Mechanism Of Cyr61Regulated Vascular Smooth Muscle Cell Migration Pathway

Atherosclerosis, one type of vascular disease involving hardening of the arteries, is a major cause of heart attacks and stroke. In westernized societies, it is the underlying cause of about50%of all deaths. Atherosclerosis is characterized by the accumulation of lipids, macrophages, vascular smooth muscle cells (VSMC), T lymphocytes and fibrous tissue in the inner-most layer of large-and medium-sized arteries. The most important step in the atherosclerosis is the lesion formation.Vascular smooth muscle cell migration is a crucial process in the lesion formation, but the mechanism is still not fully understood.Cysteine-rich61(Cyr61) has been reported to overexpress in atherosclerosis lesion, induce angiogenesis and promote cell proliferation, migration, adhesion, and differentiation. Cyr61is a member of CCN (Cyr61, CTGF and Nov) protein family, also named CCN1.Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces VSMC migration, which is an important process in atherogenesis. Platelet-derived growth factor (PDGF), a potent chemoattractant, and the specific receptor PDGFRβ were highly expressed in atherosclerotic lesions. However, how LPA or PDGF-BB induces cell migration remains elusive. Furthermore, whether Cyr61is involved in LPA or PDGF-BB triggered cell migration is also the key event. In the present study, we hypothesized that endogenous Cyr61, mediates LPA or PDGF-BB signaling, leading to VSMC migration.To determine possible matricellular protein Cyr61involvement in cell migration, first, we detect the regulation of Cyr61in LPA induced VSMC migration signaling pathway. LPA, a potent bioactive lipid found in atherosclerotic lesions, markedly induces VSMC migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced VSMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61is highly induced by LPA. We hypothesized that Cyr61mediates LP A-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61antibody blockade and siRNA inhibition both diminished LP A-induced SMC migration, indicating a novel regulatory role of Cyr61. VSMC derived from LPA receptor1(LPA1) knock-out mice lack the ability of Cyr61induction and cell migration, supporting the concept that LPA1is required for Cyr61expression and migration. By contrast, PPARy was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61accumulation. Interestingly, knockdown of Cyr61blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to VSMC migration. Our results further demonstrate that plasma membrane integrins α6β1and αyβ3transduced the LPA-Cyr61signal toward FAK activation and migration.PDGF-BB induces cell migration via mitogen-activated protein (MAP) kinase and Phosphoinositide3(PI3)-kinase/Akt pathways. However, the downstream mediators are still elusive, especially the role of extracellular mediators are largely unknown. In this study, we identified that the matricellular protein Cyr61, which is de novo synthesized in response to PDGF, is the downstream mediator of extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways, independent of p38MAPK and AKT pathways, mediating PDGF-induced smooth muscle cell migration, but not proliferation. Our results reveal that the newly synthesized Cyr61by PDGF is promptly translocated to extracellular matrix and physically interacts with integrins α6β1and αvβ3. We further demonstrate that Cyr61and integrins are integrate components of PDGF signaling pathway via an "outside-in" pathway to activate intracellular focal adhesion kinase (FAK) activation, leading to cell migration.Taken together, this study provides the first evidence that LPA or PDGF-induced endogenous extracellular matrix component Cyr61is a key mediator in VSMC migration pathway. Therefore extracellular Cyr61convergence with LPA or PDGF-BB signaling and integrin/FAK signaling is a new mechanism of VSMC migration. The discovered signaling pathway provides new insights into mechanisms underlying cell migration and may represent an important therapeutically target in atherosclerosis or cell migration/invasion related vascular diseases and tumorigenesis.