Formin Homology Domain–Containing Protein 1 Regulates Smooth Muscle Cell Phenotype

Abstract

Objective-Our goal was to test whether formin homology protein 1 (FHOD1) plays a significant role in the regulation of smooth muscle cell (SMC) differentiation and, if so, whether Rho kinase (ROCK)-dependent phosphorylation in the diaphanous autoinhibitory domain is an important signaling mechanism that controls FHOD1 activity in SMC. Methods and Results-FHOD1 is highly expressed in aortic SMCs and in tissues with a significant SMC component.Exogenous expression of constitutively active FHOD1, but not wild-type, strongly activated SMC-specific gene expression in 10T1/2 cells. Treatment of SMC with the RhoA activator sphingosine-1-phosphate increased FHOD1 phosphorylation at Thr1141, and this effect was completely prevented by inhibition of ROCK with Y-27632. Phosphomimetic mutations to ROCK target residues enhanced FHOD1 activity, suggesting that phosphorylation interferes with FHOD1 autoinhibition. Importantly, knockdown of FHOD1 in SMC strongly inhibited sphingosine-1-phosphate-dependent increases in SMC differentiation marker gene expression and actin polymerization, suggesting that FHOD1 plays a major role in RhoA-dependent signaling in SMC. Key Words: biology, developmental Ⅲ molecular biology Ⅲ signal transduction Ⅲ vascular biology Ⅲ vascular muscle S mooth muscle cell (SMC) differentiation is an important component of vascular development, and defective control of this process in adult animals has been shown to contribute to a variety of cardiovascular pathologies, including atherosclerosis and restenosis (see 1 for review). It is well known that SMC differentiation marker gene expression is regulated by serum response factor binding to conserved CArG cis elements within the SMC-specific promoters. The myocardin family of serum response factor cofactors (myocardin and the myocardin-related transcription factor-[MRTF]-A/ megakaryoblastic leukemia-1 and MRTF-B/megakaryoblastic leukemia-2) are also critical and have been shown to be required for SMC differentiation marker gene expression in a variety of in vitro and in vivo models. 2-5 Thus, identifying the signaling mechanisms by which extrinsic cues regulate serum response factor/myocardin factor activity will be critical for our understanding of the control of SMC phenotype. Conclusion-OurMiralles et al were the first to demonstrate that MRTF-A activity is controlled by the small GTPase RhoA and that MRTF-A nuclear localization was enhanced by RhoAdependent actin polymerization. 6 Studies from our laboratory and others have shown that regulation of MRTF-A and MRTF-B by this mechanism plays an important role in the regulation of SMC phenotype in at least some SMC subtypes. [7][8][9] Of the RhoA effector proteins, Rho kinase (ROCK) is the most well studied and has been shown to enhance actin polymerization through LIM kinase-mediated inhibition of cofilin and to stimulate contractility by inhibiting myosin phosphatase. Although the ROCK inhibitor Y-27632 attenuates SMC-specific transcription, 10,11 it is clear that other RhoA effectors are also involved.Ac...

Loss of Sarcomere-associated Formins Disrupts Z-line Organization, but does not Prevent Thin Filament Assembly in Caenorhabditis elegans Muscle

“…One possible mechanism for such global effects is that formins might influence production of muscle-specific proteins, as occurs with FHOD-1-related mammalian FHOD1 and CYK-1-related mammalian Dia1 and Dia2 formins in smooth muscle cells [28,29]. Actin polymerization driven by these formins can indirectly trigger myocardin-related transcription factor-dependent expression of smooth muscle cell genes, such as those encoding smooth muscle α-actin or SM22.…”

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

“…FHOD1, a formin protein, is essential for proper deposition of the actin cytoskeleton. The formin FHOD1 plays an important role in formation and organization of the vascular smooth muscle cell contractile apparatus and a lack of FHOD1 results in fewer and grossly disorganized actin filaments (53). The second potential reason for the arterial pathology is that the mutation may cause defects in maintaining the contractile apparatus in a functional organized state once assembly has occurred.…”

FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers

“…Another common feature of formins consists of their ability to induce the nuclear transcription serum response factor (SRF) to modulate the expression of specific target genes (Tominaga et al, 2000). This effect mirrors formin-mediated regulation of actin dynamics since enhanced actin polymerization liberates myocardin-related transcription factor (MRTF) cofactors from G-actin to facilitate their import into the nucleus and subsequent activation of transcription (Jurmeister et al, 2012;Staus et al, 2011). As activated FHOD1 acts as potent inducer of SRF-mediated transcription (Gasteier et al, 2003;Westendorf, 2001) without inducing de novo actin polymerization, our results indicate that reducing G-actin pools, e.g.…”