The actin depolymerizing factor destrin serves as a negative feedback inhibitor of smooth muscle cell differentiation.

We have previously shown that several components of the RhoA signaling pathway control smooth muscle cell (SMC) phenotype by altering serum response factor (SRF)-dependent gene expression. Because our genome-wide analyses of chromatin structure and transcription factor binding suggested that the actin depolymerizing factor, destrin (DSTN), was regulated in a SMC-selective fashion, the goals of the current study were to identify the transcription mechanisms that control DSTN expression in SMC and to test whether it regulates SMC function. Immunohistochemical analyses revealed strong and at least partially SMC-selective expression of DSTN in many mouse tissues, a result consistent with human data from the genotype-tissue expression (GTEx) consortium. We identified several regulatory regions that control DSTN expression including a SMC-selective enhancer that was activated by myocardin-related transcription factor-A (MRTF-A), recombination signal binding protein for immunoglobulin κ-J region (RBPJ), and the SMAD transcription factors. Indeed, enhancer activity and endogenous DSTN expression were upregulated by RhoA and transforming growth factor-ß (TGF-ß) signaling and downregulated by inhibition of Notch cleavage. We also showed that DSTN expression was decreased in vivo by carotid artery injury and in cultured SMC cells by platelet-derived growth factor-BB (PDGF-BB) treatment. siRNA-mediated depletion of DSTN significantly enhanced MRTF-A nuclear localization and SMC differentiation marker gene expression, decreased SMC migration in scratch wound assays, and decreased SMC proliferation, as measured by cell number and cyclin-E expression. Taken together our data indicate that DSTN is a negative feedback inhibitor of RhoA/SRF-dependent gene expression in SMC that coordinately promotes SMC phenotypic modulation. Interventions that target DSTN expression or activity could serve as potential therapies for atherosclerosis and restenosis.NEW & NOTEWORTHY First, DSTN is selectively expressed in SMC in RhoA/SRF-dependent manner. Second, a SMC-selective enhancer just upstream of DSTN TSS harbors functional SRF, SMAD, and Notch/RBPJ binding elements. Third, DSTN depletion increased SRF-dependent SMC marker gene expression while inhibiting SMC migration and proliferation. Taken together, our data suggest that DSTN is a critical negative feedback inhibitor of SMC differentiation.

Transcriptional and posttranscriptional regulation of the SMC-selective blood pressure-associated gene, ARHGAP42.

We previously showed that ARHGAP42 is a smooth muscle cell (SMC)-selective, RhoA-specific GTPase activating protein that regulates blood pressure and that a minor allele single nucleotide variation within a DNAse hypersensitive regulatory element in intron1 (Int1DHS) increased ARHGAP42 expression by promoting serum response factor binding. The goal of the current study was to identify additional transcriptional and posttranscriptional mechanisms that control ARHGAP42 expression. Using deletion/mutation, gel shift, and chromatin immunoprecipitation experiments, we showed that recombination signal binding protein for immunoglobulin κ-J region (RBPJ) and TEA domain family member 1 (TEAD1) binding to a conserved core region was required for full IntDHS transcriptional activity. Importantly, overexpression of the notch intracellular domain (NICD) or plating SMCs on recombinant jagged-1 increased IntDHS activity and endogenous ARHGAP42 expression while siRNA-mediated knockdown of TEAD1 inhibited ARHGAP42 mRNA levels. Re-chromatin immunoprecipitation experiments indicated that RBPJ and TEAD1 were bound to the Int1DHS enhancer at the same time, and coimmunoprecipitation assays indicated that these factors interacted physically. Our results also suggest TEAD1 and RBPJ bound cooperatively to the Int1DHS and that the presence of TEAD1 promoted the recruitment of NICD by RBPJ. Finally, we showed that ARHGAP42 expression was inhibited by micro-RNA 505 (miR505) which interacted with the ARHGAP42 3'-untranslated region (UTR) to facilitate its degradation and by AK124326, a long noncoding RNA that overlaps with the ARHGAP42 transcription start site on the opposite DNA strand. Since siRNA-mediated depletion of AK124326 was associated with increased H3K9 acetylation and RNA Pol-II binding at the ARHGAP42 gene, it is likely that AK124326 inhibits ARHGAP42 transcription.NEW & NOTEWORTHY First, RBPJ and TEAD1 converge at an intronic enhancer to regulate ARHGAP42 expression in SMCs. Second, TEAD1 and RBPJ interact physically and bind cooperatively to the ARHGAP42 enhancer. Third, miR505 interacts with the ARHGAP42 3'-UTR to facilitate its degradation. Finally, LncRNA, AK124326, inhibits ARHGAP42 transcription.

RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF.

Given our previous demonstration that RBPJ binds a methylated repressor element and regulates smooth muscle cell (SMC)-specific gene expression, we used genome-wide approaches to identify RBPJ binding regions in human aortic SMC and to assess RBPJ's effects on chromatin structure and gene expression. RBPJ bound to consensus cis elements, but also to TCmGGGA sequences within Alu repeats that were less transcriptionally active as assessed by DNAse hypersensitivity, H3K9 acetylation, and Notch3 and RNA Pol II binding. Interestingly, RBPJ binding was frequently detected at the borders of open chromatin, and a large fraction of genes induced or repressed by RBPJ depletion were associated with this cluster of RBPJ binding sites. RBPJ binding dramatically co-localized with serum response factor (SRF) and RNA seq experiments in RBPJ- and SRF-depleted SMC demonstrated that these factors interact functionally to regulate the contraction and inflammatory gene programs that help define SMC phenotype. Finally, we showed that RBPJ bound preferentially to phased nucleosomes independent of active chromatin marks and to cis elements positioned at the beginning and middle of the nucleosome dyad. These novel findings add important insight into RBPJ's role in chromatin structure and gene expression in SMC.

Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth.

Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux.

Inhibition of Diaphanous Formin Signaling In Vivo Impairs Cardiovascular Development and Alters Smooth Muscle Cell Phenotype.

OBJECTIVE: We and others have previously shown that RhoA-dependent stimulation of myocardin-related transcription factor nuclear localization promotes smooth muscle cell (SMC) marker gene expression. The goal of this study was to provide direct in vivo evidence that actin polymerization by the diaphanous-related formins contributes to the regulation of SMC differentiation and phenotype. APPROACH AND RESULTS: Conditional Cre-based genetic approaches were used to overexpress a well-characterized dominant-negative variant of mDia1 (DNmDia) in SMC. DNmDia expression in SM22-expressing cells resulted in embryonic and perinatal lethality in ≈20% of mice because of defects in myocardial development and SMC investment of peripheral vessels. Although most DNmDia(+)/SM22Cre(+) mice exhibited no overt phenotype, the re-expression of SMC differentiation marker gene expression that occurs after carotid artery ligation was delayed, and this effect was accompanied by a significant decrease in myocardin-related transcription factor-A nuclear localization. Interestingly, neointima growth was inhibited by expression of DNmDia in SMC and this was likely because of a defect in directional SMC migration and not to defects in SMC proliferation or survival. Finally, by using the tamoxifen-inducible SM MHC-CreER(T2) line, we showed that SMC-specific induction of DNmDia in adult mice decreased SMC marker gene expression. CONCLUSIONS: Our demonstration that diaphanous-related formin signaling plays a role in heart and vascular development and the maintenance of SMC phenotype provides important new evidence that Rho/actin/myocardin-related transcription factor signaling plays a critical role in cardiovascular function.

GRAF1 promotes ferlin-dependent myoblast fusion.

Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.

DNA methylation of a GC repressor element in the smooth muscle myosin heavy chain promoter facilitates binding of the Notch-associated transcription factor, RBPJ/CSL1.

OBJECTIVE: The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (SMC) differentiation marker gene expression. APPROACH AND RESULTS: We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM myosin heavy chain (MHC) promoter was highly methylated in cultured aortic SMC but not in the aorta, and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, recombination signal binding protein for immunoglobulin κ J region (RBPJ), as a methylated GC repressor-binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by platelet-derived growth factor-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did overexpression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of serum response factor and Pol II to the SMC-specific promoters. In contrast, the depletion of RBPJ in differentiated transforming growth factor-ß-treated SMC inhibited SMC-specific gene activation, supporting the idea that the effects of RBPJ/Notch signaling are context dependent. CONCLUSIONS: Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.

Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.).

Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy.

Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription.

We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure.

GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

Targeted focal adhesion kinase activation in cardiomyocytes protects the heart from ischemia/reperfusion injury.

OBJECTIVE: We previously reported that cardiac-restricted deletion of focal adhesion kinase (FAK) exacerbated myocyte death following ischemia/reperfusion (I/R). Here, we interrogated whether targeted elevation of myocardial FAK activity could protect the heart from I/R injury. METHODS AND RESULTS: Transgenic mice were generated with myocyte-specific expression of a FAK variant (termed SuperFAK) that conferred elevated allosteric activation. FAK activity in unstressed transgenic hearts was modestly elevated, but this had no discernable effect on anabolic heart growth or cardiac function. Importantly, SuperFAK hearts exhibited a dramatic increase in FAK activity and a reduction in myocyte apoptosis and infarct size 24 to 72 hours following I/R. Moreover, serial echocardiography revealed that the transgenic mice were protected from cardiac decompensation for up to 8 weeks following surgery. Mechanistic studies revealed that elevated FAK activity protected cardiomyocytes from I/R-induced apoptosis by enhancing nuclear factor-κB (NF-κB)-dependent survival signaling during the early period of reperfusion (30 and 60 minutes). Moreover, adenoviral-mediated expression of SuperFAK in cultured cardiomyocytes attenuated H(2)O(2) or hypoxia/reoxygenation-induced apoptosis, whereas blockade of the NF-κB pathway using a pharmacological inhibitor or small interfering RNAs completely abolished the beneficial effect of SuperFAK. CONCLUSIONS: Enhancing cardiac FAK activity attenuates I/R-induced myocyte apoptosis through activation of the prosurvival NF-κB pathway and may represent a novel therapeutic strategy for ischemic heart diseases.

GRAF1 Regulates Brown and Beige Adipose Differentiation and Function.

Adipose tissue, which is crucial for the regulation of energy within the body, contains both white and brown adipocytes. White adipose tissue (WAT) primarily stores energy, while brown adipose tissue (BAT) plays a critical role in energy dissipation as heat, offering potential for therapies aimed at enhancing metabolic health. Regulation of the RhoA/ROCK pathway is crucial for appropriate specification, differentiation and maturation of both white and brown adipocytes. However, our knowledge of how this pathway is controlled within specific adipose depots remains unclear, and to date a RhoA regulator that selectively controls adipocyte browning has not been identified. Our study shows that expression of GRAF1, a RhoGAP highly expressed in metabolically active tissues, closely correlates with brown adipocyte differentiation in culture and in vivo. Mice with either global or adipocyte-specific GRAF1 deficiency exhibit impaired BAT maturation, reduced capacity for WAT browning, and compromised cold-induced thermogenesis. Moreover, defects in differentiation of mouse or human GRAF1-deficient brown preadipocytes can be rescued by treatment with a Rho kinase inhibitor. Collectively, these studies indicate that GRAF1 can selectively induce brown and beige adipocyte differentiation and suggest that manipulating GRAF1 activity may hold promise for the future treatment of diseases related to metabolic dysfunction.

Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility.

Based upon our demonstration that the smooth muscle cell-selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6's role in SMCs in vivo using cell type-specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation-sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest.

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK(-/-) but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium.

Skeletal muscle differentiation and fusion are regulated by the BAR-containing Rho-GTPase-activating protein (Rho-GAP), GRAF1.

Although RhoA activity is necessary for promoting myogenic mesenchymal stem cell fates, recent studies in cultured cells suggest that down-regulation of RhoA activity in specified myoblasts is required for subsequent differentiation and myotube formation. However, whether this phenomenon occurs in vivo and which Rho modifiers control these later events remain unclear. We found that expression of the Rho-GTPase-activating protein, GRAF1, was transiently up-regulated during myogenesis, and studies in C2C12 cells revealed that GRAF1 is necessary and sufficient for mediating RhoA down-regulation and inducing muscle differentiation. Moreover, forced expression of GRAF1 in pre-differentiated myoblasts drives robust muscle fusion by a process that requires GTPase-activating protein-dependent actin remodeling and BAR-dependent membrane binding or sculpting. Moreover, morpholino-based knockdown studies in Xenopus laevis determined that GRAF1 expression is critical for muscle development. GRAF1-depleted embryos exhibited elevated RhoA activity and defective myofibrillogenesis that resulted in progressive muscle degeneration, defective motility, and embryonic lethality. Our results are the first to identify a GTPase-activating protein that regulates muscle maturation and to highlight the functional importance of BAR domains in myotube formation.

Signaling mechanisms that regulate smooth muscle cell differentiation.

Extensive studies over the last 30 years have demonstrated that vascular smooth muscle cell (SMC) differentiation and phenotypic modulation is controlled by a dynamic array of environmental cues. The identification of the signaling mechanisms by which these environmental cues regulate SMC phenotype has been more difficult because of our incomplete knowledge of the transcription mechanisms that regulate SMC-specific gene expression. However, recent advances in this area have provided significant insight, and the goal of this review is to summarize the signaling mechanisms by which extrinsic cues control SMC differentiation.

Formin homology domain-containing protein 1 regulates smooth muscle cell phenotype.

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. CONCLUSIONS: Our results indicate that FHOD1 is a critical regulator of SMC phenotype and is regulated by ROCK-dependent phosphorylation. Thus, additional studies on the role of FHOD1 during development and the progression of cardiovascular disease will be important.

Focal adhesion kinase regulates smooth muscle cell recruitment to the developing vasculature.

OBJECTIVE: The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. METHODS AND RESULTS: We crossed fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1, and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in stimulated extracellular matrix degradation. CONCLUSIONS: FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.