Direct active Fyn-paxillin interaction regulates vascular smooth muscle cell migration

Abstract

Vascular smooth muscle cell (VSMC) migration plays an important role in cardiovascular diseases, including atherosclerotic plaque formation and restenosis after vascular intervention. The mechanisms involved in VSMC migration are complex and have not been fully elucidated. Recently, we discovered a novel interaction, direct binding of active Fyn-paxillin at focal adhesions, which plays an important role in actin stress fiber formation and migration in VSMCs. In this review, we highlight paxillin as an intermediate signaling molecule that mediates actin stress fiber formation and VSMC migration through the Fyn/paxillin/Rho-kinase signaling pathway by directly binding to active Fyn. We also discuss the inhibition of VSMC migration by blocking the active Fyn-paxillin interaction and the potential of this interaction as a therapeutic target for cardiovascular diseases.

Introduction

Vascular smooth muscle cell (VSMC) migration is a normally occurring process that plays an important role in vascular development and tissue repair after vascular injury. The migration of VSMCs involves a complex series of processes, including detachment from the extracellular matrix, polarization, protrusion formation, and translocation (1, 2). Aberrant VSMC migration contributes to the development of various vascular diseases, such as atherosclerosis and restenosis (2). To date, the exact role of VSMC migration and its regulatory mechanism have not been fully elucidated. Therefore, understanding the mechanisms underlying VSMC migration is essential for developing new therapeutic strategies to treat these diseases.

Vascular Smooth Muscle Cell Migration in Pathological Conditions

Vascular smooth muscle cells (VSMCs) have two phenotypes, the contractile phenotype and the synthetic phenotype (2, 3). Under physiological conditions, VSMCs are of the contractile phenotype and perform the functions of controlling vascular contractility to maintain blood pressure and regulate blood flow in healthy vessels (4, 5). During the repair response to vascular injury, cells with a contractile phenotype can transform into cells with a synthetic phenotype, causing cell proliferation and migration (6, 7).

VSMC migration is an important process that occurs in various pathological conditions, including atherosclerosis (8, 9), restenosis (9,10,11), and aneurysm formation (12, 13). Under these pathological conditions, VSMCs undergo a phenotypic switch from a contractile phenotype to a synthetic phenotype, and cells of the synthetic phenotype can migrate toward the intima of vessels under the regulation of multiple signaling pathways, promoting atherosclerotic plaques, restenosis, and aneurysm formation (14,15,16). Multiple factors and mechanisms contribute to VSMC migration. A large number of studies have shown that epidermal growth factor (1, 17), fibroblast growth factor (18, 19), insulin-like growth factor (1, 20), platelet-derived growth factor (PDGF) (21, 22), transforming growth factor (TGF) (22), vascular endothelial growth factor (VEGF) (23, 24), angiotensin II (Ang II) (25, 26), and sphingosylphosphorylcholine (SPC) (27, 28) are involved in the migration of VSMCs. In atherosclerosis, the migration of VSMCs is associated with the activation of the Rho GTPase/Rho-kinase pathway (29, 30) and the upregulation of cell adhesion molecules (31) and matrix metalloproteinases (32, 33). In restenosis, the proliferation and migration of VSMCs following injury to the arterial wall is regulated by the PDGF pathway (34). In aneurysm formation, VSMC migration and proliferation are regulated by the TGF-β pathway (35).

Rho-kinase and Vascular Smooth Muscle Cell Migration

Rho-kinase regulates the various functions of VSMCs, including VSMC contraction (36,37,38,39) and migration (38, 40). During cell migration, actin stress fibers are thought to play a role in this process by linking the leading edge of the cell to the substrate, helping to generate traction and maintain cell shape (41). Actin stress fibers are structures formed by the polymerization of actin filaments in the cytoplasm of cells (42) and are anchored to focal adhesions, which connect the extracellular matrix to the actin cytoskeleton (43). Small GTPases such as RhoA, Rac1, and Cdc42 regulate the formation and dynamics of actin stress fibers and play important roles in cell migration (44). Rho-kinase, a downstream effector of RhoA, is activated to lead to actin stress fiber formation (45) and trigger cell migration (41). The Rho-kinase-mediated formation of bundled stress fibers and cell migration also require Src tyrosine kinase activity (46). The mDia family of Rho effectors can bind Src and cooperate with Rho-kinase to form bundled stress fibers, and the synergistic effect of mDia on stress fiber formation requires Src kinase activity (47). In addition, Rho-kinase is reported to be involved in VSMC migration through myosin light chain phosphorylation-dependent and phosphorylation-independent pathways (48). Rho-kinase is therefore considered an important therapeutic target for cardiovascular diseases, including atherosclerosis, aortic aneurysm, and vascular stenosis (38, 49,50,51,52,53).

Active Fyn-paxillin Interaction regulates Vascular Smooth Muscle Cell Migration

Fyn is a type of Src family kinase that was discovered in the regulation of VSMC contraction in our previous study (54). Our studies have also shown that Fyn activation can stimulate the formation of actin stress fibers (55, 56) and promote VSMC migration (57). Activation of Fyn has been shown to increase RhoA activity (55), which is a key regulator of actin cytoskeleton organization. Fyn activation can also regulate the expression of genes involved in cell migration, such as matrix metalloproteinases and adhesion molecules (58). Additionally, Fyn has been shown to interact with other signaling pathways that play a role in cell migration, such as the PI3K/Akt pathway and the MAPK pathway (59, 60). This interaction may allow Fyn to integrate signals from multiple pathways and regulate the behavior of VSMCs in response to various stimuli. Our previous study demonstrated that constitutively active Fyn is located at the ends of actin stress fibers and regulates the formation of actin stress fibers (27, 55, 56).

Paxillin is a cytoskeleton-associated protein involved in the regulation of various cellular processes, including cell adhesion (61, 62), migration (62, 63), and polarity (62, 64). The N-terminal region of paxillin contains five LD motifs that facilitate interactions with other proteins (61, 63, 65, 66), while the C-terminal region includes four LIM domains that are essential for targeting paxillin to focal adhesion sites (63, 66). Paxillin binds to multiple signaling and structural proteins through different domains, thereby mediating signal transduction and participating in various cellular functions. Paxillin associates with Raf and MEK at focal adhesions, forming a complex that facilitates localized ERK activation (67). Additionally, paxillin acts as a scaffold for ERK recruitment, coordinating the activation of FAK and Rac (68). Paxillin also mediates Rho signaling through the paxillin-p42/44MAPK-GEF-H1 complex (69). Therefore, paxillin is recognized as a key platform for multiple signaling and structural proteins, acting as a mediator for the transmission of signals in cellular communication pathways.

The mechanism underlying the migration of VSMCs into the intimal layer in pathological conditions remains incompletely understood. Recently, we discovered that the Fyn/paxillin/Rho-kinase signaling pathway regulates the migration of VSMCs (27). Paxillin, a protein that directly binds to active Fyn, regulates Rho-kinase-mediated actin stress fiber formation and VSMC migration (Fig. 1).

Fig. 1.

Direct active Fyn-paxillin interaction regulates Rho-kinase-mediated actin stress fiber formation and vascular smooth muscle cell (VSMC) migration.

Our findings demonstrate that the interaction between active Fyn and paxillin regulates the migration of VSMCs through two main mechanisms: (1) direct binding between active Fyn and the N-terminus of paxillin (N-pax) and (2) localization of the active Fyn-paxillin complex at the ends of actin stress fibers (27). The direct binding of active Fyn and N-pax is critical for actin stress fiber formation and cell migration. The downregulation of the expression of paxillin reduces active Fyn-paxillin binding, leading to impaired actin stress fiber formation and cell migration. Similarly, the downregulation of Fyn or a dominant-negative Fyn mutant inhibits VSMC migration (57). While the overexpression of constitutively active Fyn alone in paxillin knockdown cells cannot induce actin stress fiber formation and migration, the coexpression of constitutively active Fyn and paxillin results in successful stress fiber formation and cell migration. Additionally, the inability of the C-terminus of paxillin to bind to active Fyn prevented the rescue of actin stress fiber formation and cell migration. These results indicate that direct binding of active Fyn and paxillin is necessary for actin stress fiber formation and subsequent cell migration. Interestingly, our results also showed that the overexpression of N-pax alone is insufficient to rescue actin stress fiber formation and cell migration (27). This led us to investigate the second necessary condition for actin stress fiber formation and migration in VSMCs, which is the localization of Fyn-paxillin at the ends of actin stress fibers. We observed that constitutively active Fyn and full-length paxillin colocalize at the ends of actin stress fibers, whereas constitutively active Fyn and N-pax colocalize in the cytoplasm near the nucleus (27), suggesting that the localization of the Fyn-paxillin complex at the ends of actin stress fibers is another key mechanism of VSMC migration. Further studies revealed that overexpressed N-pax competitively binds to active Fyn, reducing active Fyn-paxillin binding and leading to impaired actin stress fiber formation and cell migration (27). These findings indicate that the direct interaction between active Fyn and paxillin could be a potential target for regulating the migration of VSMCs.

The Fyn/Paxillin/Rho-kinase Signaling Pathway Mediates VSMC Migration

Rho-kinase is a well-known regulatory factor that mediates the formation of actin stress fibers and cell migration (45, 70, 71). Fyn knockdown and dominant negative-Fyn inhibit Rho-kinase activity and Rho-kinase-mediated actin stress fiber formation (55,56,57), indicating that Fyn acts as an upstream signaling molecule of Rho-kinase. Paxillin knockdown also inhibits Rho-kinase activity and Rho-kinase-mediated actin stress fiber formation, and paxillin re-expression rescues these effects (27), indicating that paxillin is required for Rho-kinase activation. On the other hand, the association of active Fyn and paxillin suggests that paxillin, as a downstream molecule of active Fyn, regulates Fyn-mediated VSMC migration. Additionally, our recent data showed that paxillin knockdown had no impact on Fyn activation (unpublished data). Taken together, these results indicate that paxillin acts as an intermediate signaling molecule, mediating actin stress fiber formation and VSMC migration via the Fyn/paxillin/Rho-kinase signaling pathway.

Conclusions and Perspectives

Active Fyn-paxillin binding at focal adhesions plays an important role in Rho-kinase-mediated actin stress fiber formation and migration in VSMCs. Targeting of the active Fyn-paxillin interaction represents a promising therapeutic avenue for various vascular diseases. By regulating this interaction, it may be possible to modulate the behavior of VSMCs and enhance treatment outcomes. However, additional research is required to fully realize the therapeutic potential of this approach and to develop safe and effective drugs that can specifically target this interaction.

Conflict of Interest

The authors declare no conflicts of interest in association with the present study.