Serum Response Factor Activity Research Articles

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN–SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation.

Background: Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. The striated muscle activator of Rho signaling (STARS) is an actin binding protein that regulates the transcription of genes involved in muscle cell growth, structure and function via the stimulation of actin polymerization and activation of serum-response factor (SRF) signaling. STARS mediates cell proliferation in smooth and cardiac muscle models; however, whether STARS overexpression enhances cell proliferation and differentiation has not been investigated in skeletal muscle cells.Results: We demonstrate for the first time that STARS overexpression enhances differentiation but not proliferation in C2C12 mouse skeletal muscle cells. Increased differentiation was associated with an increase in the gene levels of the myogenic differentiation markers Ckm, Ckmt2 and Myh4, the differentiation factor Igf2 and the myogenic regulatory factors (MRFs) Myf5 and Myf6. Exposing C2C12 cells to CCG-1423, a pharmacological inhibitor of SRF preventing the nuclear translocation of its co-factor MRTF-A, had no effect on myotube differentiation rate, suggesting that STARS regulates differentiation via a MRTF-A independent mechanism.Conclusion: These findings position STARS as an important regulator of skeletal muscle growth and regeneration.

Regulation of myofibroblast differentiation by cardiac glycosides.

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na(+)-K(+)-ATPase, resulting in increased intracellular [Na(+)]-to-[K(+)] ratio in cells. Microarray analysis suggested that increased intracellular [Na(+)]/[K(+)] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na(+)/Ca(2+) exchanger, KB-R4943. Ouabain inhibited transforming growth factor-β (TGF-β)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na(+)]/[K(+)] ratio through the treatment of cells with K(+)-free medium or with digoxin. Although inhibition of COX-2 or of the Na(+)/Ca(2+) exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-β-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na(+)]/[K(+)] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-β. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-β by ouabain, suggesting that additional mechanisms must exist.

Ligand-dependent serum response factor activation by the human CC chemokine receptors CCR2a and CCR2b is mediated by G proteins of the Gq family.

Expression of the human CCR2 receptors, CCR2a and CCR2b, in mammalian cells results in ligand-dependent changes in the activity of multiple cellular signal transduction pathways, mediated in most cases by pertussis toxin-sensitive heterotrimeric G proteins of the Gi/o subfamily. In addition, CCR2a and CCR2b receptors have been shown to couple to Gq family members, triggering the canonical activation of phospholipase Cβ isoenzymes. Activation of pertussis toxin-insensitive Gq proteins by cell-surface receptors is not only coupled to activation of phospholipase isoenzymes but also to Rho guanine nucleotide exchange factors, which in turn mediate activation of the Rho GTPases. Activated Rho GTPases regulate numerous cellular functions, including the organization of the actin cytoskeleton and gene transcription, such as the transcription factor serum response factor. These findings prompted us to investigate whether CCR2a and/or CCR2b stimulate serum response factor activity. The results presented herein demonstrate that stimulation of human CCR2a- or CCR2b-expressing COS-7 cells caused a vigorous induction of serum response factor activity. This effect was specifically mediated by Gq and/or G14, as well as Rho A and/or a closely related Rho GTPase. Furthermore, the stimulatory effect of CCR2a and CCR2b and Gαq was sensitive to coexpression of the Gαq-interacting leukemia-associated Rho guanine nucleotide exchange factor. The findings of the work indicate a role for Gαq and/or Gα14 and in CCR2a/CCR2b-stimulated Rho A GTPase-mediated serum response factor activation and introduce a noncanonical pathway activated by CCR2 receptors by coupling to Gq proteins.

Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription

Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.

Fine-tuned SRF activity controls asymmetrical neuronal outgrowth: implications for cortical migration, neural tissue lamination and circuit assembly.

The stimulus-regulated transcription factor Serum Response Factor (SRF) plays an important role in diverse neurodevelopmental processes related to structural plasticity and motile functions, although its precise mechanism of action has not yet been established. To further define the role of SRF in neural development and distinguish between cell-autonomous and non cell-autonomous effects, we bidirectionally manipulated SRF activity through gene transduction assays that allow the visualization of individual neurons and their comparison with neighboring control cells. In vitro assays showed that SRF promotes survival and filopodia formation and is required for normal asymmetric neurite outgrowth, indicating that its activation favors dendrite enlargement versus branching. In turn, in vivo experiments demonstrated that SRF-dependent regulation of neuronal morphology has important consequences in the developing cortex and retina, affecting neuronal migration, dendritic and axonal arborization and cell positioning in these laminated tissues. Overall, our results show that the controlled and timely activation of SRF is essential for the coordinated growth of neuronal processes, suggesting that this event regulates the switch between neuronal growth and branching during developmental processes.

Proteomic analysis of SRF associated transcription complexes identified TFII-I as modulator of SRF function in neurons

The serum response factor (SRF) is a neuronal activity regulated transcription factor (TF) mediating an immediate early (IEGs, e.g. cFos, Egr1) and actin cytoskeletal gene response. SRF activity is adjusted by two competing cofactor families, ternary complex factors (TCF; e.g. Elk-1) and myocardin related transcription factors (MRTF).We investigated mechanisms of SRF activation upon seizure associated neuronal activity in mice. SRF serine 103 phosphorylation or promoter binding was not obviously changed upon neuronal activation. In contrast, a SRF directed proteomic analysis uncovered established and potentially novel components of SRF associated protein complexes whose abundance was modified by neuronal activity. This included the general transcription factor TFII-I for which we provide a first functional analysis in neurons. TFII-I modulated neuronal SRF target gene expression, enhanced nerve fiber growth and modulated the shape of neuronal growth cones. Interestingly, TFII-I modulated two SRF cofactors, Elk-1 and MRTF-A, in opposite directions. TFII-I and Elk-1 co-expression enhanced SRF target gene abundance, SRF promoter binding and neurite growth, whereas TFII-I and MRTF-A resulted in opposite outcomes.In summary, we provide a first proteome wide analysis of SRF associated proteins. We characterized TFII-I as modulator of SRF activity by imposing differential impact on two SRF cofactors.

Filamin A interacts with the coactivator MKL1 to promote the activity of the transcription factor SRF and cell migration.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor (SRF) that promotes the expression of genes associated with cell proliferation, motility, adhesion, and differentiation-processes that also involve dynamic cytoskeletal changes in the cell. MKL1 is inactive when bound to monomeric globular actin (G-actin), but signals that activate the small guanosine triphosphatase RhoA cause actin polymerization and MKL1 dissociation from G-actin. We found a new mechanism of MKL1 activation that is mediated through its binding to filamin A (FLNA), a protein that binds filamentous actin (F-actin). The interaction of FLNA and MKL1 was required for the expression of MKL1 target genes in primary fibroblasts, melanoma, mammary and hepatocellular carcinoma cells. We identified the regions of interaction between MKL1 and FLNA, and cells expressing an MKL1 mutant that was unable to bind FLNA exhibited impaired cell migration and reduced expression of MKL1-SRF target genes. Induction and repression of MKL1-SRF target genes correlated with increased or decreased MKL1-FLNA interaction, respectively. Lysophosphatidic acid-induced RhoA activation in primary human fibroblasts promoted the association of endogenous MKL1 with FLNA, whereas exposure to an actin polymerization inhibitor dissociated MKL1 from FLNA and decreased MKL1-SRF target gene expression in melanoma cells. Thus, FLNA functions as a positive cellular transducer linking actin polymerization to MKL1-SRF activity, counteracting the known repressive complex of MKL1 and monomeric G-actin.

Critical role of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy.

To investigate the expression and function of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy. The expression of serum response factor, epithelial markers and mesenchymal markers was examined in podocytes or renal cortex tissues following high glucose. Serum response factor was upregulated by its plasmids and downregulated by CCG-1423 to investigate how it influenced podocyte epithelial-mesenchymal transition in diabetic nephropathy. Streptozotocin was used to generate diabetes mellitus in rats. In podocytes after high glucose treatment, serum response factor and mesenchymal markers increased, while epithelial markers declined. Similar changes were observed in vivo. Serum response factor overexpression in podocytes induced expression of Snail, an important transcription factor mediating epithelial-mesenchymal transition. Blockade of serum response factor reduced Snail induction, protected podocytes from epithelial-mesenchymal transition and ameliorated proteinuria. Together, increased serum response factor activity provokes podocytes' epithelial-mesenchymal transition and dysfunction in diabetic nephropathy. Targeting serum response factor by small-molecule inhibitor may be an attractive therapeutic strategy for diabetic nephropathy.

CMKLR1 and GPR1 mediate chemerin signaling through the RhoA/ROCK pathway

Chemerin is an adipose-derived hormone that regulates immunity and energy homesotasis. To date, all known chemerin functions have been attributed to activation of the G protein-coupled receptor chemokine-like receptor-1 (CMKLR1). Chemerin is also the only known ligand for a second receptor, G protein-coupled receptor-1 (GPR1), whose signaling and function remains unknown. This study investigated the in vitro signal transduction mechanisms of CMKLR1 and GPR1 using a panel of luciferase-reporters and pathway-specific inhibitors. Herein we report the novel finding that chemerin signals through a RhoA and rho-associated protein kinase (ROCK)-dependent pathway for activation of the transcriptional regulator serum-response factor (SRF). Despite similarities in RhoA/ROCK, Gαi/o, and MAPK signaling, we also demonstrate species-specific and receptor-dependent variations in GPR1 and CMKLR1 signaling and expression of the SRF target genes EGR1, FOS and VCL. Moreover, we demonstrate that signaling through p38, Gαi/o, RhoA, and ROCK is required for chemerin-mediated chemotaxis of L1.2 lymphocytes and AGS gastric adenocarcinoma cells. These results provide, to our knowledge, the first empirical evidence that GPR1 is a functional chemerin receptor and identify RhoA/SRF as a novel chemerin-signaling axis via both CMKLR1 and GPR1.

The systemic control of circadian gene expression.

The mammalian circadian timing system consists of a central pacemaker in the brain's suprachiasmatic nucleus (SCN) and subsidiary oscillators in nearly all body cells. The SCN clock, which is adjusted to geophysical time by the photoperiod, synchronizes peripheral clocks through a wide variety of systemic cues. The latter include signals depending on feeding cycles, glucocorticoid hormones, rhythmic blood-borne signals eliciting daily changes in actin dynamics and serum response factor (SRF) activity, and sensors of body temperature rhythms, such as heat shock transcription factors and the cold-inducible RNA-binding protein CIRP. To study these systemic signalling pathways, we designed and engineered a novel, highly photosensitive apparatus, dubbed RT-Biolumicorder. This device enables us to record circadian luciferase reporter gene expression in the liver and other organs of freely moving mice over months in real time. Owing to the multitude of systemic signalling pathway involved in the phase resetting of peripheral clocks the disruption of any particular one has only minor effects on the steady state phase of circadian gene expression in organs such as the liver. Nonetheless, the implication of specific pathways in the synchronization of clock gene expression can readily be assessed by monitoring the phase-shifting kinetics using the RT-Biolumicorder.

Abstract 4194: APC and DIAPH1 collaborate to suppress myelodysplastic syndrome (MDS) pathogenesis

Abstract Myelodysplastic syndromes (MDS) are characterized by complex cytogenetic abnormalities, including loss of all or part of the long arm of chromosome 5 (5q). Known 5q tumor suppressors include APC1 and EGR1. APC is associated with familial colon cancer where it functions to suppress canonical Wnt signaling. Egr1 is a transcription factor that governs expression of tumor suppressor genes ranging from the p53 family to PTEN1. Adjacent to EGR1 is the DIAPH1 gene for the Rho GTPase effector formin DIAPH1. Knockout of Diaph1, Egr, or Apc in mice creates a range of MDS phenotypes. Likewise, mice with the multiple intestinal neoplasia (min/Apcmin) allele develop anemia and MDS most similar to the Diaph1 knockout phenotype. DIAPH1 builds linear actin filaments and stabilizes microtubule dynamics to establish and maintain cell polarity during adhesion, migration, and asymmetric cell division when activated by Rho GTPases, such as RhoB. Combinatorial deficiencies in RhoB (also a known tumor susceptibility gene) with Diaph1 exacerbates the Diaph1 MDS phenotype but not the rate of pathogenesis. RhoB-activated DIAPHs also signal and activate new gene expression by the myocardin-related (MRTF) and Mcm10/Agamous deficiens/ [serum response factor (SRF)] (MADS)-box transcription factors. MRTF-MADS registers changes in actin dynamics downstream of DIAPH1 to activate EGR1 expression through multiple serum response elements (SREs; recognized by SRF) within the EGR1 promoter. DIAPH1 also collaborates with APC in cytoskeletal remodeling as both proteins physically associate with and stabilize microtubules. APC can also nucleate new actin filaments that become processively elongated by DIAPH1. We examined interactions between APC and DIAPH1 signaling to SRF and in suppressing MDS pathogenesis. First, we found that both DIAPH1 and APC activate SRF- regulated gene expression. Consistent with biochemical data showing that DIAPH1 elongates APC-nucleated filaments, activation of SRF by APC relies upon DIAPH1. We bred Apcmin mice to our Diaph1 knockout mice and found that the MDS phenotype is significantly accelerated in Apcmin/Diaph1 knockout mice when compared to either the Apcmin or Diaph1 mutant phenotype. Pathological defects (dysplasia and anemia in peripheral blood and myeloid organs (spleen/bone marrow) requires at least 200 days to develop in either single mutant; combined mutations create aberrant peripheral blood cell pathology by 100 days of age. Consistent with early disease onset, mortality is accelerated in Apcmin /Diaph1-deficient mice. These novel studies show that genetic defects in Diaph1 and Apc synergize to effectively mimic 5q- MDS. Further, the data supports the idea that RhoB - DIAPH1/APC and MRTF-MADS comprise a novel tumor suppression pathway that is essential for hematopoiesis. Apcmin/Diaph1-deficient mice may prove to be a useful pre-clinical model for the development of more effective theranostic tools to attack MDS. Citation Format: Julie D. Turner, Susan M. Kitchen-Goosen, Andrew M. Howard, Heather L. Schumacher, Arthur S. Alberts. APC and DIAPH1 collaborate to suppress myelodysplastic syndrome (MDS) pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2015-4194

The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression.

The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.

Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells.

Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells.

Actin-mediated gene expression depends on RhoA and Rac1 signaling in proximal tubular epithelial cells.

Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses.

Abstract 17543: DMPK E Disrupts Sarcomere Structure by Inhibition of SRF Transcriptional Activity

Background: Heart failure is associated with elongation of cardiomyocytes and loss of sarcomeres. Although the transcription factor myocyte enhancer factor-2 (MEF2) has an important role in this adverse remodeling, the mechanism underlying the structural changes of cardiomyocytes remains to be elucidated. In a screen for MEF2 target genes, we have recently identified myotonic dystrophy protein kinase (DMPK) as a potential mediator of adverse cardiomyocyte remodeling. However, it remains to be determined whether DMPK levels are increased in failing hearts and if DMPK is sufficient to induce structural remodeling of cardiomyocytes. Methods and Results: Since the DMPK gene is subject to extensive alternative splicing, we performed RT-PCR and QPCR analysis of known DMPK splice variants in hearts from mice subjected to transverse aortic constriction (TAC) or sham surgery. This demonstrated a 1.6 fold increase in the DMPK E isoform in failing mouse hearts compared to controls (P&lt;0.05). To test the role of this specific splice isoform, we generated adenoviruses expressing DMPK E or a kinase dead mutant DMPK E. Overexpression of wildtype DMPK E, but not of the kinase dead mutant, in cardiomyocytes resulted in severe loss of sarcomeric structure. Moreover, QPCR analysis showed a decrease in mRNA levels for several sarcomeric genes after overexpression of DMPK E. Since these genes are known targets of the transcription factor serum response factor (SRF) and DMPK is known to phosphorylate SRF, we tested the effect of DMPK E on SRF activity. Luciferase experiments demonstrated that DMPK E is an inhibitor of SRF transcriptional activity. Finally, immunostaining revealed a translocation of SRF from the nucleus to the cytosol upon DMPK E overexpression. Conclusion: Our data indicate that the expression of DMPK E is increased in heart failure. Moreover, increased expression of this DMPK splice variant results in a decrease in sarcomeric gene expression by translocation and inhibition of SRF. Together, these results assign a novel function to DMPK in adverse cardiomyocyte remodeling during heart failure development.

Serum response factor accelerates the high glucose-induced Epithelial-to-Mesenchymal Transition (EMT) via snail signaling in human peritoneal mesothelial cells.

BackgroundEpithelial-to-Mesenchymal Transition (EMT) induced by glucose in human peritoneal mesothelial cells (HPMCs) is a major cause of peritoneal membrane (PM) fibrosis and dysfunction.MethodsTo investigate serum response factor (SRF) impacts on EMT-derived fibrosis in PM, we isolated HPMCs from the effluents of patients with end-stage renal disease (ESRD) to analyze alterations during peritoneal dialysis (PD) and observe the response of PM to SRF in a rat model.ResultsOur results demonstrated the activation and translocation of SRF into the nuclei of HPMCs under extensive periods of PD. Accordingly, HPMCs lost their epithelial morphology with a decrease in E-cadherin expression and an increase in α-smooth muscle actin (α-SMA) expression, implying a transition in phenotype. PD with 4.25% glucose solution significantly induced SRF up-regulation and increased peritoneal thickness. In immortal HPMCs, high glucose (HG, 60 mmol/L) stimulated SRF overexpression in transformed fibroblastic HPMCs. SRF-siRNA preserved HPMC morphology, while transfection of SRF plasmid into HPMCs caused the opposite effects. Evidence from electrophoretic mobility shift, chromatin immunoprecipitation and reporter assays further supported that SRF transcriptionally regulated Snail, a potent inducer of EMT, by directly binding to its promoter.ConclusionsOur data suggested that activation of SRF/Snail pathway might contribute to the progressive PM fibrosis during PD.

Pak1 is required to maintain ventricular Ca²⁺ homeostasis and electrophysiological stability through SERCA2a regulation in mice.

Impaired sarcoplasmic reticular Ca(2+) uptake resulting from decreased sarcoplasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) expression or activity is a characteristic of heart failure with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and the activity as novel approaches to heart failure management. We here explore the role of Pak1 in maintaining ventricular Ca(2+) homeostasis and electrophysiological stability under both normal physiological and acute and chronic β-adrenergic stress conditions. Mice with a cardiomyocyte-specific Pak1 deletion (Pak1(cko)), but not controls (Pak1(f/f)), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute β-adrenergic or chronic β-adrenergic stress leading to hypertrophy, induced by isoproterenol. Isolated Pak1(cko) ventricular myocytes correspondingly showed aberrant cellular Ca(2+) homeostasis. Pak1(cko) hearts showed an associated impairment of SERCA2a function and downregulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demonstrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels after phenylephrine stress in cultured neonatal rat cardiomyocytes. This was abolished by the Pak1-knockdown in Ad-shPak1-infected neonatal rat cardiomyocytes and increased by constitutive overexpression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor, a transcriptional factor well known for its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a. These findings indicate that Pak1 is required to maintain ventricular Ca(2+) homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis.

Binding of the WASP/N-WASP-interacting protein WIP to actin regulates focal adhesion assembly and adhesion.

The actin cytoskeleton is essential for cell adhesion and migration, functions important for tumor invasion. In addition to binding N-WASP/WASP, WIP binds and stabilizes F-actin. WIP(-/-) fibroblasts were used to test the role of WIP in F-actin function. WIP(-/-) cells had defective focal adhesion (FA), stress fiber assembly, and adherence to substrates, functions that were restored by transduction of wild-type WIP. Protein and mRNA levels of several FA constituents regulated by the myocardin-related transcription factor (MRTF)–serum response factor (SRF) transcription factor complex were reduced in WIP(-/-) fibroblasts. The level of G-actin, which sequesters MRTF in the cytoplasm, was increased, and nuclear localization of MRTF-A and SRF was reduced, in WIP(-/-) fibroblasts. Transfection of an MRTF-A mutant that constitutively translocates to the nucleus or transfection of constitutively active SRF restored FA and stress fiber assembly. Fibroblasts from knock-in mice expressing a WIP mutant that fails to bind actin phenocopied WIP(-/-) fibroblasts. Thus, WIP is a novel regulator of FA assembly and cell adhesion.