Myocardin-related Transcription Factor Research Articles

Essential Role of Cortactin in Myogenic Differentiation: Regulating Actin Dynamics and Myocardin-Related Transcription Factor A-Serum Response Factor (MRTFA-SRF) Signaling.

Cortactin (CTTN) is an actin-binding protein regulating actin polymerization and stabilization, which are vital processes for maintaining skeletal muscle homeostasis. Despite the established function of CTTN in actin cytoskeletal dynamics, its role in the myogenic differentiation of progenitor cells remains largely unexplored. In this study, we investigated the role of CTTN in the myogenic differentiation of C2C12 myoblasts by analyzing its effects on actin cytoskeletal remodeling, myocardin-related transcription factor A (MRTFA) nuclear translocation, serum response factor (SRF) activation, expression of myogenic transcription factors, and myotube formation. CTTN expression declined during myogenic differentiation, paralleling the reduction in MyoD, suggesting a potential role in the early stages of myogenesis. We also found that CTTN knockdown in C2C12 myoblasts reduced filamentous actin, enhanced globular actin levels, and inhibited the nuclear translocation of MRTFA, resulting in suppressed SRF activity. This led to the subsequent downregulation of myogenic regulatory factors, such as MyoD and MyoG. Furthermore, CTTN knockdown reduced the nuclear localization of YAP1, a mechanosensitive transcription factor, further supporting its regulatory roles in cell cycle and proliferation. Consequently, CTTN depletion impeded proliferation, differentiation, and myotube formation in C2C12 myoblasts, highlighting its dual role in the coordination of cell cycle regulation and myogenic differentiation of progenitor cells during myogenesis. This study identifies CTTN as an essential regulator of myogenic differentiation via affecting the actin remodeling-MRTFA-SRF signaling axis and cell proliferation.

Anti-Scar Effects of Micropatterned Hydrogel after Glaucoma Drainage Device Implantation.

Excessive fibrosis is the primary factor for the failure of glaucoma drainage device (GDD) implantation. Thus, strategies to suppress scar formation in GDD implantation are crucial. Although it is known that in implanted medical devices, microscale modification of the implant surface can modulate cell behavior and reduce the incidence of fibrosis, in the field of ophthalmic implants, especially the modification and effects of hydrogel micropatterns have rarely been reported. Here, we designed the patterned gelatin/acrylamide double network hydrogel and developed an innovative GDD with micropattern to suppress inflammatory and fibroblast activation after GDD implantation. Pattern topography suppressed F-actin expression and mitigated actin-dependent nuclear migration of myocardin-related transcription factor A (MRTF-A) during the proliferative phase after GDD implantation. Ultimately, the expression of α-smooth muscle actin (α-SMA), a key fibrosis-related gene product, was suppressed. Moreover, the modified GDD effectively controlled intraocular pressure (IOP), mitigated fibrous formation, and remodeled extracellular matrix (ECM) collagen distribution invivo. Therefore, the novel GDD with surface patterning interventions provides a promising strategy to inhibit scar formation after GDD implantation and raise the efficacy of GDD implantation.

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL (receptor activator for nuclear factor κB)-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that Myocardin-related transcription factor (MRTF) in breast cancer cells plays an important role in paracrine modulation of RANKL-induced OCL differentiation. This is partly attributed to MRTFs' critical role in maintaining the basal cellular expression of connective tissue growth factor (CTGF), findings that align with a strong positive correlation between CTGF expression and MRTF-A gene signature in the human disease context. Luminex analyses reveal that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extend beyond CTGF. Experimental metastasis studies demonstrate that MRTF depletion diminishes OCL abundance and bone colonizationof breast cancer cells in vivo, suggesting that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Discovery of GS-2278, a Potent and Selective LPAR1 Antagonist for the Treatment of Idiopathic Pulmonary Fibrosis.

We describe the discovery and preclinical characterization of a potent and selective lysophosphatidic acid receptor 1 (LPAR1) antagonist with a direct-acting antifibrotic mechanism. 18a was initially identified as a potent non-carboxylic acid LPAR1 antagonist in an LPA-induced myocardin-related transcription factor A (MRTF-A) nuclear translocation assay. Modifications to the aromatic elements in the structure allowed for improvements in metabolic stability and the mitigation of GSH adduct formation, but in vitro to in vivo clearance disconnects were observed with several potent sulfonamides (e.g., 27b) across preclinical species. Through modification of the sulfonamide, 42 (GS-2278) emerged as a potent LPAR1 antagonist with a suitable in vitro profile and desirable pharmacokinetic properties for oral QD dosing. GS-2278 dose-dependently blocked LPA-induced histamine release and demonstrated efficacy in an interventional model of bleomycin-induced lung fibrosis. However, CNS-related toxicity was observed in dogs, and based on these findings, the clinical development of GS-2278 for IPF was halted.

Abstract B037: Expression of MRTFA facilitates an immunosuppressive tumor microenvironment via upregulation of VSIR

Abstract Breast cancer, the second highest cause of cancer-related deaths in women, accounts for 31% of female cancer diagnoses. African American women have lower survival rates than White American women, influenced by both biological factors (age, family history, genetic factors, tumor biology) and social determinants (socio-economic status, insurance status, health care access). However, precise biological determinants remain unclear. The primary reason for breast cancer mortality is metastasis to organs like the lung, liver, and brain, with transcription factors playing a key role in this process. Myocardin related transcription factor A/B (MRTFA/B) are co-activators of serum-response factor (SRF), and the MRTF-SRF complex promotes breast cancer cell migration, invasion, and metastasis by regulating genes involved in actin cytoskeleton remodeling. MRTFA also supports tumor growth by modulating cancer-associated fibroblasts (CAFs) and remodeling the extracellular matrix (ECM) in the tumor microenvironment. While the pro-metastatic role of MRTFA/B is established in vitro and in mice, their impact on human cancer progression and metastasis requires further investigation. Therefore, understanding MRTFA's role in breast cancer metastasis is crucial for improving patient outcomes, making it essential to investigate MRTFA/B expression patterns in both tumor cells and the tumor microenvironment (TME). To explore the clinical, demographic, tumor-intrinsic, and tumor microenvironmental patterns of MRTFA/B’s expression and activation, we utilized multiplex imaging methods on multiple racially diverse tissue microarrays (TMA) and bioinformatics analyses of The Cancer Genome Atlas (TCGA), Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and available single-cell RNA sequencing datasets. Strikingly, MRTFA expression levels were higher in African American patients than in White American patients. Next, we investigated MRTFA expression patterns in the TME by using single-cell sequencing databases and found more abundant MRTFA expression in dendritic cells (DCs) within the TME compared to any other cell subtype. Moreover, our multiplex-imaging of tumor microarray (TMA) samples showed prominent MRTFA expression in HLA-DRA+CD45+ cells, indicative of antigen-presenting cells (APC). Notably, public single-cell RNA sequencing data indicated that breast cancer DCs strongly express the V-set immunoregulatory receptor (VSIR), an immune checkpoint protein, implying the immunosuppressive nature of the TME and we found that this increased VSIR expression is mediated by the MRTF-SRF complex. These findings suggest that DCs expressing MRTFA in breast cancer microenvironment contribute to creating the immunosuppressive TME via upregulation of VSIR expression, resulting in racial breast cancer disparities. Citation Format: Kihak Lee, Stephanie M. Wilk, Alexa M. Gajda, Mohamed Haloul, Virgilia Macias, Elizabeth L. Wiley, Zhengjia Chen, Xinyi Liu, Xiaowei Wang, Maria Sverdlov, Kent F. Hoskins, Ekrem Emrah Er. Expression of MRTFA facilitates an immunosuppressive tumor microenvironment via upregulation of VSIR [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B037.

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

MRTF, a transcriptional coactivator of SRF, is known to promote breast cancer progression through its tumor-cell-intrinsic function. Whether and how MRTF activity in tumor cells modulates other types of cells in the tumor microenvironment are not clearly understood.This study uncovers a novel tumor-cell-extrinsic function of MRTF in breast cancer cells in promoting osteoclast differentiation partly through CTGF regulation, and further demonstrates MRTF's requirement for bone colonization of breast cancer cells in vivo.Our studies suggest that MRTF inhibition could be an effective strategy to diminish osteoclast formation and skeletal involvement in metastatic breast cancer.

FBLN7 mediates vascular smooth muscle cell phenotype switching and vascular remodeling in hypertension.

Rationale: Arterial remodeling serves as a pivotal mechanism underlying the development of diseases such as hypertension. Fibulin-7 (FBLN7), an adhesion protein, remains enigmatic regarding its role in these pathological processes. This study aims to explore whether FBLN7 influences vascular remodeling and its underlying mechanisms. Methods: We generated FBLN7 knockout mice and smooth muscle-specific FBLN7 overexpression mice. Vascular remodeling models were established by administering angiotensin II (Ang II) for 28 days. RNA sequencing, western blot, and immunofluorescence assays were employed to investigate the biological function of FBLN7 in vascular smooth muscle cells (VSMCs). The interaction mechanism between FBLN7 and cell membrane receptors was explored through mass spectrometry analysis, co-immunoprecipitation techniques and molecular dynamics simulations. Results: Bioinformatics analysis revealed an upregulation of FBLN7 expression in the vascular remodeling model, with FBLN7 predominantly localized in VSMCs. Subsequent in vivo validation demonstrated that FBLN7 knockout attenuated Ang II-induced vascular remodeling, reducing aortic wall thickness and collagen formation. Conversely, VSMC-specific overexpression of FBLN7 via AAV vectors exacerbating the remodeling phenotype. Functionally speaking, FBLN7 potentiates Ang II-mediated phenotypic transformation. Mechanistically, FBLN7 interacts with the extracellular and transmembrane domains of syndecan-4 (SDC4) via its C-terminal region, affecting SDC4 signaling and dimer formation. This interaction inhibits SDC4-mediated activation of the Rho-associated protein kinase pathway, subsequently reducing nuclear translocation of myocardin-related transcription factor A, leading to decreased transcription of genes associated with the contractile VSMCs phenotype. Conclusions: These findings reveal FBLN7 promotes the transition of VSMCs from a contractile to a synthetic phenotype, thereby aggravating vascular remodeling. This provides further insights into the pathogenesis of vascular remodeling and potential therapeutic strategies.

Mechanical confinement matters: Unveiling the effect of two-photon polymerized 2.5D and 3D microarchitectures on neuronal YAP expression and neurite outgrowth

The effect of mechanical cues on cellular behaviour has been reported in multiple studies so far, and a specific aspect of interest is the role of mechanotransductive proteins in neuronal development. Among these, yes-associated protein (YAP) is responsible for multiple functions in neuronal development such as neuronal progenitor cells migration and differentiation while myocardin-related transcription factor A (MRTFA) facilitates neurite outgrowth and axonal pathfinding. Both proteins have indirectly intertwined fates via their signalling pathways. There is little literature investigating the roles of YAP and MRTFA in vitro concerning neurite outgrowth in mechanically confined microenvironments. Moreover, our understanding of their relationship in immature neurons cultured within engineered confined microenvironments is still lacking. In this study, we fabricated, via two-photon polymerization (2PP), 2.5D microgrooves and 3D polymeric microchannels, with a diameter range from 5 to 30 μm. We cultured SH-SY5Y cells and differentiated them into immature neuron-like cells on both 2.5D and 3D microstructures to investigate the effect of mechanical confinement on cell morphology and protein expression. In 2.5D microgrooves, both YAP and MRTFA nuclear/cytoplasmic (N/C) ratios exhibited maxima in the 10 μm grooves indicating a strong relation with mechanical-stress-inducing confinement. In 3D microchannels, both proteins’ N/C ratio exhibited minima in presence of 5 or 10 μm channels, a behaviour that was opposite to the ones observed in the 2.5D microgrooves and that indicates how the geometry and mechanical confinement of 3D microenvironments are unique compared to 2.5D ones due to focal adhesion, actin, and nuclear polarization. Further, especially in presence of 2.5D microgrooves, cells featured an inversely proportional relationship between YAP N/C ratio and the average neurite length. Finally, we also cultured human induced pluripotent stem cells (hiPSCs) and differentiated them into cortical neurons on the microstructures for up to 2 weeks. Interestingly, YAP and MRTFA N/C ratios also showed a maximum around the 10 μm 2.5D microgrooves, indicating the physiological relevance of our study. Our results elucidate the possible differences induced by 2.5D and 3D confining microenvironments in neuronal development and paves the way for understanding the intricate interplay between mechanotransductive proteins and their effect on neural cell fate within engineered cell microenvironments.

Inhibition of the Rho/MRTF pathway improves the response of BRAF-resistant melanoma to PD1/PDL1 blockade.

Metastatic cutaneous melanoma is a fatal skin cancer. Resistance to targeted and immune therapies limits the benefits of current treatments. Identifying and adding anti-resistance agents to current treatment protocols can potentially improve clinical responses. Myocardin-related transcription factor (MRTF) is a transcriptional coactivator whose activity is indirectly regulated by actin and the Rho family of GTPases. We previously demonstrated that development of BRAF inhibitor (BRAFi) resistance frequently activates the Rho/MRTF pathway in human and mouse BRAFV600E melanomas. In clinical trials, pretreatment with BRAFi reduces the benefit of immune therapies. We aimed to test the efficacy of concurrent treatment with our MRTF pathway inhibitor CCG-257081 and anti-PD1 in vivo and to examine its effects on the melanoma immune microenvironment. Because MRTF pathway activation upregulates the expression of immune checkpoint inhibitor genes/proteins, we asked whether CCG-257081 can improve the response to immune checkpoint blockade. CCG-257081 reduced the expression of PDL1 in BRAFi-resistant melanoma cells and decreased surface PDL1 levels on both BRAFi-sensitive and -resistant melanoma cells. Using our recently described murine vemurafenib-resistant melanoma model, we found that CCG-257081, in combination with anti-PD1 immune therapy, reduced tumor growth and increased survival. Moreover, anti-PD1/CCG-257081 co-treatment increased infiltration of CD8+ T cells and B cells into the tumor microenvironment and reduced tumor-associated macrophages. Here, we propose CCG-257081 as an anti-resistance and immune therapy-enhancing anti-melanoma agent.

Anti-fibrogenic effect of umbilical cord-derived mesenchymal stem cell-conditioned media in human esophageal fibroblasts.

Esophageal fibrosis can develop due to caustic or radiation injuries. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are known to mitigate fibrosis in various organs. However, the potential effects of UC-MSCs on human esophageal fibrosis remain underexplored. This study investigated the anti-fibrogenic properties and mechanisms of UC-MSC-derived conditioned media (UC-MSC-CM) on human esophageal fibroblasts (HEFs). HEFs were treated with TGF-β1 and then cultured with UC-MSC-CM, and the expression levels of extracellular matrix (ECM) components, RhoA, myocardin related transcription factor A (MRTF-A), serum response factor (SRF), Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) were measured. UC-MSC-CM suppressed TGF-β1-induced fibrogenic activation in HEFs, as evidenced by the downregulation of ECM. UC-MSC-CM diminished the expression of RhoA, MRTF-A, and SRF triggered by TGF-β1. In TGF-β1-stimulated HEFs, UC-MSC-CM decreased the nuclear localization of MRTF-A and YAP. Additionally, UC-MSC-CM diminished the TGF-β1-induced nuclear expressions of YAP and TAZ, while concurrently enhancing the cytoplasmic presence of phosphorylated YAP. Furthermore, UC-MSC-CM reduced TGF-β1-induced phosphorylation of Smad2. These findings suggest that UC-MSC-CM may inhibit TGF-β1-induced fibrogenic activation in HEFs by targeting the Rho-mediated MRTF/SRF and YAP/TAZ pathways, as well as the Smad2 pathway. This indicates its potential as a stem cell therapy for esophageal fibrosis.

Inhibition of Myocardin-related Transcription Factor 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 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.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine filamentous actin (F-actin) structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of myocardin-related transcription factor, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G protein-coupled receptor Smog, G protein αq subunit, Rho1 guanosine triphosphatase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand folded gastrulation to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, an NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

MICAL2 Is a Super Enhancer Associated Gene that Promotes Pancreatic Cancer Growth and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo . This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.

Regulation of the RhoA exchange factor GEF-H1 by profibrotic stimuli through a positive feedback loop involving RhoA, MRTF, and Sp1.

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

Suppression of smooth muscle cell inflammation by myocardin-related transcription factors involves inactivation of TANK-binding kinase 1

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.

Myocardin related transcription factor and galectin-3 drive lipid accumulation in human blood vessels

ObjectiveDiabetes and hypertension are important risk factors for vascular disease, including atherosclerosis. A driving factor in this process is lipid accumulation in smooth muscle cells of the vascular wall. The glucose- and mechano-sensitive transcriptional coactivator, myocardin-related transcription factor A (MRTF-A/MKL1) can promote lipid accumulation in cultured human smooth muscle cells and contribute to the formation of smooth muscle-derived foam cells. The purpose of this study was to determine if intact human blood vessels ex vivo can be used to evaluate lipid accumulation in the vascular wall, and if this process is dependent on MRTF and/or galectin-3/LGALS3. Galectin-3 is an early marker of smooth muscle transdifferentiation and a potential mediator for foam cell formation and atherosclerosis. Approach and resultsHuman mammary arteries and saphenous veins were exposed to altered cholesterol and glucose levels in an organ culture model. Accumulation of lipids, quantified by Oil Red O, was increased by cholesterol loading and elevated glucose concentrations. Pharmacological inhibition of MRTF with CCG-203971 decreased lipid accumulation, whereas adenoviral-mediated overexpression of MRTF-A had the opposite effect. Cholesterol-induced expression of galectin-3 was decreased after inhibition of MRTF. Importantly, pharmacological inhibition of galectin-3 with GB1107 reduced lipid accumulation in the vascular wall after cholesterol loading. ConclusionEx vivo organ culture of human arteries and veins can be used to evaluate lipid accumulation in the intact vascular wall, as well as adenoviral transduction and pharmacological inhibition. Although MRTF and galectin-3 may have beneficial, anti-inflammatory effects under certain circumstances, our results, which demonstrate a significant decrease in lipid accumulation, support further evaluation of MRTF- and galectin-3-inhibitors for therapeutic intervention against atherosclerotic vascular disease.

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

BackgroundAlzheimer’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.MethodsnRNA-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.ResultsSix 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.ConclusionsThe 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.

Targeting F-actin stress fibers to suppress the dedifferentiated phenotype in chondrocytes

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

Structural anisotropy results in mechano-directional transport of proteins across nuclear pores

The nuclear pore complex regulates nucleocytoplasmic transport by means of a tightly synchronized suite of biochemical reactions. The physicochemical properties of the translocating cargos are emerging as master regulators of their shuttling dynamics. As well as being affected by molecular weight and surface-exposed amino acids, the kinetics of the nuclear translocation of protein cargos also depend on their nanomechanical properties, yet the mechanisms underpinning the mechanoselectivity of the nuclear pore complex are unclear. Here we show that proteins with locally soft regions in the vicinity of the nuclear-localization sequence exhibit higher nuclear-import rates, and that such mechanoselectivity is specifically impaired upon knocking down nucleoporin 153, a key protein in the nuclear pore complex. This allows us to design a short, easy-to-express and chemically inert unstructured peptide tag that accelerates the nuclear-import rate of stiff protein cargos. We also show that U2OS osteosarcoma cells expressing the peptide-tagged myocardin-related transcription factor import this mechanosensitive protein to the nucleus at higher rates and display faster motility. Locally unstructured regions lower the free-energy barrier of protein translocation and might offer a control mechanism for nuclear mechanotransduction.