Inhibition of MRTF-A Ameliorates Pathological Remodeling of the Pressure-loaded Right Ventricle.

Right ventricular (RV) fibrosis is associated with RV dysfunction in a variety of RV pressure-loading conditions where RV mechanical stress is increased, but the underlying mechanisms driving RV fibrosis are incompletely understood. In pulmonary and cardiovascular diseases characterized by elevated mechanical stress and transforming growth factor - beta-1 (TGF-ß1) signaling, myocardin-related transcription factor A (MRTF-A) is a mechanosensitive protein critical to driving myofibroblast transition and fibrosis. Here we investigated whether MRTF-A inhibition improves RV pro-fibrotic remodeling and function in response to a pulmonary artery banding (PAB) model of RV pressure-loading. Rats were assigned into either 1) sham or 2) PAB groups. MRTF-A inhibitor CCG-1423 was administered daily at 0.75mg/kg in a subset of PAB animals. Echocardiography and pressure-volume hemodynamics were obtained at a terminal experiment 6-weeks later. RV myocardial samples were analyzed for fibrosis, cardiomyocyte hypertrophy, and pro-fibrotic signaling. MRTF-A inhibition slightly reduced systolic dysfunction in PAB rats reflected by increased lateral tricuspid annulus peak systolic velocity, while diastolic function parameters were not significantly improved. RV remodeling was attenuated in PAB rats with MRTF-A inhibition, displaying reduced fibrosis. This was accompanied with a reduction in PAB-induced upregulation of yes-associated protein (YAP) and its paralog transcriptional co-activator with PDZ-binding motif (TAZ). We also confirmed using a second-generation MRTF-A inhibitor CCG-203971 that MRTF-A is critical in driving RV fibroblast expression of TAZ and markers of myofibroblast transition in response to TGF-ß1 stress and RhoA activation. These studies identify RhoA, MRTF-A, and YAP/TAZ as interconnected regulators of pro-fibrotic signaling in RV pressure-loading, and as potential targets to improve RV pro-fibrotic remodeling.

Single-nucleus analysis reveals microenvironment-specific neuron and glial cell enrichment in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complicated neurodegenerative disease. Neuron-glial cell interactions are an important but not fully understood process in the progression of AD. We used bioinformatic methods to analyze single-nucleus RNA sequencing (snRNA-seq) data to investigate the cellular and molecular biological processes of AD. METHOD: snRNA-seq data were downloaded from Gene Expression Omnibus (GEO) datasets and reprocessed to identify 240,804 single nuclei from healthy controls and patients with AD. The cellular composition of AD was further explored using Uniform Manifold Approximation and Projection (UMAP). Enrichment analysis for the functions of the DEGs was conducted and cell development trajectory analyses were used to reveal underlying cell fate decisions. iTALK was performed to identify ligand-receptor pairs among various cell types in the pathological ecological microenvironment of AD. RESULTS: Six cell types and multiple subclusters were identified based on the snRNA-seq data. A subcluster of neuron and glial cells co-expressing lncRNA-SNHG14, myocardin-related transcription factor A (MRTFA), and MRTFB was found to be more abundant in the AD group. This subcluster was enriched in mitogen-activated protein kinase (MAPK)-, immune-, and apoptosis-related pathways. Through molecular docking, we found that lncRNA-SNHG14 may bind MRTFA and MRTFB, resulting in an interaction between neurons and glial cells. CONCLUSIONS: The findings of this study describe a regulatory relationship between lncRNA-SNHG14, MRTFA, and MRTFB in the six main cell types of AD. This relationship may contribute to microenvironment remodeling in AD and provide a theoretical basis for a more in-depth analysis of AD.

RASSF1A is required for the maintenance of nuclear actin levels.

Nuclear actin participates in many essential cellular processes including gene transcription, chromatin remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin-9 and exportin-6, but how this transport is regulated remains unclear. Here, we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin-6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleocytoplasmic actin transport and the concomitant regulation of myocardin-related transcription factor A (MRTF-A), a co-activator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MRTF-A/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MRTF-A/SRF in tumour suppression.

MRTF-A alleviates myocardial ischemia reperfusion injury by inhibiting the inflammatory response and inducing autophagy.

Myocardin-related transcription factor A (MRTF-A) has an inhibitory effect on myocardial infarction; however, the mechanism is not clear. This study reveals the mechanism by which MRTF-A regulates autophagy to alleviate myocardial infarct-mediated inflammation, and the effect of silent information regulator 1 (SIRT1) on the myocardial protective effect of MRTF-A was also verified. MRTF-A significantly decreased cardiac damage induced by myocardial ischemia. In addition, MRTF-A decreased NLRP3 inflammasome activity, and significantly increased the expression of autophagy protein in myocardial ischemia tissue. Lipopolysaccharide (LPS) and 3-methyladenine (3-MA) eliminated the protective effects of MRTF-A. Furthermore, simultaneous overexpression of MRTF-A and SIRT1 effectively reduced the injury caused by myocardial ischemia; this was associated with downregulation of inflammatory factor proteins and when upregulation of autophagy-related proteins. Inhibition of SIRT1 activity partially suppressed these MRTF-A-induced cardioprotective effects. SIRT1 has a synergistic effect with MRTF-A to inhibit myocardial ischemia injury through reducing the inflammation response and inducing autophagy.

p38MAPK silencing attenuates scar proliferation after cleft palate repair surgery in rats via MRTF-A/SRF pathway.

Scarring is the primary factor of maxilla growth restriction among people who have undergone cleft palate repair surgery. p38 mitogen-activated protein kinase (p38MAPK) promotes fibrosis in a variety of organs. However, its role in post-surgery scarring on the hard palate has not been fully understood. This study is designed to investigate the role of p38MAPK in scar formation and maxilla growth of rats. We removed the mucosa on the hard palate of rats and applied the p38MAPK silencing adenovirus vector on it two weeks after surgery. Then the scarring tissue and maxilla growth were evaluated by histological and morphological examination. The effect of p38MAPK silencing on scarring-related genes in fibroblasts was also studied. We found that local injection of Ad-p38MAPK-1 in vivo effectively reduces the expression of p38MAPK and scarring-related proteins and weakens the impact of scarring on the width of the hard palate. Mechanistically, p38MAPK silencing inhibits the expression of α-smooth muscle actin (α-SMA) via mediating the production and nuclear localization of myocardin-related transcription factor A (MRTF-A) in fibroblasts. These results reveal a molecular pathway of scar formation involving p38MAPK/MRTF-A stimulation and support targeting p38MAPK as a potentially effective treatment for post-surgery scarring on the hard palate.

MRTF-A/SRF signaling suppresses invasion of oral squamous cell carcinoma.

BACKGROUND: Serum response factor (SRF) and myocardial-associated transcription factor-A (MRTF-A) had different regulatory effects on the tumorigenesis and development in different cancers. However, the role of MRTF-A/SRF in oral squamous cell carcinoma (OSCC) remains to be determined. METHODS: CCK-8 assay, cell scratch experiment, and transwell invasion assay were conducted to investigate the effects of MRTF-A/SRF on biological behavior of OSCC cells. The expression pattern and prognostic value of MRTF-A/SRF in OSCC were analyzed based on cBioPortal website and TCGA database. Protein-protein interaction network was visualized to identify protein functions. Go and KEGG pathway analyses were performed to investigate related pathways. The effect of MRTF-A/SRF on epithelial-mesenchymal transformation (EMT) of OSCC cells was explored by western blot assay. RESULTS: Overexpression of MRTF-A/SRF inhibited the proliferation, migration, and invasion of OSCC cells in vitro. High expression of SRF was related to better prognosis of OSCC patients on hard palate, alveolar ridge, and oral tongue. Besides, overexpression of MRTF-A/SRF inhibited the EMT of OSCC cells. CONCLUSION: SRF was closely related to the prognosis of OSCC. High expression of SRF and its co-activator MRTF-A inhibited proliferation, migration, and invasion of OSCC cells in vitro, possibly via EMT suppression.

Interdependence of Angiogenesis and Arteriogenesis in Development and Disease.

The structure of arterial networks is optimized to allow efficient flow delivery to metabolically active tissues. Optimization of flow delivery is a continuous process involving synchronization of the structure and function of the microcirculation with the upstream arterial network. Risk factors for ischemic cardiovascular diseases, such as diabetes mellitus and hyperlipidemia, adversely affect endothelial function, induce capillary regression, and disrupt the micro- to macrocirculation cross-talk. We provide evidence showing that this loss of synchronization reduces arterial collateral network recruitment upon arterial stenosis, and the long-term clinical outcome of current revascularization strategies in these patient cohorts. We describe mechanisms and signals contributing to synchronized growth of micro- and macrocirculation in development and upon ischemic challenges in the adult organism and identify potential therapeutic targets. We conclude that a long-term successful revascularization strategy should aim at both removing obstructions in the proximal part of the arterial tree and restoring "bottom-up" vascular communication.

Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes.

Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF-MRTF-Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF-MRTFA-actin axis in cardiac remodeling.

Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells.

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

MRTF-A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration.

Myocardin-related transcription factor-A/serum response factor (MRTF-A/SRF), a well-known transcriptional programme, has been proposed to play crucial roles in skeletal muscle development and function. However, whether MRTF-A participates in muscle regeneration and the molecular mechanisms are not completely understood. Here, we show that MRTF-A levels are highly correlated with myogenic genes using a RNA-seq assay, which reveal that MRTF-A knockdown in C2C12 cells significantly reduces PAX7 expression. Subsequent in vitro and in vivo data show that MRTF-A and PAX7 present identical expression patterns during myoblast differentiation and CTX-induced muscle injury and repair. Remarkably, MRTF-A overexpression promotes myoblast proliferation, while inhibiting cell differentiation and the expression of MyoD and MyoG. MRTF-A loss of function produces the opposite effect. Moreover, mice with lentivirus (MRTF-A) injection possesses more PAX7+ satellite cells, but less differentiating MyoD+ and MyoG+ cells, leading subsequently to diminished muscle regeneration. Our mechanistic results reveal that MRTF-A contributes to PAX7-mediated myoblast self-renewal, proliferation, and differentiation by binding to its distal CArG box. Overall, we propose that MRTF-A functions as a novel PAX7 regulator upon myoblast commitment to differentiation, which could provide pathways for dictating muscle stem cell fate and open new avenues to explore stem cell-based therapy for muscle degenerative diseases.

Hypoxia suppresses myofibroblast differentiation by changing RhoA activity.

Fibroblasts show a high range of phenotypic plasticity, including transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor ß can induce the myofibroblastic program, whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemia-induced tissue remodeling and the fibrotic response.This article has an associated First Person interview with the first author of the paper.

MMP9 Differentially Regulates Proteins Involved in Actin Polymerization and Cell Migration during TGF-ß-Induced EMT in the Lens.

Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-ß)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-ß-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (ß-actin) (ACTB) mRNA between the lenses of TGF-ß-overexpressing (TGF-ßtg) mice and TGF-ßtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-ß-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar differential regulation of cortactin, FAK, LIMK1 and MLC2 as observed in the array. Finally, a reduction in the nuclear localization of MRTF-A, a master regulator of cytoskeletal remodeling during EMT, was observed in rat LECs co-treated with JNJ0966 and TGF-ß. In conclusion, MMP9 deficiency results in differential regulation of proteins involved in actin polymerization and cell migration, and this in turn prevents TGF-ß-induced EMT in the lens.

A hypothesis for insulin resistance in primary human adipocytes involving MRTF-A and suppression of PPARγ.

Obesity is the main risk factor behind insulin resistance and type 2 diabetes. Still, the mechanism behind adipocyte dysfunction is not yet resolved. Recently, we reported that rapid actin remodeling correlates with adipose cell size changes after short-term overfeeding. Therefore, we hypothesized that the actin-driven myocardin-related transcription factor (MRTF-A) contributes to impaired mature adipocyte function. Primary human adipocytes were subjected to adenoviral overexpression of MRTF-A or MRTF-B, followed by Western blot analysis and tracer glucose uptake assay. Further, we assessed cell size distribution, insulin response, MRTF-A localization, actin organization and degree of polymerization in adipocytes isolated from Ob/Ob mice. Overexpression of MRTF-A, but not MRTF-B, markedly suppressed PPARγ expression. Further, MRTF-A expression resulted in decreased IRS-1 level, shifted phosphorylation of Akt (pS473/pT308), IRS-1 (pS302) and AS160 (pT642), and lowered insulin-stimulated glucose uptake. Hypertrophic adipocytes from Ob/Ob mice displayed an increased proportion of polymerized actin, and increased nuclear translocation of MRTF-A compared with control (Ob/+). Similar with human adipocytes overexpressing MRTF-A, adipocytes isolated from Ob/Ob mice had reduced expression of IRS-1 and PPARγ, as well as impaired insulin response. Together, these data demonstrate that MRTF-A negatively influences insulin sensitivity and the expression of key targets in fully mature human adipocytes. This suggests that MRTF-A is poised to exert a transcriptional response in hypertrophic adipocytes, contributing to adipocyte dysfunction and insulin resistance.

The MRTF-A/miR-155/SOX1 pathway mediates gastric cancer migration and invasion.

BACKGROUND: Gastric cancer (GC) is the leading cause of death worldwide and is closely related to metastasis. MRTF-A is one of the most well-characterized genetic markers in cancer. However, the mechanism whereby MRTF-A mediate gastric cancer (GC) tumorigenesis is not fully clear. Increasing evidence has confirmed that miRNA dysregulation is involved in MRTF-A-mediated tumorigenesis, supporting their potential as therapeutic targets for cancer. Although miR-155 has been reported as an upregulated miRNA, the interplay between miR-155 and MRTF-A-mediated gastric cancer progression remain largely elusive. METHODS: Real-time PCR was performed to determine miR-155 expression after transfected with MRTF-A encoding plasmids and siRNA. Potential target genes were identified by Western blot and luciferase reporter assay. Chip assay was proved that MRTF-A binds in the promoter region of miR-155. Transwell assay and Scratch-healing migration assay was used to investigate the role of MRTF-A and SOX1 in gastric cancer cell migration and invasion. RESULTS: MRTF-A can interact with the miR-155 promoter to promote histone acetylation and RNA polymerase II recruitment via the Wnt-ß-catenin pathway. miR-155 promotes gastric cancer cell migration by suppressing SOX1 expressiom by targeting its 3'UTR in vitro and in vivo. MRTF-A inhibited the inhibitory effects of SOX1 on gastric cancer cell migration by promoting the express -ion of miR-155. CONCLUSION: Our data therefore provide important and novel insights into how the MRTF-A/miR-155/SOX1 pathway mediates migration and invasion in GC.

Sphingosine 1-phosphate signaling induces SNAI2 expression to promote cell invasion in breast cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical process implicated in the initial stage of cancer metastasis, which is the major cause of tumor recurrence and mortality. Although key transcription factors that regulate EMT, such as snail family transcriptional repressor 2 (SNAI2), are well characterized, the upstream signaling pathways controlling these transcriptional mediators are largely unknown, which limits therapeutic strategies. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator, generated by sphingosine kinases (SPHK1 and SPHK2), that mainly exerts its effects by binding to the following 5 GPCRs: S1P1 to S1P5. S1P signaling has been reported to regulate different aspects of cancer progression including cell proliferation, apoptosis, and migration; nevertheless, its role in cancer metastasis, specifically via EMT, is not established. Here we show that SPHK1 expression correlates significantly with EMT score in breast cancer cell lines, and with SNAI2 in patient-derived breast tumors. Cell-based assays demonstrate that S1P can rapidly up-regulate the expression of SNAI2 in breast cancer cells via the activation of cognate receptors S1P2 and S1P3. Knockdown studies suggest that S1P2 and S1P3 mediate this effect by activating myocardin-related transcription factor A (MRTF-A) and yes-associated protein (YAP), respectively. Michigan Cancer Foundation 7 cells stably overexpressing S1P2 or S1P3 exhibit a more invasive phenotype, when compared to control cells. Taken together, our findings suggest that S1P produced by SPHK1 induces SNAI2 expression via S1P2-YAP and S1P3-MRTF-A pathways, leading to enhanced cell invasion. Cumulatively, this study reveals a novel mechanism by which S1P activates parallel pathways that regulate the expression of SNAI2, a master regulator of EMT, and provides new insights into druggable therapeutic targets that may limit cancer metastasis. Wang, W., Hind, T., Lam, B. W. S., Herr, D. R. Sphingosine 1-phosphate signaling induces SNAI2 expression to promote cell invasion in breast cancer cells.

RXRα and MRTF-A have a synergistic effect in the retinoic acid-induced neural-like differentiation of adult bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have multilineage differentiation potential and can transform into neuron cells under an appropriate environment. Retinoic acid (RA) facilitates the neuronal differentiation of MSCs. We found that RXRα, a RA receptor, was significantly upregulated in RA-induced process. Here, we show that RXRα collaborated with myocardin-related transcription factor-A (MRTF-A) to strongly promote the RA-induced process as evidenced by the increase in NF-H expression and NF-H promoter transcription activity. Our studies reveal that RXRα and MRTF-A exhibit protein interactions and synergistically inhibit the MSCs apoptosis by enhancing the P21 expression. Furthermore, RXRα and MRTF-A can activate P21 transcription by affecting the formation of the MRTF-A/RXRα/RARE complex. These findings reveal the important roles of RXRα and MRTF-A signaling in RA-induced neural-like differentiation of MSCs and describe a new mechanism underlying the synergistic interaction of RXRα and MRTF-A.

p75 neurotrophin receptor regulates NGF-induced myofibroblast differentiation and collagen synthesis through MRTF-A.

Myofibroblasts are characterized by de novo expression of α-smooth muscle actin (α-SMA) and play a key role in tissue repair and remodeling. In addition to TGF-ß1, recent studies have shown that nerve growth factor (NGF) has effects on myofibroblast differentiation and collagen synthesis. However, the regulatory mechanism remains poorly defined. NGF effects are mediated by the specific expression of the NGF neurotrophic tropomyosin-receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR). Using NIH/3T3 fibroblast cell lines, we examined the induction of myofibroblast differentiation stimulated by NGF. Our findings showed that p75NTR was in keeping with the expression of α-SMA. Herein, we investigated the role of p75NTR in NGF-induced myofibroblast differentiation and collagen synthesis in these cells using lentivirus transfection to overexpress and knock down. Our results showed that p75NTR was preferentially expressed and was sufficient to induce actin cytoskeleton remodeling, which was required for NGF-induced α-SMA expression. Furthermore, NGF induced nuclear translocation of MRTF-A, an effect that was regulated by p75NTR, and required for α-SMA and collagen-I expression in myofibroblasts. Using a novel MRTF-A pathway inhibitor, CCG-203971, we further demonstrated the requirement of MRTF-A nuclear localization and activity in NGF-induced α-SMA expression. In conclusion, we conclude that p75NTR regulates NGF-induced myofibroblast differentiation and collagen synthesis through MRTF-A. Regulation of NGF-p75NTR interactions represents a promising therapy for fibrotic disorders.

Mechanical strain induces a pro-fibrotic phenotype in human mitral valvular interstitial cells through RhoC/ROCK/MRTF-A and Erk1/2 signaling pathways.

The mitral valve is a complex multilayered structure populated by fibroblast-like cells, valvular interstitial cells (VIC) which are embedded in an extracellular matrix (ECM) scaffold and are submitted to the mechanical deformations affecting valve at each heartbeat, for an average of 40 million times per year. Myxomatous mitral valve (MMV) is the most frequent heart valve disease characterized by disruption of several valvular structures due to alterations of their ECM preventing the complete closure of the valve resulting in symptoms of prolapse and regurgitation. VIC and their ECM exhibit reciprocal dynamic processes between the mechanical signals issued from the ECM and the modulation of VIC phenotype responsible for ECM homeostasis of the valve. Abnormal perception and responsiveness of VIC to mechanical stress may induce an inappropriate adaptative remodeling of the valve progressively leading to MMV. To investigate the response of human VIC to mechanical strain and identify the molecular mechanisms of mechano-transduction in these cells, a cyclic equibiaxial elongation of 14% at the cardiac frequency of 1.16 Hz was applied to VIC by using a Flexercell-4000 T™ apparatus for increasing time (from 1 h to 8 h). We showed that cyclic stretch induces an early (1 h) and transient over-expression of TGFß2 and αSMA. CTGF, a profibrotic growth factor promoting the synthesis of ECM components, was strongly induced after 1 and 2 h of stretching and still upregulated at 8 h. The mechanical stress-induced CTGF up-regulation was dependent on RhoC, but not RhoA, as demonstrated by siRNA-mediated silencing approaches, and further supported by evidencing RhoC activation upon cell stretching and suppression of cell response by pharmacological inhibition of the effector ROCK1/2. It was also dependent on the MEK/Erk1/2 pathway which was activated by mechanical stress independently of RhoC and ROCK. Finally, mechanical stretching induced the nuclear translocation of myocardin related transcription factor-A (MRTF-A) which forms a transcriptional complex with SRF to promote the expression of target genes, notably CTGF. Treatment of stretched cultures with inhibitors of the identified pathways (ROCK1/2, MEK/Erk1/2, MRTF-A translocation) blocked CTGF overexpression and abrogated the increased MRTF-A nuclear translocation. CTGF is up-regulated in many pathological processes involving mechanically challenged organs, promotes ECM accumulation and is considered as a hallmark of fibrotic diseases. Pharmacological targeting of MRTF-A by newly developed inhibitors may represent a relevant therapy for MMV.

MRTF-A regulates proliferation and survival properties of pro-atherogenic macrophages.

We have previously reported that promoter polymorphism of myocardin-related transcription factor A (MRTF-A) is associated with coronary atherosclerosis. However, the contribution of MRTF-A to the development of atherosclerosis remains unknown. Macrophages are known to be important mediators of atherosclerosis. It has been demonstrated that local proliferation and survival of macrophages are atherogenic. In this study, we found that MRTF-A was highly expressed in lesional macrophages in human carotid atherosclerotic plaque. We then investigated the role of macrophagic MRTF-A in the pathogenesis of atherosclerosis. ApoE null MRTF-A transgenic mice (ApoE-/-/MRTF-Atg/+), in which human MRTF-A was specifically overexpressed in monocytes/macrophages, were established and fed with normal diet to examine the progression of atherosclerosis. We found that ApoE-/-/MRTF-Atg/+ aggravated atherosclerosis and lesional macrophages were more prominently accumulated in the aortic sinus of ApoE-/-/MRTF-Atg/+ than in that of ApoE-/- littermates. We also found that MRTF-A promoted proliferation and mitigated apoptosis of macrophages both in vitro and in vivo, and down regulated the expression of cyclin-dependent kinase inhibitors. From these findings, we conclude that MRTF-A modulates functional properties of pro-atherogenic macrophages. Our study may play a valuable role in understanding the pathological role of macrophagic MRTF-A in the progression of atherosclerosis.

RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFß-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFß-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 (col1a1). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFß-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFß-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFß signaling and have implications for our understanding of MSC function in fibrosis.