Molecular Mechanisms By Which LPA Induces The Matricellular Protein CCN2 Expression In Vascular Smooth Muscle Cells

Cardiovascular diseases(CVD),such as acute myocardial infarction,hypertension and stroke,are now the leading causes of death worldwide..The main underlying cause of coronary artery disease(CAD),the most common form of cardiovascular disease,is atherosclerosis(AS).In the pathogenesis of atherosclerosis,low-density lipoprotein(LDL)is oxidatively modified to oxidized LDL(ox-LDL).Ox-LDL can be efficiently trapped by differentiated microphage-like vascular smooth muscle cells(SMC),thereby promoting foam cell formation.In the middle stages of atherosclerosis,SMCs migrate to the intima and proliferate there.Lysophosphatidic acid(LPA),which is highly accumulated in human atherosclerotic lesions,can affect the functions of cells involved in this pathology,such as promoting SMC proliferation and migration.Communication network factor 2(CCN2),a member of the extracellular matrix(ECM)family of regulatory proteins,is associated with cellular processes such as proliferation,cell adhesion and adherence,motility/migration,and synthesis of the extracellular matrix.Therefore,understanding the CCN2 expression signaling pathway can provide a new research perspective underlying the mechanisms of atherogenesis and provide a new research perspective for the prevention and diagnosis of atherosclerosis as well as the development of new medicines for the treatment of the disease.To investigate the molecular mechanism by which LPA induces CCN2 expression in SMCs,we took a cellular immunofluorescence approach using a confocal microscope to reveal the spatial and temporal dynamics of the matricellular protein CCN2 expression in SMCs in response to LPA stimulation.We monitored the localization of CCN2 protein in the cellular specific compartments of SMCs in response to LPA stimulation by the immunofluorescence assay.The results showed that LPA-induced expression of CCN2 protein reached its peak time at 3 h in vascular smooth muscle cells.We observed that LPA-induced massive CCN2 protein rapidly accumulated in the SMC Golgi apparatus and then translocated to the extracellular matrix.Our previous studies showed that LPA significantly and time-dependently induced activation of Extracellular-signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and p38.To investigate the role of these kinases in the LPA-induced CCN2 expression in SMC,we took gene knockdown and immunoblotting approaches to determine which specific isoforms of ERK and JNK contributes to LPA-induced CCN2 expression.Our results showed that deletion of ERK2 and JNK1 protein expression,but not ERK1 and JNK2,largely reduced LPA-induced CCN2 expression,indicating that ERK2 and JNK1 mediated LPA-induced CCN2 expression in vascular smooth muscle cells.Furthermore,we observed knockout of ERK2 blocked JNK activation,indicating that ERK is upstream of JNK,this result challenges current dogma that is ERK and JNK are parallel kinases.To investigate the interrelationship between the two MAPK molecules,ERK and JNK,we used immunoprecipitation(IP)and proximity ligation(PLA)techniques to determine whether ERK and JNK interreact with each other.Interestingly,our results demonstrated that ERK and JNK molecules interact each other in our IP assay and PLA analysis,while MAPK ERK and p38 do not interact with each other.These results reveal a novel interaction relationship between ERK and JNK,which has not been recognized previously.Previous studies showed that CCN2 mediates biological functions such as cell proliferation,migration,adhesion,wound healing and angiogenesis in various types of cells.To investigate the functional role of LPA-induced CCN2 expression in SMCs,we used the specific si RNA of CCN2 to deplete CCN2 expression in SMCs.Cell migration and proliferation was determined by Boyden chamber assay,DNA synthesis assay and conventional cell counting method.Our results showed that depletion of CCN2 protein levels significantly reduced LPA-induced SMC proliferation,but not migration.In summary,our study elucidated the functional role of LPA-induced matricellular protein CCN2 expression in vascular smooth muscle cells,that is LPA-induced CCN2 mediating SMC proliferation.Our data also reveal that ERK2 is the upstream component,which mediates JNK1 activation,leading LPA-induced CCN2 expression in SMCs.Furthermore,we discovered a previous unknown interaction mechanism between ERK and JNK,and p-ERK and p-JNK.Together these results revealed new targets and signaling pathways,which would contribute to new approaches for the treatment of cardiovascular disease.