Coactivator Of Serum Response Factor Research Articles

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.

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 heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also 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.

Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-β induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-β stimulation at nucleus, suggesting that TGF-β facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

BackgroundVascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease.MethodsWire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype.ResultsPhenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation.ConclusionsThe present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.Graphical abstract

LPA receptor 1 (LPAR1) is a novel interaction partner of Filamin A that promotes Filamin A phosphorylation, MRTF-A transcriptional activity and oncogene-induced senescence

Myocardin-related transcription factors A and B (MRTFs) are coactivators of Serum Response Factor (SRF), which controls fundamental biological processes such as cell growth, migration, and differentiation. MRTF and SRF transcriptional activity play an important role in hepatocellular carcinoma (HCC) growth, which represents the second leading cause of cancer-related mortality in humans worldwide. We, therefore, searched for druggable targets in HCC that regulate MRTF/SRF transcriptional activity and can be exploited therapeutically for HCC therapy. We identified the G protein-coupled lysophosphatidic acid receptor 1 (LPAR1) as a novel interaction partner of MRTF-A and Filamin A (FLNA) using fluorescence resonance energy transfer-(FRET) and proximity ligation assay (PLA) in vitro in HCC cells and in vivo in organoids. We found that LPAR1 promotes FLNA phosphorylation at S2152 which enhances the complex formation of FLNA and MRTF-A, actin polymerization, and MRTF transcriptional activity. Pharmacological blockade or depletion of LPAR1 prevents FLNA phosphorylation and complex formation with MRTF-A, resulting in reduced MRTF/SRF target gene expression and oncogene-induced senescence. Thus, inhibition of the LPAR1–FLNA–MRTF-A interaction represents a promising strategy for HCC therapy.

SRF/MRTF-A and liver cirrhosis: Pathologic associations.

Liver cirrhosis results from prolonged and extensive liver fibrosis in which fibrotic tissues replace functional hepatic cells. Chronic liver disease due to various viral, chemical, or metabolic factors initiates hepatic fibrogenesis. Cirrhosis is associated with multiple clinical complications and a poor patient prognosis; therefore, developing novel antifibrotic therapies to prevent cirrhosis is of high priority. Mounting evidence points to the key role of serum response factor (SRF) and myocardin-related transcription factor (MRTF)-A in the pathogenesis of liver fibrosis. SRF is a transcription factor and MRTF-A is a co-activator of SRF and normally resides in the cytoplasm. Upon the induction of fibrotic pathways, MRTF-A translocates into the nucleus and forms the active SRF/MRTF-A complex, leading to the expression of a multitude of fibrotic proteins and components of extracellular matrix. Silencing or inhibiting MRTF-A impedes hepatic stellate cell transdifferentiation into myofibroblasts and slows down the deposition of extracellular matrix in the liver, making it a potential therapeutic target. Here, we review the recent findings regarding the role of the SRF/MRTF-A complex in liver fibrosis and its therapeutic potential for the management of cirrhosis.

Temporal control of PDGFRα regulates the fibroblast-to-myofibroblast transition in wound healing.

SUMMARYFibroblasts differentiate into myofibroblasts by acquiring new contractile function. This is important for tissue repair, but it also contributes to organ fibrosis. Platelet-derived growth factor (PDGF) promotes tissue repair and fibrosis, but the relationship between PDGF and myofibroblasts is unclear. Using mice with lineage tracing linked to PDGF receptor α (PDGFRα) gene mutations, we examine cell fates during skin wound healing. Elevated PDGFRα signaling increases proliferation but unexpectedly delays the fibroblast-to-myofibroblast transition, suggesting that PDGFRα must be downregulated for myofibroblast differentiation. In contrast, deletion of PDGFRα decreases proliferation and myofibroblast differentiation by reducing serum response factor (SRF) nuclear localization. Consequences of SRF deletion resemble PDGFRα deletion, but deletion of two SRF coactivators, MRTFA and MRTFB, specifically eliminates myofibroblasts. Our findings suggest a scenario where PDGFRα signaling initially supports proliferation of fibroblast progenitors to expand their number during early wound healing but, later, PDGFRα downregulation facilitates fibroblast differentiation into myofibroblasts.

Lamina-associated polypeptide 2\u03b1 is required for intranuclear MRTF-A activity

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

A myocardin-adjacent lncRNA balances SRF-dependent gene transcription in the heart.

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.

Cyclase-associated protein 2 (CAP2) controls MRTF-A localization and SRF activity in mouse embryonic fibroblasts

Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity in CAP2 mutant MEFs was associated with altered actin turnover, a shift in the actin equilibrium towards monomeric actin (G-actin) as well as and reduced nuclear levels of myocardin-related transcription factor A (MRTF-A), a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation with jasplakinolide restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actin-dependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.

Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue:https://doi.org/10.1111/jnc.15067.

Expression of SOLOIST/MRTFB i4, a novel neuronal isoform of the mouse serum response factor coactivator myocardin-related transcription factor-B, negatively regulates dendritic complexity in cortical neurons.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB), a serum response factor (SRF) coactivator, is an important regulator of gene expression and neuronal morphology. Here, we show that different mouse MRTFB splice isoforms, including a novel fourth MRTFB isoform named spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4), play distinct roles in this process. SOLOIST/MRTFB i4 has a short exon that encodes 21 amino acid residues ahead of the first RPXXXEL (RPEL) motif in MRTFB isoform 3. Quantitative PCR revealed that SOLOIST/MRTFB i4 and isoform 1 were enriched in the forebrain and neurons, and up-regulated during brain development. Conversely, isoform 3 was detected in various tissues, including both neurons and astrocytes, and was down-regulated in the developing brain. Reporter assays supported the SRF-coactivator function of SOLOIST/MRTFB i4 as well as isoform 1. Acute expression of MRTFB isoform 1, but not isoform 3 or SOLOIST/MRTFB i4, in neuronal cells within 24hr drastically increased endogenous immediate early gene [c-fos, egr1, and activity-regulated cytoskeleton-associated protein] expression, but not endogenous actinin α1, β-actin, gelsolin, or srf gene expression measured by qPCR. Over-expression of SOLOIST/MRTFB i4 reduced the dendritic complexity of cortical neurons, whereas over-expression of isoform 1 increased this complexity. Co-expression of isoform 1 and SOLOIST/MRTFB i4 in cortical neurons revealed that isoform 1 competitively counteracted down-regulation by SOLOIST/MRTFB i4. Our findings indicate that MRTFB isoforms have unique expression patterns and differential effects on gene expression and dendritic complexity, which contribute to shaping neuronal circuits, at least in part.

Exercise-dependent increases in protein synthesis are accompanied by chromatin modifications and increased MRTF-SRF signalling.

AimResistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well‐defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise.MethodsWe performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans.ResultsWe identified a new phosphorylation site on Myocardin‐Related Transcription Factor B (MRTF‐B), a co‐activator of serum response factor (SRF). Phosphorylation of MRTF‐B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high‐intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF‐target genes, and prevent increases in protein synthesis after exercise.ConclusionOur results identify a new exercise signalling fingerprint in vivo, instrumental for exercise‐induced protein synthesis and potentially muscle growth.

Long non-coding RNA PVT1 regulates the migration of hepatocellular carcinoma HepG2 cells via miR-3619-5p/MKL1 axis.

Hepatocellular carcinoma (HCC) is the third most common malignant tumor of the digestive system. Plasma cell tumor heterotopic gene 1 (PVT1) is an intergenic long non-coding RNA that is aberrantly expressed in different cancers. Myocardin-related transcription factor A or megakaryoblastic leukemia 1 (MKL1) is a transcriptional coactivator of serum response factor that has been shown to promote cancer cell migration and invasion. In this study, we investigated the relationship between PVT1 and MKL1 as a novel regulatory mechanism underlying HCC progression. We used HepG2 and Cos-7 cell lines. Transfection experiments with miR-3619-5p mimics/inhibitor, PVT1, siRNA-PVT1, MKL1, or siRNA-MKL1 were performed. RNA and protein levels were analyzed by quantitative reverse transcription PCR and Western blot, respectively. Cell migration was assessed by transwell assay. Luciferase assays, RNA-FISH, RNA immunoprecipitation, and chromatin immunoprecipitation assays were performed to confirm the interaction between PVT1, miR-3619-5p, and MKL1 in HCC cells. Overexpression of PVT1 was positively correlated with MKL1 upregulation, which promoted HepG2 cell migration. miR-3619-5p inhibited MKL1 expression in HCC cells by acting on its 3′-UTR. Furthermore, PVT1 promoted MKL1 expression and migration in HCC cells by directly binding to miR-3619-5p. In a positive feedback loop, MKL1 could activate PVT1 transcription by binding to the CArG box in the promoter region. Our findings may provide a basis for the development of novel targeted therapies in HCC.

Inhibition of TRPM7 blocks MRTF/SRF-dependent transcriptional and tumorigenic activity.

Myocardin-related transcription factors A and B (MRTFs) are coactivators of Serum Response Factor (SRF) that mediates the expression of genes involved in cell proliferation, migration and differentiation. There is mounting evidence that MRTFs and SRF represent promising targets for hepatocellular carcinoma (HCC) growth. Since MRTF-A nuclear localization is a prerequisite for its transcriptional activity and oncogenic properties, we searched for pharmacologically active compounds able to redistribute MRTF-A to the cytoplasm. We identified NS8593, a negative gating modulator of the transient receptor potential cation channel TRPM7, as a novel inhibitor of MRTF-A nuclear localization and transcriptional activity. Using a pharmacological approach and targeted genome editing, we investigated the functional contribution of TRPM7, a unique ion channel containing a serine-threonine kinase domain, to MRTF transcriptional and tumorigenic activity. We found that TRPM7 function regulates RhoA activity and subsequently actin polymerization, MRTF-A-Filamin A complex formation and MRTF-A/SRF target gene expression. Mechanistically, TRPM7 signaling relies on TRPM7 channel-mediated Mg2+ influx and phosphorylation of RhoA by TRPM7 kinase. Pharmacological blockade of TRPM7 results in oncogene-induced senescence of hepatocellular carcinoma (HCC) cells in vitro and in vivo in HCC xenografts. Hence, inhibition of the TRPM7/MRTF axis emerges as a promising strategy to curb HCC growth.

Critical role of CRAG, a splicing variant of centaurin-\u03b33/AGAP3, in ELK1-dependent SRF activation at PML bodies

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3–knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.

Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Cells of the adult nucleus pulposus (NP) are critically important in maintaining overall disc health and function. NP cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Such changes result in physical cues of matrix stiffness that may be potent regulators of NP cell phenotype and may contribute to a transition toward a senescent and fibroblastic NP cell with a limited capacity for repair. Here, we investigate the mechanosignaling cues generated from changes in matrix stiffness in directing NP cell phenotype and identify mechanisms that can potentially preserve a biosynthetically active, juvenile NP cell phenotype. Using a laminin-functionalized polyethylene glycol hydrogel, we show that when NP cells form rounded, multicell clusters, they are able to maintain cytosolic localization of myocardin-related transcription factor (MRTF)-A, a coactivator of serum-response factor (SRF), known to promote fibroblast-like behaviors in many cells. Upon preservation of a rounded shape, human NP cells similarly showed cytosolic retention of transcriptional coactivator Yes-associated protein (YAP) and its paralogue PDZ-binding motif (TAZ) with associated decline in activation of its transcription factor TEA domain family member–binding domain (TEAD). When changes in cell shape occur, leading to a more spread, fibrotic morphology associated with stronger F-actin alignment, SRF and TEAD are up-regulated. However, targeted deletion of either cofactor was not sufficient to overcome shape-mediated changes observed in transcriptional activation of SRF or TEAD. Findings show that substrate stiffness-induced promotion of F-actin alignment occurs concomitantly with a flattened, spread morphology, decreased NP marker expression, and reduced biosynthetic activity. This work indicates cell shape is a stronger indicator of SRF and TEAD mechanosignaling pathways than coactivators MRTF-A and YAP/TAZ, respectively, and may play a role in the degeneration-associated loss of NP cellularity and phenotype.—Fearing, B. V., Jing, L., Barcellona, M. N., Witte, S. E., Buchowski, J. M., Zebala, L. P., Kelly, M. P., Luhmann, S., Gupta, M. C., Pathak, A., Setton, L. A. Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

An intronic deletion in megakaryoblastic leukemia 1 is associated with hyperproliferation of B cells in triplets with Hodgkin lymphoma.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo. This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Role of integrin-linked kinase in the hypoxia-induced phenotypic transition of pulmonary artery smooth muscle cells: Implications for hypoxic pulmonary hypertension

The phenotypic transition of pulmonary artery smooth muscle cells (PASMCs) from a contractile/differentiated to synthetic/de-differentiated phenotype is an important mechanism for the occurrence and development of hypoxic pulmonary hypertension (HPH). Integrin-linked kinase (ILK) is an early hypoxic response factor whose kinase activity is significantly affected during early hypoxia. Myocardin and ETS-like protein 1 (Elk-1) are co-activators of serum response factor (SRF) and can bind to SRF to mediate the phenotypic transition of PASMCs. However, little is known about the role of ILK on the phenotypic transition of these PASMCs. Thus, in our study, we explored the role of ILK in this process. We found that the expression of ILK and myocardin decreased gradually with the increase in hypoxia exposure time in the pulmonary arteries of rats. We observed that hypoxia exposure for 1 h caused an increase in the phosphorylation of Elk-1 but did not affect the expression of ILK, myocardin, or SRF. Exposure to hypoxic treatment for 1 h decreased ILK kinase activity and caused Elk-1 to suppress myocardin binding to SRF and the smooth muscle (SM) α-actin gene promoters. In addition, hypoxia exposure for 24 h decreased the expression of ILK, myocardin, SM α-actin, and calponin but increased the expression of osteopontin. Silencing of the myocardin gene significantly decreased the expression of SM α-actin and calponin but increased the expression of osteopontin. Silencing of the ILK gene significantly decreased the expression of myocardin, SM α-actin, and calponin but increased the expression of osteopontin. ILK overexpression reversed the effects of 24 h of hypoxia on the expression of myocardin, SM α-actin, calponin, and osteopontin and reversed the decrease in binding of myocardin to the SM α-actin promoter caused by 24 h of hypoxia exposure. Thus, our results suggest that ILK initiates the phenotypic transition of PASMCs. The underlying mechanism may involve hypoxia downregulating ILK kinase activity and protein expression, causing Elk-1 to compete with myocardin for binding to the SM α-actin promoter, which downregulates the expression of the downstream target myocardin and results in the phenotypic transition of PASMCs from a contractile to a synthetic phenotype. This may be an important mechanism in the development of HPH.

Myocardin ablation in a cardiac-renal rat model

Cardiorenal syndrome is defined by primary heart failure conditions influencing or leading to renal injury or dysfunction. Dilated cardiomyopathy (DCM) is a major co-existing form of heart failure (HF) with renal diseases. Myocardin (MYOCD), a cardiac-specific co-activator of serum response factor (SRF), is increased in DCM porcine and patient cardiac tissues and plays a crucial role in the pathophysiology of DCM. Inhibiting the increased MYOCD has shown to be partially rescuing the DCM phenotype in porcine model. However, expression levels of MYOCD in the cardiac tissues of the cardiorenal syndromic patients and the effect of inhibiting MYOCD in a cardiorenal syndrome model remains to be explored. Here, we analyzed the expression levels of MYOCD in the DCM patients with and without renal diseases. We also explored, whether cardiac specific silencing of MYOCD expression could ameliorate the cardiac remodeling and improve cardiac function in a renal artery ligated rat model (RAL). We observed an increase in MYOCD levels in the endomyocardial biopsies of DCM patients associated with renal failure compared to DCM alone. Silencing of MYOCD in RAL rats by a cardiac homing peptide conjugated MYOCD siRNA resulted in attenuation of cardiac hypertrophy, fibrosis and restoration of the left ventricular functions. Our data suggest hyper-activation of MYOCD in the pathogenesis of the cardiorenal failure cases. Also, MYOCD silencing showed beneficial effects by rescuing cardiac hypertrophy, fibrosis, size and function in a cardiorenal rat model.