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Myocardin is a master regulator of smooth muscle cell (SMC) differentiation, which induces the expression of smooth-muscle-specific genes through its direct association with serum response factor (SRF). During the past two decades, significant insights have been obtained regarding the regulatory control of myocardin expression and transcriptional activity at the transcriptional, post-transcriptional, and post-translational levels. However, whether and how SUMOylation plays important roles in modulating myocardin function remain elusive. In this study, we found that myocardin is modified by SUMO-1 at lysine 573, which can be reversibly de-conjugated by SENP2. SUMO-1 modification promotes myocardin protein stability, whereas SENP2 facilitates its proteasome-dependent degradation. Moreover, we found that PIAS4 is the SUMO E3 ligase that enhances the SUMOylation and protein stability of myocardin. Most importantly, we found that SENP2 promotes phenotypic switching of VSMC. We therefore concluded that SENP2 promotes VSMC phenotypic switching via de-SUMOylation of myocardin and regulation of its protein stability.

Serum response factor (SRF) controls gene transcription in vascular smooth muscle cells (VSMCs) and regulates VSMC phenotypic switch from a contractile to a synthetic state, which plays a key role in the pathogenesis of cardiovascular diseases (CVD). It is not known how post-translational SUMOylation regulates the SRF activity in CVD. Here we show that Senp1 deficiency in VSMCs increased SUMOylated SRF and the SRF-ELK complex, leading to augmented vascular remodeling and neointimal formation in mice. Mechanistically, SENP1 deficiency in VSMCs increases SRF SUMOylation at lysine 143, reducing SRF lysosomal localization concomitant with increased nuclear accumulation and switching a contractile phenotype-responsive SRF-myocardin complex to a synthetic phenotype-responsive SRF-ELK1 complex. SUMOylated SRF and phospho-ELK1 are increased in VSMCs from coronary arteries of CVD patients. Importantly, ELK inhibitor AZD6244 prevents the shift from SRF-myocardin to SRF-ELK complex, attenuating VSMC synthetic phenotypes and neointimal formation in Senp 1-deficient mice. Therefore, targeting the SRF complex may have a therapeutic potential for the treatment of CVD. How post-translational SUMOylation regulates the SRF activity in cardiovascular disease is unclear. Here, the authors report that SRF SUMOylation increased by SENP1 deficiency switches vascular smooth muscle cells from a healthy contractile phenotype to a disease-associated synthetic phenotype.

microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.

Abstract Rationale Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR-II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF-β (transforming growth factor β) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])-2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH. Objectives We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin-related transcription factor), which drives muscle protein expression. Methods SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats. Measurements and Main Results SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF-β treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF-β–treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR-II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF-β–induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2-silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats. Conclusions Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and—via MRTF disinhibition—hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype.

Right ventricular (RV) fibrosis is associated with RV dysfunction in a variety of RV pressure-loading conditions in which 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-β1 signaling, myocardin-related transcription factor A (MRTF-A) is a mechanosensitive protein critical to driving myofibroblast transition and fibrosis. In this study, we investigated whether MRTF-A inhibition improves RV profibrotic remodeling and function in response to a pulmonary artery banding (PAB) model of RV pressure loading. Rats were assigned into either sham or PAB groups. MRTF-A inhibitor CCG-1423 was administered daily at 0.75 mg/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 profibrotic signaling. MRTF-A inhibition slightly reduced systolic dysfunction in PAB rats reflected by increased lateral tricuspid annulus peak systolic velocity, whereas 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 coactivator 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 transforming growth factor-β1 stress and RhoA activation. These studies identify RhoA, MRTF-A, and YAP/TAZ as interconnected regulators of profibrotic signaling in RV pressure loading and as potential targets to improve RV profibrotic remodeling.

Vascular smooth muscle cell (VSMC) phenotype transformation significantly contributes to vascular intimal hyperplasia. However, effective preventive and therapeutic measures are lacking. Colchicine, a binary alkaloid derived from Colchicum autumnale , is traditionally used for treating inflammatory diseases. Its role in neointima formation is not fully understood. Here, we investigated the role of colchicine in vascular intimal hyperplasia. We found that colchicine significantly reduced vascular intimal hyperplasia in an animal model at 7, 14, and 28 days post carotid artery ligation and increased the number of contractile-phenotype VSMCs (SMA-positive cells) in the neointimal areas. In vitro experiments demonstrated that colchicine facilitated the transition of VSMCs from a proliferative phenotype to a contractile phenotype. Additionally, colchicine attenuated PDGF-BB-induced phenotypic conversion and upregulated the expression of serum response factor (SRF) and myocardin (MYOCD). Further molecular mechanistic studies revealed that colchicine inhibited the expression of forkhead box protein O3A (FOXO3A) to increase the activation of the SRF‒MYOCD complex. FOXO3A can bind to MSX1/2, thereby inhibiting the expression of SRF–MYOCD and contractile genes. Moreover, colchicine maintains vascular homeostasis and stabilizes the contractile phenotype by affecting the expression of autophagy-related genes (LC3II, p62, and Beclin-1) induced by FOXO3A. Additionally, colchicine inhibited monocyte/macrophage infiltration and inflammatory cytokine expression. In summary, this study suggests that colchicine inhibits vascular intimal hyperplasia by modulating FOXO3A-mediated SRF-MYOCD activation and autophagy, providing new insights for future therapeutic approaches targeting occlusive vascular diseases.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile–proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

Previous studies have reported that the Ras homolog family member A (RhoA)/myocardin-related transcription factor A (MRTF-A) nuclear translocation axis positively regulates fibrogenesis induced by mechanical forces in various organ systems. The aim of the present study was to determine whether this signaling pathway was involved in the pathogenesis of nucleus pulposus (NP) fibrosis induced by mechanical overload during the progression of intervertebral disc degeneration (IVDD) and to confirm the alleviating effect of an MRTF-A inhibitor in the treatment of IVDD. NP cells (NPCs) were cultured on substrates of different stiffness (2.9 and 41.7 KPa), which mimicked normal and overloaded microenvironments, and were treated with an inhibitor of MRTF-A nuclear import, CCG-1423. In addition, bipedal rats were established by clipping the forelimbs of rats at 1 month and gradually elevating the feeding trough, and in order to establish a long-term overload-induced model of IVDD, and their intervertebral discs were injected with CCG-1423 in situ. Cell viability was determined by Cell Counting Kit-8 assay, and protein expression was determined by western blotting, immunofluorescence and immunohistochemical staining. The results demonstrated that the viability of NPCs was not affected by the application of force or the inhibitor. In NPCs cultured on stiff matrices, MRTF-A was mostly localized in the nucleus, and the expression levels of fibrotic proteins, including type I collagen, connective tissue growth factor and α-smooth muscle cell actin, were upregulated compared with those in NPCs cultured on soft matrices. The levels of these proteins were reduced by CCG-1423 treatment. In rats, 6 months of upright posture activated MRTF-A nuclear-cytoplasmic trafficking and fibrogenesis in the NP and induced IVDD; these effects were alleviated by CCG-1423 treatment. In conclusion, the results of the present study demonstrated that the RhoA/MRTF-A translocation pathway may promote mechanical overload-induced fibrogenic activity in NP tissue and partially elucidated the molecular mechanisms underlying the occurrence of IVDD.

Background Breast cancer progression and metastasis involve the action of multiple transcription factors in tumors and in the cells of the tumor microenvironment (TME) and understanding how these transcription factors are coordinated can guide novel therapeutic strategies. Myocardin-related transcription factors A and B (MRTFA/B also known as MKL1/2) are two related transcription factors that redundantly control cancer cell invasion and metastasis in mouse models of breast cancer, but their roles in human cancer are incompletely understood. Here, we investigated the expression and activation of these transcription factors to better assess their tumorigenic and metastatic impact on breast cancer and cells of the tumor microenvironment. Methods We used a multiplexed immunofluorescence approach to label MRTFA, MRTFB, tumor cells by using pan Cytokeratin, endothelial cells by using CD31, and antigen presenting cells (APCs) by using HLA-DRA on two different breast cancer tissue microarrays (TMA): The breast cancer progression TMA provided by the Cooperative Human Tissue Network (CHTN_BrCaProg3) and the University of Illinois Breast Cancer Working Group (TMA BCWG UIC-001-TMA) that included primary tumor and lymph node metastases from patients residing in the West Side and South Side of Chicago. We also used bioinformatics analyses of the TCGA and METABRIC databases and the Broad Institute’s single-cell RNA sequencing portal to investigate MRTFA/B expression patterns in the cells of the tumor microenvironment (TME). Results We found that in human tumors, MRTFA/B are concurrently activated in cancer cells, but they show distinct patterns of expression across different histological subtypes and in the cells of the TME. Importantly, MRTFA expression was elevated in metastatic tumors of African American patients, who disproportionately die from breast cancer. Interestingly, in contrast to publicly available mRNA expression data, MRTFA was similarly expressed across estrogen receptor (ER) positive and negative breast tumors, while MRTFB expression was highest in ER+ breast tumors. Furthermore, MRTFA was specifically expressed in the perivascular antigen-presenting cells (APCs), which has been previously associated with immune suppression and breast cancer progression. We also found that MRTFA expression correlated with the expression of the immune checkpoint protein V-set immunoregulatory receptor (VSIR) in the TCGA data and found that MRTFA activity promotes VSIR expression in THP-1 monocytes and cultured HEK293 cells. Conclusions Our results provide unique insights into how MRTFA and MRTFB promote metastasis in human cancer, the differences of their expression patterns, and their immune suppressive function within the breast cancer TME. Our results will guide future studies on targeting MRTFA/B transcriptional activity and the resulting immune suppression in breast cancer. Graphical Abstract

The acetylation of the lysine 40 residue of α-tubulin was described more than 30 years ago and has been the subject of intense research ever since. Although the exact function of this covalent modification of tubulin in the cell remains unknown, it has been established that tubulin acetylation confers resilience to mechanical stress on the microtubules. Formins have a dual role in the fate of the actin and tubulin cytoskeletons. On the one hand, they catalyze the formation of actin filaments, and on the other, they bind microtubules, act on their stability, and regulate their acetylation and alignment with actin fibers. Recent evidence indicates that formins coordinate the actin cytoskeleton and tubulin acetylation by modulating the levels of free globular actin (G-actin). G-actin, in turn, controls the activity of the myocardin-related transcription factor-serum response factor transcriptional complex that regulates the expression of the α - tubulin acetyltransferase 1 (α- TAT1 ) gene, which encodes the main enzyme responsible for tubulin acetylation. The effect of formins on tubulin acetylation is the combined result of their ability to activate α- TAT1 gene transcription and of their capacity to regulate microtubule stabilization. The contribution of these two mechanisms in different formins is discussed, particularly with respect to INF2, a formin that is mutated in hereditary human renal and neurodegenerative disorders.