Myocardin-related Transcription Factor A Research Articles

Severe Molecular Defects Exhibited by the R179H Mutation in Human Vascular Smooth Muscle α-Actin

Mutations in vascular smooth muscle α-actin (SM α-actin), encoded by ACTA2, are the most common cause of familial thoracic aortic aneurysms that lead to dissection (TAAD). The R179H mutation has a poor patient prognosis and is unique in causing multisystemic smooth muscle dysfunction (Milewicz, D. M., Østergaard, J. R., Ala-Kokko, L. M., Khan, N., Grange, D. K., Mendoza-Londono, R., Bradley, T. J., Olney, A. H., Ades, L., Maher, J. F., Guo, D., Buja, L. M., Kim, D., Hyland, J. C., and Regalado, E. S. (2010) Am. J. Med. Genet. A 152A, 2437-2443). Here, we characterize this mutation in expressed human SM α-actin. R179H actin shows severe polymerization defects, with a 40-fold higher critical concentration for assembly than WT SM α-actin, driven by a high disassembly rate. The mutant filaments are more readily severed by cofilin. Both defects are attenuated by copolymerization with WT. The R179H monomer binds more tightly to profilin, and formin binding suppresses nucleation and slows polymerization rates. Linear filaments will thus not be readily formed, and cells expressing R179H actin will likely have increased levels of monomeric G-actin. The cotranscription factor myocardin-related transcription factor-A, which affects cellular phenotype, binds R179H actin with less cooperativity than WT actin. Smooth muscle myosin moves R179H filaments more slowly than WT, even when copolymerized with equimolar amounts of WT. The marked decrease in the ability to form filaments may contribute to the poor patient prognosis and explain why R179H disrupts even visceral smooth muscle cell function where the SM α-actin isoform is present in low amounts. The R179H mutation has the potential to affect actin structure and function in both the contractile domain of the cell and the more dynamic cytoskeletal pool of actin, both of which are required for contraction.

Myocardin-related transcription factor A (MRTF-A) activity-dependent cell adhesion is correlated to focal adhesion kinase (FAK) activity.

The regulation of cell-substrate adhesion is tightly linked to the malignant phenotype of tumor cells and plays a role in their migration, invasion, and metastasis. Focal adhesions (FAs) are dynamic adhesion structures that anchor the cell to the extracellular matrix. Myocardin-related transcription factors (MRTFs), co-regulators of the serum response factor (SRF), regulate expression of a set of genes encoding actin cytoskeletal/FA-related proteins. Here we demonstrated that the forced expression of a constitutively active MRTF-A (CA-MRTF-A) in B16F10 melanoma cells induced the up-regulation of actin cytoskeletal and FA proteins, resulting in FA reorganization and the suppression of cell migration. Expression of CA-MRTF-A markedly increased phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important components for FA dynamics. Notably, FAK activation was triggered by the clustering of up-regulated integrins. Our results revealed that the MRTF-SRF-dependent regulation of cell migration requires both the up-regulation of actin cytoskeletal/FA proteins and the integrin-mediated regulation of FA components via the FAK/Src pathway. We also demonstrated that activation of the MRTF-dependent transcription correlates FAK activation in various tumor cells. The elucidation of the correlation between MRTF and FAK activities would be an effective therapeutic target in focus of tumor cell migration.

Myocardin-related transcription factor A (MRTFA) regulates the fate of bone marrow mesenchymal stem cells and its absence in mice leads to osteopenia

ObjectiveArising from common progenitors in the bone marrow, adipogenesis and osteogenesis are closely associated yet mutually exclusive during bone marrow mesenchymal stem cell (BMSC) development. Previous studies have shown that morphological changes can affect the early commitment of pluripotent BMSCs to the adipose versus osteoblastic lineage via modulation of RhoA activity. The RhoA pathway regulates actin polymerization to promote the incorporation of globular actin (G-actin) into filamentous actin (F-actin). In doing so, myocardin-related transcription factors (MRTFs) dissociate from bound G-actin and enter the nucleus to co-activate serum response factor (SRF) target gene expression. In this study, we investigated whether MRTFA/SRF is acting downstream of the RhoA pathway to regulate BMSC commitment in mice. MethodsThe effects of knocking out MRTFA on skeletal homeostasis was studied in MRTFA KO mice using micro-CT, QPCR and western blot assays. To determine how MRTFA affects the mechanisms regulating BMSC fate decisions, primary bone marrow stromal cells from WT and MRTFA KO mice as well as C3H10T1/2 cell lines were analyzed in vitro. ResultsGlobal MRTFA KO mice have lower whole body weight, shorter femoral and tibial lengths as well as significantly decreased bone mass in their femurs. BMSCs isolated from the KO mice show increased adipogenesis and reduced osteogenesis when compared to WT littermates. KO mice, particularly females, develop osteopenia with age, and this was enhanced by a high fat diet. Over-expression of MRTFA or SRF enhances osteogenesis in CH310T1/2 cell lines. Sca1+, CD45− cells from KO marrow express lower amounts of smooth muscle actin (SMA) and TAZ/YAP target genes compared to WT counterparts. ConclusionThis study identified MRTFA as a novel regulator of skeletal homeostasis by regulating the balance between adipogenic and osteogenic differentiation of BMSCs. We propose that MRTFA promotes the osteogenic activity of TAZ/YAP by maintaining SMA production in BMSCs.

Stress-dependent phosphorylation of myocardin-related transcription factor A (MRTF-A) by the p38MAPK/MK2 axis

Myocardin-related transcription factor A (MRTF-A) is a known actin-regulated transcriptional coactivator of serum response factor (SRF). Stimulation of actin polymerization activates MRTF-A by releasing it from G-actin and thus allowing it to bind to and activate SRF. Here, we compared protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2 in two independent phosphoproteomic approaches using anisomycin-treated MEF cells and LPS-stimulated mouse macrophages, respectively. Two MRTF-A sites, Ser351 (corresponding to Ser312 in human) and Ser371 (Ser333 in human), showed significantly stronger phosphorylation (12-fold and 6-fold increase) in the cells expressing MK2. MRTF-A is phosphorylated at these sites in a stress-, but not in a mitogen-induced manner, and p38MAPK/MK2 catalytic activities are indispensable for this phosphorylation. MK2-mediated phosphorylation of MRTF-A at Ser312 and Ser333 was further confirmed in an in vitro kinase assay and using the phospho-protein kinase-D (PKD)-consensus motif antibody (anti-LXRXXpS/pT), the p38MAPK inhibitor BIRB-796, MK2/3-deficient cells and MRTF-A phospho-site mutants. Unexpectedly, dimerization, subcellular localization and translocation, interaction with actin, SRF or SMAD3 and transactivating potential of MRTF-A seem to be unaffected by manipulating the p38MAPK/MK2-dependent phosphorylations. Hence, MRTF-A is stress-dependently phosphorylated by MK2 at Ser312 and Ser333 with so far undetected functional and physiological consequences.

Cooperative signaling by TGF-β1 and WNT-11 drives sm-α-actin expression in smooth muscle via Rho kinase-actin-MRTF-A signaling.

Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-β1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-β1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-β1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-β1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-β1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn mediates sm-α-actin expression. Finally, we demonstrate that TGF-β1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study thus demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.

The role of myocardin-related transcription factor-A in Aβ25–35 induced neuron apoptosis and synapse injury

Myocardin-related transcription factor-A (MRTF-A) highly expressed in brain has been demonstrated to promote neuronal survival via regulating the transcription of related target genes as a powerful co-activator of serum response factor (SRF). However, the role of MRTF-A in Alzheimer's disease (AD) is still unclear. Here, we showed that MRTF-A was significantly downregulated in cortex of the Aβ25–35-induced AD rats, which played a key role in Aβ25–35 induced cerebral neuronal degeneration in vitro. Bilateral intracerebroventricular injection of Aβ25–35 caused significantly MRTF–A expression decline in cortex of rats, along with significant neuron apoptosis and plasticity damage. In vitro, transfection of MRTF-A into primary cultured cortical neurons prevented Aβ25–35 induced neuronal apoptosis and synapses injury. And luciferase reporter assay determined that MRTF-A could bind to and enhance the transactivity of the Mcl-1 (Myeloid cell leukemia-1) and Arc (activity-regulated cytoskeletal-associated protein) promoters by activating the key CArG box element. These data demonstrated that the decreasing of endogenous MRTF-A expression might contribute to the development of AD, whereas the upregulation MRTF-A in neurons could effectively reduce Aβ25–35 induced synapse injury and cell apoptosis. And the underlying mechanism might be partially due to MRTF-A-mediated the transcription and expression of Mcl-1 and Arc by triggering the CArG box.

Redox-Sensitive Regulation of Myocardin-Related Transcription Factor (MRTF-A) Phosphorylation via Palladin in Vascular Smooth Muscle Cell Differentiation Marker Gene Expression.

Vascular smooth muscle cells (VSMCs) undergo a phenotypic switch from a differentiated to synthetic phenotype in cardiovascular diseases such as atherosclerosis and restenosis. Our previous studies indicate that transforming growth factor-β (TGF-β) helps to maintain the differentiated phenotype by regulating expression of pro-differentiation genes such as smooth muscle α-actin (SMA) and Calponin (CNN) through reactive oxygen species (ROS) derived from NADPH oxidase 4 (Nox4) in VSMCs. In this study, we investigated the relationship between Nox4 and myocardin-related transcription factor-A (MRTF-A), a transcription factor known to be important in expression of smooth muscle marker genes. Previous work has shown that MRTF-A interacts with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear, as is the role of phosphorylation in MRTF-A activity. We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression. Knockdown of Nox4 using siRNA decreases TGF-β -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway. Knockdown of palladin also decreases MRTF-A phosphorylation. These data suggest that Nox4-dependent palladin expression and ROCK regulate phosphorylation of MRTF-A, a critical factor in the regulation of SRF responsive gene expression.

Myocardin-Related Transcription Factor A and Yes-Associated Protein Exert Dual Control in G Protein-Coupled Receptor- and RhoA-Mediated Transcriptional Regulation and Cell Proliferation.

The ability of a subset of G protein-coupled receptors (GPCRs) to activate RhoA endows them with unique growth-regulatory properties. Two transcriptional pathways are activated through GPCRs and RhoA, one utilizing the transcriptional coactivator myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF) and the other using the transcriptional coactivator Yes-associated protein (YAP) and TEA domain family members (TEAD). These pathways have not been compared for their relative levels of importance and potential interactions in RhoA target gene expression. GPCRs for thrombin and sphingosine-1-phosphate (S1P) on human glioblastoma cells robustly couple to RhoA and induce the matricelluar protein CCN1. Knockdown of either MRTF-A or YAP abrogates S1P-stimulated CCN1 expression, demonstrating that both coactivators are required. MRTF-A and YAP are also both required for transcriptional control of other S1P-regulated genes in various cell types and for S1P-stimulated glioblastoma cell proliferation. Interactions between MRTF-A and YAP are suggested by their synergistic effects on SRE.L- and TEAD-luciferase expression. Moreover, MRTF-A and YAP associate in coimmunoprecipitations from S1P-stimulated cells. Chromatin immunoprecipitation (ChIP) analysis of the CCN1 gene promoter demonstrated that S1P increases coactivator binding at the canonical transcription factor sequences. Unexpectedly, S1P also enhances MRTF-A binding at TEA sites. Our findings reveal that GPCR- and RhoA-regulated gene expression requires dual input and integration of two distinct transcriptional pathways.

RhoA/ROCK signaling regulates TGFβ-induced epithelial-mesenchymal transition of lens epithelial cells through MRTF-A.

Transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) leads to the formation of ocular fibrotic pathologies, such as anterior subcapsular cataract and posterior capsule opacification. Remodeling of the actin cytoskeleton, mediated by the Rho family of GTPases, plays a key role in EMT, however, how actin dynamics affect downstream markers of EMT has not been fully determined. Our previous work suggests that myocardin related transcription factor A (MRTF-A), an actin-binding protein, might be an important mediator of TGFβ-induced EMT in lens epithelial cells. The aim of the current study was to determine the requirement of RhoA/ROCK signaling in mediating TGFβ-induced nuclear accumulation of MRTF-A, and ultimate expression of α-smooth muscle actin (αSMA), a marker of a contractile, myofibroblast phenotype. Using rat lens epithelial explants, we demonstrate that ROCK inhibition using Y-27632 prevents TGFβ-induced nuclear accumulation of MRTF-A, E-cadherin/β-catenin complex disassembly, and αSMA expression. Using a novel inhibitor specifically targeting MRTF-A signaling, CCG-203971, we further demonstrate the requirement of MRTF-A nuclear localization and activity in the induction of αSMA expression. Overall, our findings suggest that TGFβ-induced cytoskeletal reorganization through RhoA/ROCK/MRTF-A signaling is critical to EMT of lens epithelial cells.

Myocardin-related transcription factor A (MRTF-A) plays an essential role in hepatic stellate cell activation by epigenetically modulating TGF-β signaling

Fibrosis following injury is a common adaptive response in the liver, which can lead to irreparable and life-threatening cirrhosis and hepatocellular carcinoma without effectual intervention. The molecular mechanisms underlying fibrogenic response in the liver remains poorly understood. Here we report that mice with deficiency in myocardin-related transcription factor A (MRTF-A) showed resistance to thioacetamide (TAA)-induced liver fibrosis with significantly reduced expression of pro-fibrogenic genes when compared to wild type littermates. Over-expression of MRTF-A enhanced whereas depletion of MRTF-A alleviated pro-fibrogenic transcription induced by TGF-β, a major pro-fibrogenic factor in hepatic stellate cells (HSCs). Mechanistically, MRTF-A silencing in HSCs impacted the chromatin structure by reducing the deposition of methylated histone H3K4 on the promoters of pro-fibrogenic genes. Further analyses revealed that MRTF-A interacted with and recruited several key epigenetic factors involved in H3K4 methylation, including ASH2, WDR5, and SET1, to the promoters of pro-fibrogenic genes in response to TGF-β treatment. Over-expression of ASH2, WDR5, or SET1 enhanced the transactivation of pro-fibrogenic gene promoters by TGF-β in an MRTF-A-dependent manner. In conclusion, MRTF-A regulates liver fibrosis by epigenetically tuning the TGF-β signaling pathway in HSCs.

2015 AHA Late-Breaking Basic Science Abstracts.

### 23060 ### Validation Of Circulating MicroRnas As Biomarkers In Heart Failure In Two Large Independent Cohorts Antoni Bayes-Genis1, David Lanfear2, Maurice de Ronde3, Josep Lupon1, Joost Leenders4, Elisabet Zamora1, L. Keoki Williams2, De Antonio Marta5, Zhen Liu4, Koos Zwinderman3, Sara-Joan Pinto-Sietsma3, Yigal Pinto3; 1Universitat Autonoma de Barcelona, Barcelona, Spain; 2Henry Ford Hospital, Detroit, MI; 3Academic Med Cntr, Univ of Amsterdam, Amsterdam, Netherlands; 4Mirnext BV, Amsterdam, Netherlands; 5Germans Trias i Pujol Hosp, Badalona, Spain Background: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF. Methods: We measured 15 circmiRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification signals. Results: In general, between 20–40% of measurements were “invalid”, while miR-499a\_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a\_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II). …

Baicalin inhibiting cerebral ischemia/hypoxia-induced neuronal apoptosis via MRTF-A-mediated transactivity

Baicalin has been shown to provide the neuroprotective effect by alleviating cerebral ischemia injury. However, little′s known about the underlying mechanism. Here, a cerebral artery occlusion (MACO)/reperfusion rat model and rat primary cortical neuron culture exposed to hydrogen peroxide (H2O2) were established to evaluate the effect of baicalin on ischemia-induced neuronal apoptosis. We found baicalin can significantly less neurological deficit and reduced infarct volume in vivo. And it efficiently inhibited neuronal apoptosis in vivo and vitro, which was especially characterized by the enhancing of transcription and expression of myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma-2 (BCL-2) in a dose-dependent manner. Furthermore, Baicalin markedly increased myocardin-related transcription factor-A (MRTF-A) level either in ischemic hemisphere or in primary cortical neuron cultures, whiles the anti-apoptosis effect of baicalin was significantly inhibited by transfected with the small interfering RNA of MRTF-A (MRTF-A siRNA) in primary cortical neuron cultures. The luciferase assays also indicated baicalin enhanced the transactivity of MCL-1 and BCL-2 promoter by activating the key CArG box (CC [A/T] 6GG) element, which was reduced by MRTF-A siRNA, suggesting MRTF-A may participate the anti-apoptosis effect of baicalin, and MRTF-A was involved in the transcriptional activity of MCL-1 and BCL-2 that was induced by baicalin. LY294002 (phosphatidylinositol-3 kinase (PI3K) inhibitor) and PD98059 (extracellular signal regulates kinase-1/2 (ERK1/2) inhibitor) obviously reduced baicalin-induced MRTF-A expression and transactivity and expression of MCL-1 and BCL-2, which further abolished the anti-apoptotic effect of baicalin on neuronal apoptosis. Taken together, our data provided the evidence demonstrating the neuroprotective effect of baicalin partially due to MRTF-A-mediated transactivity and expression of MCL-1 and BCL-2 by triggering the CArG box, which might be controlled by the activation of PI3K and ERK1/2.

Immunodeficiency and severe susceptibility to bacterial infection associated with a loss-of-function homozygous mutation of MKL1

AbstractMegakaryoblastic leukemia 1 (MKL1), also known as MAL or myocardin-related transcription factor A (MRTF-A), is a coactivator of serum response factor, which regulates transcription of actin and actin cytoskeleton–related genes. MKL1 is known to be important for megakaryocyte differentiation and function in mice, but its role in immune cells is unexplored. Here we report a patient with a homozygous nonsense mutation in the MKL1 gene resulting in immunodeficiency characterized predominantly by susceptibility to severe bacterial infection. We show that loss of MKL1 protein expression causes a dramatic loss of filamentous actin (F-actin) content in lymphoid and myeloid lineage immune cells and widespread cytoskeletal dysfunction. MKL1-deficient neutrophils displayed reduced phagocytosis and almost complete abrogation of migration in vitro. Similarly, primary dendritic cells were unable to spread normally or to form podosomes. Silencing of MKL1 in myeloid cell lines revealed that F-actin assembly was abrogated through reduction of globular actin (G-actin) levels and disturbed expression of multiple actin-regulating genes. Impaired migration of these cells was associated with failure of uropod retraction likely due to altered contractility and adhesion, evidenced by reduced expression of the myosin light chain 9 (MYL9) component of myosin II complex and overexpression of CD11b integrin. Together, our results show that MKL1 is a nonredundant regulator of cytoskeleton-associated functions in immune cells and fibroblasts and that its depletion underlies a novel human primary immunodeficiency.

Myocardin-related transcription factor A cooperates with brahmarelated gene 1 to activate P-selectin transcription

Expression of P-selectin in injured or activated endothelia cells serves as a permissive step towards leukocyte recruitment and perpetuation of inflammation in the pathogenesis of atherosclerosis. P-selectin can be induced by pro-inflammatory stimuli via the transcription factor NF-κB, but the epigenetic mechanisms remain incompletely understood. Previously we reported that myocardin-related transcription factor A (MRTF-A) mediates the transactivation of a slew of adhesion molecules by oxidized low-density lipoprotein (oxLDL), likely through a crosstalk with brahma-related gene 1 (BRG1), a chromatin remodeling protein. Here, we show that MRTF-A was both sufficient and necessary for the transactivation of P-selectin gene in endothelial cells treated with TNF-α. Depletion of MRTF-A using small interfering RNA (siRNA) abrogated the binding of BRG1 on the P-selectin promoter. Over-expression of BRG1 up-regulated the activity of P-selectin promoter activity while BRG1 knockdown attenuated P-selectin expression. Finally, BRG1 silencing suppressed the accumulation of acetylated histone H3 and methylated histone H3K4, and altered the binding of NF-κB on the P-selectin promoter. Therefore, our data demonstrate an essential role for MRTF-A and BRG1 in P-selectin transactivation in endothelial cells.

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

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. 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. 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.

Anti-apoptotic effects of myocardin-related transcription factor-A on rat cardiomyocytes following hypoxia-induced injury.

Myocardin-related transcription factor-A (MRTF-A) can transduce both biomechanical and humoral signals, which can positively modulate cardiac damage induced by acute myocardial infarction. However, the molecular mechanism that underlies the contribution that MRTF-A provides to the myocardium is not completely understood. The objective of this study was to investigate the effects of MRTF-A on myocardium apoptosis and its mechanisms. Our experiment results showed that MRTF-A expression increased and Bcl-2 expression reduced during myocardial ischemia-reperfusion in rat. Meanwhile, primary cardiomyocytes were pretreated with wild-type MRTF-A or siRNA of MRTF-A before exposure to hypoxia. We found that overexpression of MRTF-A in myocardial cells inhibited apoptosis and the release of cytochrome c. MRTF-A enhanced Bcl-2, which contributes to MRTF-A interaction with Bcl-2 in the nuclei of cardiomyocytes. MRTF-A upregulation expression of Bcl-2 in cardiomyocytes induced by hypoxia was inhibited by PD98059, an ERK1/2 inhibitor. In conclusions, MRTF-A improved myocardial cell survival in a cardiomyocyte model of hypoxia-induced injury; this effect was correlated with the upregulation of anti-apoptotic gene Bcl-2 through the activation of ERK1/2.

Stereospecific Inhibitory Effects of CCG-1423 on the Cellular Events Mediated by Myocardin-Related Transcription Factor A.

CCG-1423 suppresses several pathological processes including cancer cell migration, tissue fibrosis, and the development of atherosclerotic lesions. These suppressions are caused by inhibition of myocardin-related transcription factor A (MRTF-A), which is a critical factor for epithelial–mesenchymal transition (EMT). CCG-1423 can therefore be a potent inhibitor for EMT. CCG-1423 and related compounds, CCG-100602 and CCG-203971 possess similar biological activities. Although these compounds are comprised of two stereoisomers, the differences in their biological activities remain to be assessed. To address this issue, we stereoselectively synthesized optically pure isomers of these compounds and validated their biological activities. The S-isomer of CCG-1423 rather than the R-isomer exhibited modestly but significantly higher inhibitory effects on the cellular events triggered by MRTF-A activation including serum response factor-mediated gene expression and cell migration of fibroblasts and B16F10 melanoma cells. Accordingly, the S-isomer of CCG-1423 more potently blocked the serum-induced nuclear import of MRTF-A than the R-isomer. No such difference was observed in cells treated with each of two stereoisomers of CCG-100602 or CCG-203971. We previously reported that the N-terminal basic domain (NB), which functions as a nuclear localization signal of MRTF-A, is a binding site for CCG-1423. Consistent with the biological activities of two stereoisomers of CCG-1423, docking simulation demonstrated that the S-isomer of CCG-1423 was more likely to bind to NB than the R-isomer. This is a first report demonstrating the stereospecific biological activities of CCG-1423.

Signs of stress on soft surfaces : A commentary on: Cui, Y., F.M. Hameed, B. Yang, K. Lee, C.Q. Pan, S. Park, and M. Sheetz. 2015. Cyclic stretching of soft substrates induces spreading and growth. Nat Commun. 6:6333.

Cells experience mechanical stimuli during growth and differentiation and transduce these stimuli into biochemical signals that in turn regulate cell responses to the imposed forces. Reduced spreading and impaired stress fiber formation are indicators of the mechano-response to growth on soft elastic culture substrates. However, Cui and coworkers demonstrate that cell spreading and stress fiber formation on soft substrates is possible if simultaneous cyclic stretching compensates for the lack of substrate stiffness-induced cell stress. The stress(ed) response is dependent on cyclic stretch amplitude and frequency and, at least in part, mediated by myocardin related transcription factor A (MRTF-A) and Yes-associated protein (YAP). The study thus provides novel insight into the mechanisms of cell mechanosensing and how materials can be designed to mimic mechanical conditions of body tissues.

Myocardin-related transcription factor-A-overexpressing bone marrow stem cells protect cardiomyocytes and alleviate cardiac damage in a rat model of acute myocardial infarction.

Myocardin-related transcription factor-A (MRTF-A) can transduce biomechanical and humoral signals, which can positively modulate cardiac damage induced by acute myocardial infarction (AMI). In the clinic, bone marrow stem cell (BMSC) therapy is being increasingly utilized for AMI; however, the effects of BMSC transplantation remain to be optimized. Therefore, a novel strategy to enhance BMSC‑directed myocardial repair is particularly important. The present study was performed to assess the efficacy of MRTF‑A-overexpressing BMSCs in a rat model of AMI. Primary cardiomyocytes were prepared from neonatal Sprague-Dawley rats and BMSCs were isolated from male Sprague-Dawley rats (aged 8-12 weeks). Annexin V-phycoerythrin/7-actinomycin D staining was used to evaluate BMSC and cardiomyocyte survival after exposure to hydrogen peroxide in vitro. B-cell lymphoma 2 (Bcl-2) protein expression was measured by flow cytometric and western blot analyses. The effects of MRTF-A‑overexpressing BMSCs in a rat model of AMI were investigated by hematoxylin and eosin staining and western blot analysis of Bcl-2 expression in myocardial tissue sections. MRTF-A enhanced the migration of BMSCs, and overexpression of MRTF-A in BMSCs prevented hydrogen peroxide-induced apoptosis in primary cardiomyocytes ex vivo. In addition, co-culture of cardiomyocytes with MRTF‑A-overexpressing BMSCs inhibited hydrogen peroxide-induced apoptosis and the enhanced expression of Bcl-2. Furthermore, in vivo, enhanced cell survival was observed in the MRTF-A-modified BMSC group compared with that in the control group. These observations indicated that MRTF-A-overexpressing BMSCs have the potential to exert cardioprotective effects against hydrogen peroxide-induced injury and that treatment with MRTF‑A‑modified BMSCs is able to reverse cardiac dysfunction after AMI.

A novel inhibitory mechanism of MRTF-A/B on the ICAM-1 gene expression in vascular endothelial cells.

The roles of myocardin-related transcription factor A (MRTF-A) and MRTF-B in vascular endothelial cells are not completely understood. Here, we found a novel regulatory mechanism for MRTF-A/B function. MRTF-A/B tend to accumulate in the nucleus in arterial endothelial cells in vivo and human aortic endothelial cells (HAoECs) in vitro. In HAoECs, nuclear localization of MRTF-A/B was not significantly affected by Y27632 or latrunculin B, primarily due to the reduced binding of MRTF-A/B to G-actin and in part, to the low level of MRTF-A phosphorylation by ERK. MRTF-A/B downregulation by serum depletion or transfection of siRNA against MRTF-A and/or MRTF-B induced ICAM-1 expression in HAoECs. It is known that nuclear import of nuclear factor−κB (NF−κB) plays a key role in ICAM-1 gene transcription. However, nuclear accumulation of NF−κB p65 was not observed in MRTF-A/B-depleted HAoECs. Our present findings suggest that MRTF-A/B inhibit ICAM-1 mRNA expression by forming a complex with NF−κB p65 in the nucleus. Conversely, downregulation of MRTF-A/B alleviates this negative regulation without further translocation of NF−κB p65 into the nucleus. These results reveal the novel roles of MRTF-A/B in the homeostasis of vascular endothelium.