| Migration of vascular smooth muscle cell (VSMC) from media to intima is an earlier event in response to vascular injury, such as atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). VSMC migration is the biological basis of neointima formation. Many of growth factors, cytokines and extacellular matrix (ECM) are involved in the migration of VSMC toward intima. However, the signaling pathways that regulate VSMC migration are still obscure.Recent findings have shown that integrins, as ECM receptors, not only mediate the interaction of ECM-cell and cell-cell, but also integrate extracellular stimuli, then transmitte the signal into cell. Focal adhesion kinase (FAK) is the intermediator linking integrins to downstream signal proteins, and plays a key role in multiple signal pathways. FAK recruits signal proteins such as PI-3K by means of its tyrosine sites, and then results in cascade phosphylation, thereby allowing signals to transfer further into cell. The signaling pathway is required for regulating cell contraction and movement.Our previous studies have demonstrated that VSMC in vitro cultured and in arterial neointima can secrete a substantial ECM, including osteopontin (OPN) and fibronectin (FN). As the marker of synthetic VSMC, OPN promotes adhesion of VSMC, through interaction with integrinβ3, by which cell is anchored in ECM. However the signal pathway triggered by OPN in VSMC migration isn't well understood. Although many studies focus on the roles of integrin signal pathway in regulation cell migration, it cann't still determine whether integrinβ3-FAK is implicated in OPN-induced migration of VSMC. In present study, we characterized the effect of OPN on VSMC migration and investigated the relationshipbetween OPN-induced VSMC migration and the activation of integrinβ3-FAK signal pathway. By transfecting the recombinants of FAK related non-kinase (FRNK) into VSMC to inhibit FAK activation, we try to confirm whether integrinβ3-FAK signal pathway plays important roles in OPN-induced VSMC migration. 1. Relationship between expression of integrinβ3, FAK and migration of VSMC stimulated by ECMThe interaction of cell-ECM is the molecular basis of VSMC adhesion, migration, proliferation and differentiation, this interaction is mediated by ECM receptor integrins. In the study, we investigated the effect of ECM proteins (including OPN and FN) on VSMC migration and the relationship between expression of integrinβ3, FAK and migration of VSMC stimulated by ECM. The results as following:(1) VSMC migration was examined using a model of wounding injury of confluent cells. The migration activity of VSMC stimulated by OPN (5, 10, 20μg/ml) was increased by 1.04, 1.36 and 1.57 folds, compared with control VSMC, respectively. FN promoted migration of VSMC only in high dose (20μg/ml), but not in low dose. The data indicated that both of OPN and FN induced VSMC migration in dose-dependent manner, OPN was more effective than FN.(2) The results of Western blotting suggested that there was low expression level of integrinβ3 in untreated VSMC, but expression of integrinβ3 was greatly upregulated after treated by OPN and FN for 24h. The upregulation of integrinβ3 induced by OPN was more effective and was paralleled to migration induced by OPN. We speculated that integrinβ3 might be involved in VSMC migration induced by OPN and FN.(3) To test the effect of OPN and FN on the expression of migration related molecules FAK and Gax, the two genes were detected by real time RT-PCR. The mRNA level of FAK could not be detected in untreated VSMC. After stimulated by OPN and FN (20μg/ml) for 3, 6, 12 and 24h, the mRNA level of FAK was greatly increased, but not in time-dependentmanner. In addition, OPN and FN could also inhibit expression of transcription factor Gax, respectively. OPN was more effective than FN. Because Gax is capable of inhibiting cell migration, downregulation of Gax induced by OPN might be one of molecular mechanisms of OPN-induced VS...
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