Myocardin-related Transcription Factor Research Articles

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.

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

AbstractThe 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.

The actin-binding protein CAP1 represses MRTF-SRF-dependent gene expression in mouse cerebral cortex.

Serum response factor (SRF) is an essential transcription factor for brain development and function. Here, we explored how an SRF cofactor, the actin monomer-sensing myocardin-related transcription factor MRTF, is regulated in mouse cortical neurons. We found that MRTF-dependent SRF activity in vitro and in vivo was repressed by cyclase-associated protein CAP1. Inactivation of the actin-binding protein CAP1 reduced the amount of actin monomers in the cytoplasm, which promoted nuclear MRTF translocation and MRTF-SRF activation. This function was independent of cofilin1 and actin-depolymerizing factor, and CAP1 loss of function in cortical neurons was not compensated by endogenous CAP2. Transcriptomic and proteomic analyses of cerebral cortex lysates from wild-type and Cap1 knockout mice supported the role of CAP1 in repressing MRTF-SRF-dependent signaling in vivo. Bioinformatic analysis identified likely MRTF-SRF target genes, which aligned with the transcriptomic and proteomic results. Together with our previous studies that implicated CAP1 in axonal growth cone function as well as the morphology and plasticity of excitatory synapses, our findings establish CAP1 as a crucial actin regulator in the brain relevant for formation of neuronal networks.

The Mediator Med23 controls a transcriptional switch for muscle stem cell proliferation and differentiation in muscle regeneration

Muscle stem cells (MuSCs) contribute to a robust muscle regeneration process after injury, which is highly orchestrated by the sequential expression of multiple key transcription factors. However, it remains unclear how key transcription factors and cofactors such as the Mediator complex cooperate to regulate myogenesis. Here, we show that the Mediator Med23 is critically important for MuSC-mediated muscle regeneration. Med23 is increasingly expressed in activated/proliferating MuSCs on isolated myofibers or in response to muscle injury. Med23 deficiency reduced MuSC proliferation and enhanced its precocious differentiation, ultimately compromising muscle regeneration. Integrative analysis revealed that Med23 oppositely impacts Ternary complex factor (TCF)-targeted MuSC proliferation genes and myocardin-related transcription factor (MRTF)-targeted myogenic differentiation genes. Consistently, Med23 deficiency decreases the ETS-like transcription factor 1 (Elk1)/serum response factor (SRF) binding at proliferation gene promoters but promotes MRTF-A/SRF binding at myogenic gene promoters. Overall, our study reveals the important transcriptional control mechanism of Med23 in balancing MuSC proliferation and differentiation in muscle regeneration.

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 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.

Abstract 6827: Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal cancer

Abstract Background: Cancer-associated fibroblasts (CAFs) play a key role in modifying the tumor microenvironment (TME) and could be a future target for treatment. CAFs are typically categorized based on function: myofibroblastic CAFs (myCAFs) amplify extracellular matrix production that blocks therapy and immune cell infiltration while inflammatory CAFs (iCAFs) produce immuno-suppressive cytokines and chemokines. Here we sought to better understand the role and mechanisms of myCAFs in altering the TME by identifying their unique markers and potential drivers. Methods: Z-scores of ACTA2/TAGLN or ICAM1/PDPN were used to create datasets high in myCAF and iCAF markers, respectively, from TCGA data. A gene set enrichment analysis (GSEA) was performed on the resulting differential expression. Samples of patients were collected to create a cohort of colorectal cancers that were stained via immunohistochemistry (IHC) for alpha-smooth muscle actin (αSMA), transgelin (TAGLN), myocardin (MYOCD) and myocardin-related transcription factor A (MRTFA). Slides were evaluated for collagen using Masson’s trichrome stain. MYOCD and MRTFA nuclear expression was expressed as positively stained nuclei per high powered field (HPF; 400x). TAGLN, αSMA, and collagen were scored on a scale of 0-3+. Results: Of the 592 TCGA patients compiled across all disease stages, 23 (median age 63) formed the myCAF-high group (7 males, 16 females). Within this group, GSEA indicated an upregulation of myogenesis (q <0.001) with the following genes positively associated with myCAF markers: MYLK (q < 0.001, log2 fold change [FC] 1.52), DES (q < 0.001, 5.32), and MYH11 (q < 0.001, 3.57). The following were also upregulated in the myCAF high cohort: CNN1 (q < 0.001, 2.95) and MYOCD (q< 0.001, 2.27). These genes are targets of MYOCD and MRTFA transcription factors potentially driving the myCAF phenotype. To assess this potential relationship, we evaluated 306 tumor samples from 153 patients with CRC. The cohort consisted of 56 female patients and 97 male patients aged 13-90 years old across all disease stages. High amounts of nuclear MRTFA were associated with high scores for myCAF markers aSMA (average score 1.54 when MRTFA+ nuclei < 5 per HPF vs 2.06 when MRTFA + nuclei > 5 per HPF, p<0.001, Wilcoxon Rank Sum Test), TAGLN (1.37 vs 1.62, p = 0.043), and collagen (1.73 vs 2.10, p = 0.009). Likewise, for MYOCD, αSMA (1.79 vs 2.14, p = 0.001) and collagen (1.87 vs 2.15, p = 0.017) were positively correlated. Conclusions: Increased nuclear expression of MYOCD and MRTFA in stromal cells of CRC are positively correlated with myCAF gene expression, supporting our initial hypothesis of these being drivers of the phenotype. Our findings now motivate further analysis as to whether the inhibition of these transcription factors could lead to a reduction in the immune regulatory myCAF phenotype. Citation Format: Alec B. Cornelio, Katherine A. Johnson, Yousef Gadalla, Sharanya Nath, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6827.

Inhibitors of Rho/MRTF/SRF Transcription Pathway Regulate Mitochondrial Function.

RhoA-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factors (MRTFs) signaling pathway has emerged as a promising therapeutic target for pharmacological intervention in multiple diseases. Altered mitochondrial metabolism is one of the major hallmarks of cancer, therefore, this upregulation is a vulnerability that can be targeted with Rho/MRTF/SRF inhibitors. Recent advances identified a novel series of oxadiazole-thioether compounds that disrupt the SRF transcription, however, the direct molecular target of these compounds is unclear. Herein, we demonstrate the Rho/MRTF/SRF inhibition mechanism of CCG-203971 and CCG-232601 in normal cell lines of human lung fibroblasts and mouse myoblasts. Further studies investigated the role of these molecules in targeting mitochondrial function. We have shown that these molecules hyperacetylate histone H4K12 and H4K16 and regulate the genes involved in mitochondrial function and dynamics. These small molecule inhibitors regulate mitochondrial function as a compensatory mechanism by repressing oxidative phosphorylation and increasing glycolysis. Our data suggest that these CCG molecules are effective in inhibiting all the complexes of mitochondrial electron transport chains and further inducing oxidative stress. Therefore, our present findings highlight the therapeutic potential of CCG-203971 and CCG-232601, which may prove to be a promising approach to target aberrant bioenergetics.

WAVE2 Is a Vital Regulator in Myogenic Differentiation of Progenitor Cells through the Mechanosensitive MRTFA-SRF Axis.

Skeletal myogenesis is an intricate process involving the differentiation of progenitor cells into myofibers, which is regulated by actin cytoskeletal dynamics and myogenic transcription factors. Although recent studies have demonstrated the pivotal roles of actin-binding proteins (ABPs) as mechanosensors and signal transducers, the biological significance of WAVE2 (Wiskott-Aldrich syndrome protein family member 2), an ABP essential for actin polymerization, in myogenic differentiation of progenitor cells has not been investigated. Our study provides important insights into the regulatory roles played by WAVE2 in the myocardin-related transcription factor A (MRTFA)-serum response factor (SRF) signaling axis and differentiation of myoblasts. We demonstrate that WAVE2 expression is induced during myogenic differentiation and plays a pivotal role in actin cytoskeletal remodeling in C2C12 myoblasts. Knockdown of WAVE2 in C2C12 cells reduced filamentous actin levels, increased globular actin accumulation, and impaired the nuclear translocation of MRTFA. Furthermore, WAVE2 depletion in myoblasts inhibited the expression and transcriptional activity of SRF and suppressed cell proliferation in myoblasts. Consequently, WAVE2 knockdown suppressed myogenic regulatory factors (i.e., MyoD, MyoG, and SMYD1) expressions, thereby hindering the differentiation of myoblasts. Thus, this study suggests that WAVE2 is essential for myogenic differentiation of progenitor cells by modulating the mechanosensitive MRTFA-SRF axis.

Brain-derived neurotrophic factor (BDNF) downregulates mRNA levels of suppressor of cancer cell invasion (SCAI) variants in cortical neurons.

Suppressor of cancer cell invasion (SCAI) acts as a transcriptional repressor of serum response factor (SRF)-mediated gene expression by binding to megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which is an SRF transcriptional coactivator. Growing evidence suggests that SCAI is a negative regulator of neuronal morphology, whereas MKL2/MRTFB is a positive regulator. The mRNA expression of SCAI is downregulated during brain development, suggesting that a reduction in SCAI contributes to the reduced suppression of SRF-mediated gene induction, thus increasing dendritic complexity and developing neuronal circuits. In the present study, we hypothesized that brain-derived neurotrophic factor (BDNF), which is important for neuronal plasticity and development, might alter SCAI mRNA levels. We therefore investigated the effects of BDNF on SCAI mRNA levels in primary cultured cortical neurons. Furthermore, because alternative splicing generates several SCAI variants in the brain, we measured SCAI variant mRNA after BDNF stimulation. Both SCAI variant 1 and total SCAI mRNA expression levels were downregulated by BDNF. Moreover, the extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) pathway was involved in the BDNF-mediated decrease in SCAI mRNA expression. Our findings provide insights into the molecular mechanism underlying a neurotrophic factor switch for the repressive transcriptional complex that includes SCAI.

Concurrent left ventricular pressure-loading improves right ventricular function, remodeling, and molecular signaling in response to pulmonary artery banding

Abstract Background Studies have established the significance of right ventricle (RV) function in individuals with many congenital heart diseases, however the impact of pressure overload (PO) on the RV, concomitant with changes in LV pressure, has yet to be fully understood. We have previously demonstrated that pulmonary artery banding (PAB) with simultaneous LVPO improved systolic function in both ventricles and reduced myocyte hypertrophy and collagen content compared to PAB alone1. These findings suggested that increasing LV afterload attenuated maladaptive RV remodeling and molecular signaling. Purpose We investigated whether the degree of LV afterload beneficially impacts RV function, remodeling, and molecular signaling pathways. Methods We established a model of double-banding (DB) in male Sprague-Dawley rats (n = 17) where the PA and transverse aorta were constricted with 16-gauge and 14-gauge needles, respectively. At 6-weeks post-surgery, Doppler-echocardiography was used to measure the flow velocity across the aortic constriction to divide the DB rats into moderate (DBMOD) and mild (DBMILD) groups. PAB-only (n=9) and Sham (n=9) groups were used as controls. All animals underwent echocardiography and cardiac catheterization 6-weeks post-surgery to assess RV/LV structure and hemodynamic function. Heart tissue was collected for Picrosirius red (PSR) staining to assess RV collagen content. Immunoblotting and RT-qPCR were used to quantify protein and transcripts of fibrosis and mechanotransduction pathways. Results Echocardiography identified increases in RV end-diastolic and end-systolic areas alongside a decrease in tricuspid annular plane systolic excursion (TAPSE) in PAB rats compared to shams. These changes were abrogated in DBMOD rats and to a lesser extent in DBMILD rats (p<0.001). Hemodynamic analyses identified significantly increased dP/dtmax/min, and end-systolic pressure-volume relationship in the PAB group, each of which returned to near-Sham levels in the DBMOD group (p<0.0001). PSR-staining revealed a significant decrease of RV fibrosis in the DBMOD group compared to PAB rats (p<0.05). Immunoblotting in RV tissue revealed increases in myocardin-related transcription factor A, and YAP/TAZ in PAB rats; protein expression returned to near-sham levels in the DBMOD group (p<0.05) with an intermediate expression in the DBMILD group (p<0.05). RT-qPCR of RV tissue revealed significantly increased collagen 1&3, TGFβ1, and CTGF transcripts which returned to baseline in the DBMOD group (p<0.05). Conclusion Our results indicate that the degree of LV pressure provides a beneficial impact on RV function, remodeling, and both Hippo and profibrotic signaling during RV PO. Our study demonstrates the importance of ventricular interdependence from a physiological and molecular level and suggests that modulating LV pressures during diseases that impact the RV (such as pulmonary hypertension) offer a potentially attractive therapeutic route.

Matrix Nonlinear Viscoelasticity Regulates Skeletal Myogenesis through MRTF Nuclear Localization and Nuclear Mechanotransduction.

Mechanically sensitive tissues (e.g., skeletal muscles) greatly need mechanical stimuli during the development and maturation. The extracellular matrix (ECM) mediates these signals through nonlinear viscoelasticity of collagen networks that are predominant components of the ECM. However, the interactions between cells and ECM form a feedback loop, and it has not yet been possible to determine the degree to which, if any, of the features of matrix nonlinear viscoelasticity affect skeletal muscle development and regeneration. In this study, a nonlinear viscoelastic feature (i.e., strain-enhanced stress relaxation (SESR)) in normal skeletal muscles is observed, which however is almost absent in diseased muscles from Duchenne muscular dystrophy mice. It is recapitulated such SESR feature in vitro and separated the effects of mechanical strain and ECM viscoelasticity on myoblast response by developing a collagen-based hydrogel platform. Both strain and stress relaxation induce myogenic differentiation and myotube formation by C2C12 myoblasts, and myogenesis is more promoted by applying SESR. This promotion can be explained by the effects of SESR on actin polymerization-mediated myocardin related transcription factor (MRTF) nuclear localization and nuclear mechanotransduction. This study represents the first attempt to investigate the SESR phenomenon in skeletal muscles and reveal underlying mechanobiology, which will provide new opportunities for the tissue injury treatments.

Prostaglandin E2 attenuates lung fibroblast differentiation via inactivation of yes-associated protein signaling.

Prostaglandin E2 (PGE2 ) has been implicated in counteracting fibroblast differentiation by TGFβ1 during pulmonary fibrosis. However, the precise mechanism is not well understood. We show here that PGE2 via EP2 R and EP4 R inhibits the expression of mechanosensory molecules Lysyl Oxidase Like 2 (LOXL2), myocardin-related transcription factor A (MRTF-A), ECM proteins, plasminogen activation inhibitor 1 (PAI-1), fibronectin (FN), α-smooth muscle actin (α-SMA), and redox sensor (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4)) required for TGFβ1-mediated fibroblast differentiation. We further demonstrate that PGE2 inhibits fibrotic signaling via Yes-associated protein (YAP) but does so independently from its actions on SMAD phosphorylation and conserved cylindromatosis (CYLD; deubiquitinase) expression. Mechanistically, PGE2 phosphorylates/inactivates YAP downstream of EP2 R/Gαs and restrains its translocation to the nucleus, thus inhibiting its interaction with TEA domain family members (TEADs) and transcription of fibrotic genes. Importantly, pharmacological or siRNA-mediated inhibition of YAP significantly downregulates TGFβ1-mediated fibrotic gene expression and myofibroblast formation. Notably, YAP expression is upregulated in the lungs of D. farinae-treated wild type (WT) mice relative to saline-treated WT mice. Our results unravel a unique role for PGE2 -YAP interactions in fibroblast differentiation, and that PGE2 /YAP inhibition can be used as a novel therapeutic target in the treatment of pathological conditions associated with myofibroblasts like asthma.

Role of Rho/MRTF in Aggressive Vemurafenib-Resistant Murine Melanomas and Immune Checkpoint Upregulation.

Cutaneous melanoma is the deadliest skin cancer. Most have Ras-MAPK pathway (BRAFV600E or NRAS) mutations and highly effective targeted therapies exist; however, they and immune therapies are limited by resistance, in part driven by small GTPase (Rho and Rac) activation. To facilitate preclinical studies of combination therapies to provide durable responses, we describe the first mouse melanoma lines resistant to BRAF inhibitors. Treatment of mouse lines, YUMM1.7 and YUMMER, with vemurafenib (Vem), the BRAFV600E-selective inhibitor, resulted in high-level resistance (IC50 shifts 20-30-fold). Resistant cells showed enhanced activation of Rho and the downstream transcriptional coactivator, myocardin-related transcription factor (MRTF). Resistant cells exhibited increased stress fibers, nuclear translocation of MRTF-A, and an increased MRTF-A gene signature. Pharmacological inhibition of the Rho/MRTF pathway using CCG-257081 reduced viability of resistant lines and enhanced sensitivity to Vem. Remarkably, co-treatment of parental lines with Vem and CCG-257081 eliminated resistant colony development. Resistant cells grew more slowly in vitro, but they developed highly aggressive tumors with a shortened survival of tumor-bearing mice. Increased expression of immune checkpoint inhibitor proteins (ICIs) in resistant lines may contribute to aggressive in vivo behavior. Here, we introduce the first drug-resistant mouse melanoma models for assessing combinations of targeted and immune therapies.

Ac-SDKP promotes KIF3A-mediated β-catenin suppression through a ciliary mechanism to constrain silica-induced epithelial-myofibroblast transition

Kinesin family member 3 A (KIF3A) decrease have been reported in silicotic patients and rats. However, the detailed mechanisms of KIF3A in silicosis remain unknown. In this study, we demonstrated that KIF3A effectively blocked the expression of β-catenin and downstream myocardin-related transcription factor (MRTF)-A/serum response factor (SRF) signaling, thus inhibiting silica-induced epithelial−myofibroblast transition (EMyT). Moreover, KIF3A was identified as a downstream mediator of an antifibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Knockdown of KIF3A expression reactivated β-catenin/myocardin-related transcription factor (MRTF)-A/serum response factor (SRF) signaling that was attenuated by Ac-SDKP in vitro. Collectively, our findings suggest that Ac-SDKP plays its anti-fibrosis role via KIF3A-mediated β-catenin suppression, at least in part, in both in vivo model of silicosis and in vitro model of EMyT.

Persistent Hypoxia with Intermittent Aggravation Causes Imbalance in Smad3/Myocardin-Related Transcription Factor Signaling with Consequent Endothelial Senescence and Pulmonary Arterial Remodeling.

Loss of Smad3 and the consequent activation of myocardin-related transcription factor (MRTF) are associated with vascular pathologies. This study aimed to examine the impact of persistent hypoxia with intermittent aggravation (PI hypoxia) on cellular senescence and pulmonary arterial remodeling mediated by the Smad3/MRTF imbalance. We examined the effects of PI hypoxia on the Smad3/MRTF pathway and cellular senescence using human pulmonary artery endothelial cells (HPAECs) and in vivo studies in rats. The senescent degree was evaluated using β-galactosidase staining, p16 quantitation and the measurement of senescence-associated secretory phenotype. Structural data in the pathological analysis of pulmonary artery remodeling were collected. Compared to the control, HPAECs and pulmonary tissue from rats exposed to PI hypoxia showed a significantly higher senescent degree, lower expression of Smad3, and higher MRTF levels. The overexpression of Smad3 significantly mitigated HPAECs senescence in vitro. Further, treatment with CCG-203971, which inhibits MRTF, increased Smad3 levels and reduced β-galactosidase positive cells in rat lung tissue. This intervention also alleviated PI hypoxia-induced pathological changes, including remodeling indices of pulmonary arterial thickening, muscularization, and collagen formation. In conclusion, imbalanced Smad3/MRTF signaling is linked to PI hypoxia-induced senescence and pulmonary arterial remodeling, making it a potential therapeutic target for patients with sleep apnea and chronic obstructive pulmonary disease.

An N-Cyanoamide Derivative of Lithocholic Acid Co-Operates with Lysophosphatidic Acid to Promote Human Osteoblast (MG63) Differentiation.

Less-calcaemic vitamin D receptor (VDR) agonists have the potential to promote osteoblast maturation in a bone regenerative setting. The emergence of lithocholic acid (LCA) as a bona fide VDR agonist holds promise as an adjunct for arthroplasty following reports that it was less calcaemic than calcitriol (1,25D). However, LCA and some earlier derivatives, e.g., LCA acetate, had to be used at much higher concentrations than 1,25D to elicit comparable effects on osteoblasts. However, recent developments have led to the generation of far more potent LCA derivatives that even outperform the efficacy of 1,25D. These new compounds include the cyanoamide derivative, Dcha-150 (also known as AY2-79). In light of this significant development, we sought to ascertain the ability of Dcha-150 to promote human osteoblast maturation by monitoring alkaline phosphatase (ALP) and osteocalcin (OC) expression. The treatment of MG63 cells with Dcha-150 led to the production of OC. When Dcha-150 was co-administered with lysophosphatidic acid (LPA) or an LPA analogue, a synergistic increase in ALP activity occurred, with Dcha-150 showing greater potency compared to 1,25D. We also provide evidence that this synergy is likely attributed to the actions of myocardin-related transcription factor (MRTF)-serum response factor (SRF) gene transcription following LPA-receptor-induced cytoskeletal reorganisation.

Grb2-related adaptor protein GRAP is a novel regulator of liver fibrosis

AimsExcessive liver fibrosis is frequently observed in chronic liver diseases and associated with decline of liver functions. Hepatic stellate cells (HSCs) are considered the principal mediator of liver fibrosis by trans-differentiating into myofibroblasts. In the present study we investigated the role of Grb2-related adaptor protein (GRAP) in HSC activation and liver fibrosis. Methods and materialsLiver fibrosis was induced by carbon tetrachloride (CCl4) injection. Gene expression was examined by quantitative PCR. Cell proliferation was evaluated by EdU incorporation. DNA-protein interaction was examined by chromatin immunoprecipitation (ChIP). Key findingsGRAP expression was up-regulated during HSC-myofibroblast transition both in vivo and in vitro. Mechanistically, serum response factor (SRF) and myocardin-related transcription factor A (MRTF-A) formed a complex to bind to the GRAP promoter and activate GRAP transcription. Small interfering RNA (siRNA) mediated GRAP silencing blocked HSC-myofibroblast transition in vitro. Importantly, adeno-associated virus 6 (AAV6) mediated GRAP knockdown in myofibroblasts attenuated liver fibrosis in mice. Of note, inhibition of ERK signaling abrogated enhancement of HSC-myofibroblast transition by GRAP over-expression. SignificanceOur data suggest that GRAP, possibly via ERK activation, regulates HSC-myofibroblast transition and contributes to liver fibrosis. Screening for small-molecule GRAP inhibitors may yield novel therapeutic solutions against liver fibrosis.

Myocardin-related transcription factor contributes to renal fibrosis through the regulation of extracellular microenvironment surrounding fibroblasts.

Fibroblast accumulation and extracellular matrix (ECM) deposition are common critical steps for the progression of organ fibrosis, but the precise molecular mechanisms remain to be fully investigated. We have previously demonstrated that lysophosphatidic acid contributes to organ fibrosis through the production of connective tissue growth factor (CTGF) via actin cytoskeleton-dependent signaling, myocardin-related transcription factor family (MRTF) consisting of MRTF-A and MRTF-B-serum response factor (SRF) pathway. In this study, we investigated the role of the MRTF-SRF pathway in the development of renal fibrosis, focusing on the regulation of ECM-focal adhesions (FA) in renal fibroblasts. Here we showed that both MRTF-A and -B were required for the expressions of ECM-related molecules such as lysyl oxidase family members, type I procollagen and fibronectin in response to transforming growth factor (TGF)-β1 . TGF-β1 -MRTF-SRF pathway induced the expressions of various components of FA such as integrin α subunits (αv , α2 , α11 ) and β subunits (β1 , β3 , β5 ) as well as integrin-linked kinase (ILK). On the other hand, the blockade of ILK suppressed TGF-β1 -induced MRTF-SRF transcriptional activity, indicating a mutual relationship between MRTF-SRF and FA. Myofibroblast differentiation along with CTGF expression was also dependent on MRTF-SRF and FA components. Finally, global MRTF-A deficient and inducible fibroblast-specific MRTF-B deficient mice (MRTF-AKO BiFBKO mice) are protected from renal fibrosis with adenine administration. Renal expressions of ECM-FA components and CTGF as well as myofibroblast accumulation were suppressed in MRTF-AKO BiFBKO mice. These results suggest that the MRTF-SRF pathway might be a therapeutic target for renal fibrosis through the regulation of components forming ECM-FA in fibroblasts.

Recent progress on the role of cellular communication network factors (CCN) 3, 4 and 6 in regulating adiposity, liver fibrosis and pancreatic islets.

CCN/WISP (cellular communication network factors, or Wnt-inducted secreted proteins) family of proteins consists of six extracellular matrix (ECM)-associated proteins that regulate development, cell adhesion and proliferation, ECM remodeling, inflammation, and tumorigenesis. In the last two decades, metabolic regulation by these matricellular proteins has been studied extensively, several excellent reviews have covered the roles of CCN1, -2 and - 5. In this brief review, we will focus on those lesser-known members and more recent discoveries, together with other recent articles presenting a more complete picture of the current state of knowledge. We have found that CCN2, -4, and - 5 promote pancreatic islet function, while CCN3 plays a unique and negative role. CCN3 and - 4 are pro-adiposity leading to insulin resistance, but CCN5 and - 6 are anti-adiposity. While CCN2 and - 4 promote tissue fibrosis and inflammation, all other four members are clearly anti-fibrotic. As for cellular signaling, they are known to interact with integrins, other cell membrane proteins and ECM thereby regulate Akt/protein kinase B, myocardin-related transcription factor (MRTF), and focal adhesion kinase. Yet, a cohesive mechanism of action to comprehensively explain those major functions is still lacking.

An MRTF-A–ZEB1–IRF9 axis contributes to fibroblast–myofibroblast transition and renal fibrosis

Myofibroblasts, characterized by the expression of the matricellular protein periostin (Postn), mediate the profibrogenic response during tissue repair and remodeling. Previous studies have demonstrated that systemic deficiency in myocardin-related transcription factor A (MRTF-A) attenuates renal fibrosis in mice. In the present study, we investigated the myofibroblast-specific role of MRTF-A in renal fibrosis and the underlying mechanism. We report that myofibroblast-specific deletion of MRTF-A, achieved through crossbreeding Mrtfa-flox mice with Postn-CreERT2 mice, led to amelioration of renal fibrosis. RNA-seq identified zinc finger E-Box binding homeobox 1 (Zeb1) as a downstream target of MRTF-A in renal fibroblasts. MRTF-A interacts with TEA domain transcription factor 1 (TEAD1) to bind to the Zeb1 promoter and activate Zeb1 transcription. Zeb1 knockdown retarded the fibroblast–myofibroblast transition (FMyT) in vitro and dampened renal fibrosis in mice. Transcriptomic assays showed that Zeb1 might contribute to FMyT by repressing the transcription of interferon regulatory factor 9 (IRF9). IRF9 knockdown overcame the effect of Zeb1 depletion and promoted FMyT, whereas IRF9 overexpression antagonized TGF-β-induced FMyT. In conclusion, our data unveil a novel MRTF-A–Zeb1–IRF9 axis that can potentially contribute to fibroblast–myofibroblast transition and renal fibrosis. Screening for small-molecule compounds that target this axis may yield therapeutic options for the mollification of renal fibrosis.