Serum Response Factor Activity Research Articles

The Purα/Purβ Single‐Strand DNA‐Binding Proteins Attenuate Smooth‐Muscle Actin Gene Transactivation in Myofibroblasts

Expression of smooth muscle alpha-actin (SMαA) is essential for myofibroblast-mediated wound contraction following tissue injury. The Pur α/β and YB-1 transcriptional repressors govern the DNA-binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre-existing SRF complexes with SMαA core promoter DNA, whereas Pur β was more effective in disrupting SRF-DNA interaction. Pur proteins were less efficient competitors of pre-existing, core-promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFβ1-activated myofibroblasts. TGFβ1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF-A/Pur β complex during early phase myofibroblast differentiation. Pur β was replaced by Pur α in the pSmad3/MRTF-A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFβ1-activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling.

SRF Phosphorylation by Glycogen Synthase Kinase-3 Promotes Axon Growth in Hippocampal Neurons

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

Antifibrotic Effects of Noscapine through Activation of Prostaglandin E2 Receptors and Protein Kinase A

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-β-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-β-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

Association between non-coding polymorphisms of HOPX gene and syncope in hypertrophic cardiomyopathy.

Homeodomain Only Protein X (HOPX) is an unusual homeodomain protein which regulates Serum Response Factor (SRF) dependent gene expression. Due to the regulatory role of HOPX on SRF activity and the regulatory role of SRF on cardiac hypertrophy, we aimed to investigate the relationship between HOPX gene variations and hypertrophic cardiomyopathy (HCM). In this study, designed as a case-control study, we analyzed coding and flanking non-coding regions of the HOPX gene through 67 patients with HCM and 31 healty subjects. Certain regions of the gene were investigated by Single Stranded Conformation Polymorphism (SSCP) and Restriction Fragment Length Polymorphism (RFLP). Statistical analyses of genotypes and their relationship with clinical parameters were performed by chi-square, Kruskal-Wallis and the Fisher's exact test. In 5' Untranslated Region (UTR) and intronic region of the HOPX gene, we found a C>T substitution and an 8-bp insertion/deletion (In/Del) polymorphism, respectively. These two polymorphisms seemed to constitute an haplotype. While the frequency of homozygous genotypes of In/Del and C/T polymorphisms were found significantly lower in the patients with syncope (p=0.014 and p=0.017, respectively), frequency of their heterozygous genotypes were found significantly higher in the patients with syncope (p=0.048 and p=0.030, respectively). CONCLUSİON: Though there was not found any mutation in coding sequence of HOPX gene, two non-coding polymorphisms were found related to syncope in HCM patients. While homozygous status of these polymorphisms was found to be protective against the syncope, their heterozygous status seemed to be a risk factor for syncope in HCM patients. Our results suggest that HOPX may contribute to pathogenesis or manifestation of HCM as a modifier gene.

Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-β in cardiac myocytes

The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-β (transforming growth factor-β) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-β. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased βMHC (β-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-β, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.

MiR-663 and the miRaculous Vascular Smooth Muscle Phenotypic Switch

Cardiovascular diseases, such as ischemic heart disease and stroke, represent the number one cause of death worldwide.1 In most cases, atherosclerosis and hypertension constitute the underlying causes for cardiovascular disease because it leads to arterial occlusion and impaired cardiac function, respectively. Therapeutic compensation of atherosclerotic artery occlusion by bypass grafting, angioplasty, or stenting bears the risk of intimal hyperplasia and subsequent restenosis.2 In general, a prerequisite for any pathological and physiological vascular remodeling process is the phenotypic switch of vascular smooth muscle cells (VSMCs).3 Even in adult blood vessels, VSMCs retain a remarkable plasticity that is essential for any changes in the vessel wall architecture. Contractile VSMCs representing the majority of VSMCs in healthy vessels guarantee maintenance of vascular tone and thereby the systemic blood pressure and blood flow by controlling the blood vessel diameter. As outlined in the Figure, a plethora of humoral factors, such as platelet-derived growth factor-BB and biomechanical stimuli, especially chronic changes in blood flow and wall stress, is capable to trigger the phenotypic switch of VSMCs from a quiescent, contractile differentiated state to a synthetic, proliferative, and dedifferentiated state.3,4 Figure. The effect of microRNA (miR)-633 on vascular homeostasis and remodeling. Major stimuli, transcription factors, and miRs that are involved in the differentiation and phenotypic switch of vascular smooth muscle cells (VSMCs) are depicted. miR-143/miR-145 promotes the contractile state by inhibiting suppressors of myocardin/serum response factor (SRF) activity (eg, ETS domain-containing protein-1 [Elk-1] and Kruppel-like factor [KLF] 4/5). Interestingly, myocardin activity seems to be regulated by an intrinsic loop because it stabilizes the expression of miR-145, whereas miR-143 seems to inhibit myocardin expression. miR-633 is a novel player with a dual effect on the VSMC phenotype not only by attenuating proliferation, migration, and proteolytic activity, but also by limiting their …

Abstract 066: MEF2-induced Loss of Sarcomeres is Mediated by an Alternative Splice Variant of DMPK

The pathology of heart failure is characterized by poorly contracting and dilated ventricles. Although this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres, the mechanism underlying these changes in cardiomyocyte structure remains to be elucidated. We have previously identified the transcription factor myocyte enhancer factor-2 (MEF2) as important trigger for adverse cardiomyocyte remodeling. Here, we use microarray analysis and gain- and loss- of function approaches to identify MEF2 target genes involved in structural remodeling of the cardiomyocyte. Isolated neonatal rat cardiomyocytes infected with adenoviruses expressing MEF2 underwent cellular elongation associated with loss of sarcomeric structure. Microarray analysis revealed myotonic dystrophy protein kinase (DMPK) as MEF2 target gene, which we verified by chromatin immunoprecipitation experiments. siRNA mediated knockdown of DMPK prevented MEF2-induced cardiomyocyte elongation and loss of sarcomeres. Interestingly, RT-PCR analysis of known DMPK splice variants demonstrated a relative increase of the DMPK E isoform in failing mouse hearts. 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, quantitative PCR analysis showed a decrease in mRNA levels for several sarcomeric genes after overexpression of DMPK E. These genes are known targets of the transcription factor serum response factor (SRF) and DMPK is known to phosphorylate SRF. Therefore, we tested the effect of DMPK on SRF activity in luciferase experiments, which demonstrated that DMPK E is an inhibitor of SRF transcriptional activity. Our data indicate that MEF2 induces loss of sarcomeres, which is mediated by at least one specific splice variant of DMPK. Moreover, increased expression of this DMPK splice variant results in a decrease in sarcomeric gene expression, which possibly involves inhibition of SRF transcriptional activity. Together, these results assign a novel function to MEF2 and DMPK in adverse cardiomyocyte remodeling during heart failure development.

Acrolein-induced inflammatory signaling in vascular smooth muscle cells requires activation of serum response factor (SRF) and NFκB

Modulation of inflammatory signaling has been elucidated in several disease models. Acrolein, an environmental pollutant, has been linked to diseases such as atherosclerosis and to the inflammatory process involving nuclear factor κB (NFκB). Serum response factor (SRF), a transcription factor, regulates cell development, differentiation and proliferation through signaling molecules such as extracellular signal-regulated kinase 1/2 (ERK1/2) and CD36. We hypothesized that acrolein toxicity involves SRF in the process of activating NFκB and may involve CD36/ERK1/2. Vascular smooth muscle cells (VSMCs) were exposed to acrolein (0.5 μg/mL) in the presence or absence of 10 nM QNZ (NFκB inhibitor), 300 nM CCG1423 (SRF inhibitor) and 50 μM PD98059 (ERK1/2 inhibitor). Protein and RNA were isolated. Changes in expression were determined by Western blot and polymerase chain reaction (PCR) array. Subtoxic doses of acrolein increased ERK1/2, SRF and NFκB protein expression, whereas CD36 expression was unchanged. Increase in NFκB expression was accompanied by an increase in activity. ERK1/2 inhibition only blunted SRF expression. SRF inhibition blunted NFκB expression but not that of ERK1/2. CD36 expression was unchanged in the presence of either inhibitor. PCR array analysis indicated up-regulation of nine genes (>4- to 50-fold) and down-regulation of six genes (>4- to 12-fold) involved in inflammatory signaling. We propose that SRF is required in acrolein activation of NFκB and is ERK1/2 dependent.

SRF selectively controls tip cell invasive behavior in angiogenesis

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

SRF selectively controls tip cell invasive behavior in angiogenesis

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Control of Myofibroblast Differentiation by Microtubule Dynamics through a Regulated Localization of mDia2

Myofibroblast differentiation plays a critical role in wound healing and in the pathogenesis of fibrosis. We have previously shown that myofibroblast differentiation is mediated by the activity of serum response factor (SRF), which is tightly controlled by the actin polymerization state. In this study, we investigated the role of the microtubule cytoskeleton in modulating myofibroblast phenotype. Treatment of human lung fibroblasts with the microtubule-destabilizing agent, colchicine, resulted in a formation of numerous stress fibers and expression of myofibroblast differentiation marker proteins. These effects of colchicine were independent of Smad signaling but were mediated by Rho signaling and SRF, as they were attenuated by the Rho kinase inhibitor, Y27632, or by the SRF inhibitor, CCG-1423. TGF-β-induced myofibroblast differentiation was not accompanied by gross changes in the microtubule polymerization state. However, microtubule stabilization by paclitaxel attenuated TGF-β-induced myofibroblast differentiation. Paclitaxel had no effect on TGF-β-induced Smad activation and Smad-dependent gene transcription but inhibited actin polymerization, nuclear accumulation of megakaryoblastic leukemia-1 protein, and SRF activation. The microtubule-associated formin, mDIA2, localized to actin stress fibers upon treatment with TGF-β, and paclitaxel prevented this localization. Treatment with the formin inhibitor, SMI formin homology 2 domain, inhibited stress fiber formation and myofibroblast differentiation induced by TGF-β, without affecting Smad-phosphorylation or microtubule polymerization. Together, these data suggest that (a) TGF-β promotes association of mDia2 with actin stress fibers, which further drives stress fiber formation and myofibroblast differentiation, and (b) microtubule polymerization state controls myofibroblast differentiation through the regulation of mDia2 localization.

Androgen-responsive Serum Response Factor target genes regulate prostate cancer cell migration

Progression of prostate cancer (CaP) relies on androgen receptor (AR) signaling, but AR-dependent events that underlie the lethal phenotype remain unknown. Recently, an indirect mechanism of androgen action in which effects of AR on CaP cells are mediated by Serum Response Factor (SRF) has been identified. This is the first mode of androgen action to be associated with aggressive CaP and disease recurrence. The manner in which androgen-responsive SRF activity controls aggressive CaP cell behavior is unknown. Here, the contribution of two representative SRF effector genes that are underexpressed, calponin 2 (CNN2), or overexpressed, sidekick-homolog 1 (SDK1), in clinical CaP specimens is studied. AR- and SRF- dependency of CNN2 and SDK1 expression was verified using synthetic and natural androgens, antiandrogens, and small interfering RNAs targeting AR or SRF, and evaluating the kinetics of androgen induction and SRF binding to endogenously and exogenously expressed regulatory gene regions in AR-positive CaP model systems that mimic the transition from androgen-stimulated to castration-recurrent disease. Small interfering RNA-mediated deregulation of CNN2 or SDK1 expression did not affect CaP cell proliferation or apoptosis but had marked effects on CaP cell morphology and actin cytoskeleton organization. Loss of CNN2 induced cellular protrusions and increased CaP cell migration, whereas silencing of SDK1 led to cell rounding and blunted CaP cell migration. Changes in cell migration did not involve epithelial-mesenchymal transition but correlated with altered β1-integrin expression. Taken together, individual androgen-responsive SRF target genes affect CaP cell behavior by modulating cell migration, which may have implications for therapeutic intervention downstream of AR and SRF.

Fluorescent protein and epitope tagging within the helical domain of G12 subfamily proteins

Epitope and fluorescent tagging of trimeric G protein α subunits poses challenges due to potential disruption of functional regions, including the receptor‐interacting N‐ and C‐termini. In previous work, we inserted green fluorescent protein (GFP) and a Myc tag into a specific loop within the helical domain of Gα12, flanking each adduct with the sextet ser‐gly‐gly‐gly‐gly‐ser, and showed these tagged Gα12 variants to retain interaction with downstream target proteins, stimulate serum response factor, and exhibit conformational activation. Recently, we employed a similar approach for tagging Gα13, which shares 67% identity with Gα12 but has significant sequence divergence in the helical domain. Using molecular visualization software (PyMol) to superimpose the crystal structures of Gα12 and Gα13, we identified Gα13 residues corresponding to the site of tag insertion in Gα12, and made key substitutions of Gα12‐specific residues into Gα13. GFP‐tagged Gα13 is detectable in transfected cells by epifluorescence microscopy and immunoblotting, and shows robust activation of serum response factor. We are utilizing tagged Gα13 in binding assays to determine whether mutations in Gα13 disrupt binding to effectors. Furthermore, these tagged constructs are facilitating side‐by‐side comparisons of Gα12 and Gα13 in their relative affinity for common downstream effector proteins, such as Rho‐specific guanine nucleotide exchange factors, cadherins, and other targets. We acknowledge support from the North Carolina Biotechnology Center (DNB) and the Lineberger Comprehensive Cancer Center (TEM).

The zinc-binding region of IL-2 inducible T cell kinase (Itk) is required for interaction with Gα13 and activation of serum response factor

Tec family kinases play critical roles in the activation of immune cells. In particular, Itk is important for the activation of T cells via the T cell Receptor (TcR), however, molecules that cooperate with Itk to activate downstream targets remain little explored. Here we show that Itk interacts with the heterotrimeric G-protein α subunit Gα13 during TcR triggering. This interaction requires membrane localization of both partners, and is partially dependent on GDP- and GTP-bound states of Gα13. Furthermore, we find that Itk interacts with Gα13 via the zinc binding regions within its Tec homology domain. The interaction between Itk and Gα13 also results in tyrosine phosphorylation of Gα13, however this is not required for the interaction. Itk enhances Gα13 mediated activation of serum response factor (SRF) transcriptional activity dependent on its ability to interact with Gα13, but its kinase activity is not required to enhance SRF activity. These data reveal a new pathway regulated by Itk in cells, and suggest cross talk between Itk and G-protein signaling downstream of the TcR.

Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice

Members of the CUG-BP, Elav-like family (CELF) regulate alternative splicing in the heart. In MHC-CELFΔ transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an α-myosin heavy chain promoter. MHC-CELFΔ mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELFΔ mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF) network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELFΔ mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELFΔ males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor.

Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation

AbstractHow components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization. We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation.

Activation of PPAR-α induces cell cycle arrest and inhibits transforming growth factor-β1 induction of smooth muscle cell phenotype in 10T1/2 mesenchymal cells

Transforming growth factor-β1 (TGF-β1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-β1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.

Neuregulin1 signaling targets SRF and CREB and activates the muscle spindle-specific gene Egr3 through a composite SRF–CREB-binding site

Muscle spindles are sensory receptors embedded within muscle that detect changes in muscle length. Each spindle is composed of specialized muscle fibers, known as intrafusal muscle fibers, along with the endings of axons from sensory neurons that innervate these muscle fibers. Formation of muscle spindles requires neuregulin1 (NRG1), which is released by sensory axons, activating ErbB receptors in muscle cells that are contacted. In muscle cells, the transcription factor Egr3 is transcriptionally induced by NRG1, which in turn activates various target genes involved in forming the intrafusal fibers of muscle spindles. The signaling relay within the NRG1–ErbB pathway that acts to induce Egr3 is presumably critical for muscle spindle formation but for the most part has not been determined. In the current studies, we examined, using cultured muscle cells, transcriptional regulatory mechanisms by which Egr3 responds to NRG1. We identified a composite regulatory element for the Egr3 gene, consisting adjacent sites that bind cAMP response element binding protein (CREB) and serum response factor (SRF), with a role in NRG1 responsiveness. The SRF element also influences Egr3 basal expression in unstimulated myotubes, and in the absence of the SRF element, the CREB element influences basal expression. We show that NRG1 signaling, to target SRF, acts on the SRF coactivators myocardian-related transcription factor (MRTF)-A and MRTF-B, which are known to activate SRF-mediated transcription, by stimulating their translocation from the cytoplasm to the nucleus. CREB is phosphorylated, which is known to contribute to its activation, in response to NRG1. These results suggest that NRG1 induces expression of the muscle spindle-specific gene Egr3 by stimulating the transcriptional activity of CREB and SRF.

Dilated Cardiomyopathy-Associated <i>FHOD3</i> Variant Impairs the Ability to Induce Activation of Transcription Factor Serum Response Factor

Dilated cardiomyopathy (DCM) is characterized by a dilated left ventricular cavity with systolic dysfunction manifested by heart failure. It has been revealed that mutations in genes for cytoskeleton or sarcomere proteins cause DCM. However, the disease-causing mutations can be found only in far less than half of patients with a family history, indicating that there should be other disease genes for DCM. Formin homology 2 domain containing 3 (FHOD3) is a sarcomeric protein expressed in the heart that plays an essential role in sarcomere organization during myofibrillogenesis. The purpose of this study was to explore a possible novel disease gene for DCM. We analyzed 48 Japanese familial DCM patients for mutations in FHOD3, and a missense variant, Tyr1249Asn, which was predicted to modify the 3D structure and damage protein function, was found in a case with adult-onset DCM. Functional studies revealed that the DCM-associated mutation significantly reduced the ability to induce actin dynamics-dependent activation of serum response factor, although no remarkable change in the cellular localization was induced in neonatal rat cardiomyocytes transfected with a mutant construct of FHOD3. The DCM-associated FHOD3 variant may cause DCM by interfering with actin filament assembly.

Blood-Borne Circadian Signal Stimulates Daily Oscillations in Actin Dynamics and SRF Activity

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.