors of CTGF expression induced by LPA,Wild-type C57BL/6J mice,LPA₁and LPA₂gene knockout mice and stimulated by LPA.The results showed that in the SMCs of LPA receptor 1（LPA₁）and receptor2（LPA₂）gene knockout mice,the deletion of LPA₁could not induce CTGF expression,while the deletion of LPA₂did not affect the expression of CTGF.These results indicate that LPA₁mediates the secretion of CTGF induced by LPA.The experimental results finally revealed that the intracellular signaling pathway of LPA activates PKCδthrough LPA₁,and PKCδactivates both ERK and JNK.Meanwhile,ERK can also directly activate JNK.This study provides the regulation mechanism of LPA-induced CTGF expression in VSMCs and its signaling pathway was discovered.Furthermore,the important role of PKC subtype,PKCδ,in that regulation was also found.The discovered may provide new sight for the treatment of cardiovascular inflammatory diseases.