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Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

ABSTRACT: Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle ?-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins.

SUBMITTER: Shi F

PROVIDER: S-EPMC3994013 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

Shi Feng F Long Xiaochun X Hendershot Allison A Miano Joseph M JM Sottile Jane J

PloS one 20140421 4

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can ...[more]

PMID: 24752318

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Project description:ObjectiveWe examined the role of syndecan-1 in modulating the phenotype of vascular smooth muscle cells in the context of endogenous inflammatory factors and altered microenvironments that occur in disease or injury-induced vascular remodeling.Methods and resultsVascular smooth muscle cells (vSMCs) display a continuum of phenotypes that can be altered during vascular remodeling. While the syndecans have emerged as powerful and complex regulators of cell function, their role in controlling vSMC phenotype is unknown. Here, we isolated vSMCs from wild type (WT) and syndecan-1 knockout (S1KO) mice. Gene expression and western blotting studies indicated decreased levels of α-smooth muscle actin (α-SMA), calponin, and other vSMC-specific differentiation markers in S1KO relative to WT cells. The spread area of the S1KO cells was found to be greater than WT cells, with a corresponding increase in focal adhesion formation, Src phosphorylation, and alterations in actin cytoskeletal arrangement. In addition, S1KO led to increased S6RP phosphorylation and decreased AKT and PKC-α phosphorylation. To examine whether these changes were present in vivo, isolated aortae from aged WT and S1KO mice were stained for calponin. Consistent with our in-vitro findings, the WT mice aortae stained higher for calponin relative to S1KO. When exposed to the inflammatory cytokine TNF-α, WT vSMCs had an 80% reduction in syndecan-1 expression. Further, with TNF-α, S1KO vSMCs produced increased pro-inflammatory cytokines relative to WT. Finally, inhibition of interactions between syndecan-1 and integrins αvβ3 and αvβ5 using the inhibitory peptide synstatin appeared to have similar effects on vSMCs as knocking out syndecan-1, with decreased expression of vSMC differentiation markers and increased expression of inflammatory cytokines, receptors, and osteopontin.ConclusionsTaken together, our results support that syndecan-1 promotes vSMC differentiation and quiescence. Thus, the presence of syndecan-1 would have a protective effect against vSMC dedifferentiation and this activity is linked to interactions with integrins αvβ3 and αvβ5.

Project description:BackgroundOverexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.Methods and resultssiRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.ConclusionsMARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

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Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

Publications

Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

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