SRF Activity Research Articles

Influence of different fluid types on the development of fibrosis and atrophy in patients with nAMD

Aims/Purpose: Subretinal fibrosis and macular atrophy often associated with vision loss are regularly seen in patients with long‐term neovascular age‐related macular degeneration (nAMD). Apart from the influence of fluid distribution on visual acuity its effect on the development of fibrotic and atrophic alterations could give important insight for better understanding the pathogenesis of end‐stages and might influence treatment decisions. Hence, we investigated the influence of fluid distribution on fibrosis and atrophy using AI‐supported fluid quantification.Methods: Sub‐ and intra‐retinal fluid (SRF and IRF) and pigment epithelium detachment (PED) were quantified on optical coherence tomography (OCT) volumetric scans using the Vienna Fluid Monitor, Version 2 (RetInSight, Vienna, Austria) at all timepoints until the appearance of atrophy or fibrosis evaluated by the Vienna Reading Center (VRC). In total, 569 treatment naïve nAMD patients were included in the analysis. A disease activity‐dependent treat&extend‐based anti‐VEGF treatment regimen was used. A logistic regression model for fibrosis/atrophy was created.Results: Fluid distribution and dynamics showed a significant influence on the development of fibrosis/atrophy. If the standard deviation of IRF increased by 1 unit (100 nL; p = 0.006) odds to develop fibrosis increased by 4.4. Hence, patients with little IRF at baseline that increased rapidly during the following visits were likely to develop fibrosis. However, atrophy did not seem to be influenced by IRF significantly, whereas PED and SRF showed a significant influence on the development of MA.Conclusions: Automated assessment of fluid type and volume using artificial intelligence allows an objective and precise assessment of the activity of IRF, SRF and PED and therefore its influence on the development of fibrosis or atrophy. AI‐based fluid monitoring offers guidance to improve anatomical and visual outcomes in the long‐term management of nAMD in clinical routine.

Synovial fluid from end-stage osteoarthritis induces proliferation and fibrosis of articular chondrocytes via MAPK and RhoGTPase signaling

Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.

Fibrosis Without Myofibroblasts Revealed by Genetic Analysis of PDGFRα

The appearance of myofibroblasts is hallmark of fibrosis and tissue repair. Myofibroblasts are stromal cells that transition from fibroblastic progenitors by acquiring new contractile function. Platelet-derived growth factor (PDGF) promotes fibrosis and supports tissue repair, but the relationship between PDGF and the myofibroblast phenotype is unclear. Using mice with PDGF receptor α (PDGFRα) gene activation or deletion and mutation-linked lineage tracing, we examine fibroblastic progenitor cell fate during spontaneous fibrosis and wound-induced fibrosis. Unexpectedly, elevated PDGFRα signaling generates fibrosis while inhibiting myofibroblast formation. Non-canonical TGFβ-p38 signaling is blocked by PDGFRα activity, thereby inhibiting myofibroblasts. However, deletion of PDGFRα also inhibits myofibroblasts by reducing SRF activity, which leads to defective wound repair. Our findings suggest a scenario where PDGFRα initially expands the progenitor population for tissue repair and later suppression of PDGFRα in activated progenitors permits their transition to myofibroblast.

Differential Induction of SOCS Isoforms by Leishmania donovani Impairs Macrophage-T Cell Cross-Talk and Host Defense.

Immune evasion strategies adopted by Leishmania donovani involve the exploitation of suppressor of cytokine signaling (SOCS) proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow-derived macrophages (BMMфs) and CD4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macrophages and T cells, respectively, whereas the SOCS1 level remains consistently high in BMMфs and SOCS3 expression is pronounced and long lasting in T cells. Consequently, this inhibits STAT1-mediated IL-12 induction in macrophages & STAT4-mediated IFN-γ synthesis in T cells. Mechanistically, PI3K/Akt-mediated SRF activation promotes nuclear translocation and binding of Egr2 to SOCS1 promoter for its early induction in infected BMMфs. Additionally, L. donovani activates IDO/kynurenine/AHR signaling in BMMфs to maintain prolonged SOCS1 expression. Later, PGE2, secreted from infected BMMфs induces cAMP-PKA pathway by binding to the EP2/EP4 receptor of CD4+ T cells, leading to SP1, CREB, and GATA1 activation and SOCS3 expression. Small interfering RNA-mediated silencing of SOCS1 and SOCS3 in macrophage and T cells, respectively, restored IL-12 and IFN-γ cytokine levels and BMMф-T cell interaction. Vivo morpholino-mediated silencing of SOCS1 and SOCS3 resulted in protective cytokine responses, thereby reducing organ parasite burden significantly in L. donovani-infected BALB/c mice. Collectively, our results imply that L. donovani orchestrates different SOCS isoforms to impair macrophage-T cell cross-talk and preserve its own niche.

Formin Links Erythroid Cytoskeleton to Organelle Clearance through Escrt-III Complex during Reticulocyte Maturation

The final stages of mammalian terminal erythropoiesis involve cell cycle exit of orthochromatic erythroblast, enucleation of the condensed nucleus, and organelle clearance of the nascent reticulocytes. Although many critical factors in these processes have been discovered over the past years, several key questions remain unanswered. For example, what are the factors that regulate the exit of the last mitosis of erythroblast for enucleation? How does the nascent reticulocyte separate from the extruded nucleus? What are the signals involved in regulating the clearance of organelles in reticulocyte? Answers to these questions with mechanistic insights are important not only for our understanding of the basic biology of terminal erythropoiesis and pathophysiology of many red cell-related diseases, but also to provide clues for efficient strategies for in vitro or ex vivo generation of red blood cells in transfusion medicine. Our work on formin family proteins, enzymes involved in linear actin filament polymerization, in erythropoiesis may shed light on the clues to these questions. We show in our published work that mDia2, one of the diaphanous-related formins, plays critical roles in enucleation and cytokinesis of erythroblasts. However, the mechanism of how mDia2 regulates these processes is unclear. In this study, we used mDia2 hematopoietic-specific knockout mouse model and revealed that mDia2 controls the motility of the nascent reticulocyte that is required for the detachment of the pyknotic nucleus. Reticulocytes in mDia2 deficient mice are rigid with extended spectrin chains, possibly due to disrupted actin protofilaments. Indeed, a stochastic optical reconstruction (STORM) high resolution microscopy analysis revealed that actin protofilaments were completely disrupted with loss of mDia2 in the reticulocytes. Using immuno-gold stain and electron microscopy, we further found that mDia2 localized at the junctional complex, confirming its critical role in the polymerization of actin protofilaments and maintenance of erythroid cytoskeleton. In addition to the cytoskeleton defects, reticulocytes from mDia2 deficient mice also showed enlarged volume with many organelles failed to be eliminated. Flow cytometry analyses showed that several membrane proteins destined to be downregulated, as well as mitochondria and lysosome markers, remained high in mDia2 deficient reticulocytes. We also performed a tandem mass tagging (TMT) mass spectrometry, which revealed numerous chromatin-associated proteins that failed to be downregulated. Together, these results demonstrated an important role of mDia2 in the reticulocyte maturation. The erythroid phenotypes in mDia2 deficient mice, including failure of cytokinesis and organelle clearance, prompted us to investigate whether there are any defects in ESCRT complexes that are cellular components essential for these processes. Indeed, several ESCRT III complex and associated proteins, including Chmp5, Vta1, and Usp8, were significantly downregulated. mDia2 is known to function though actin polymerization to influence the transcriptional activity of SRF. We found that Chmp5 was a novel target of SRF through ChIP assay. Transplantation of mDia2 knockout c-kit positive progenitors transduced with Chmp5 into the lethally irradiated WT recipient mice dramatically reduced the percentages of bi-nuclear erythroblasts and reverted anemia. Consistent with phenotypes of Chmp5 knockout cells where increased late endosome and lysosome are commonly found, markers for late stage endosome and lysosome were significantly increased in mDia2 deficient erythroblasts and reticulocytes. These data support that mDia2 regulates endosome/MVB and lysosome discharge through Chmp5 during reticulocyte maturation. More importantly, overexpression of Chmp5 largely rescued the defects in lysosome and mitochondria clearance in mDia2 deficient reticulocytes. Our study reveals mDia2 formin as a master regulator of the late stage terminal erythropoiesis in the maintenance of erythroid cytoskeleton and organelle clearance. The novel mDia2-SRF-ESCRT III complex pathway provides the first signaling axis that connects erythroid cytoskeleton to reticulocyte maturation, which may open a new field in signaling networks that modulate enucleation to reticulocyte formation. Disclosures Ji: Longbiopharma: Consultancy.

Regulation of MRTF-A by JMY via a nucleation-independent mechanism

BackgroundMRTF-A (myocardin-related transcription factor A) is a coactivator for SRF-mediated gene expression. The activity of MRTF-A is critically dependent on the dissociation of G-actin from N-terminal RPEL motifs. MRTF-SRF induction often correlates with enhanced polymerization of F-actin. Here we investigate MRTF regulation by the multifunctional JMY protein, which contains three WASP/verprolin homology 2 (WH2/V) domains and facilitates Arp2/3-dependent and -independent actin nucleation.MethodsCo-immunoprecipitation experiments, immunofluorescence and luciferase reporter assays were combined with selective inhibitors to investigate the effect of JMY and its domains on MRTF-A in NIH 3 T3 mouse fibroblasts.ResultsJMY induced MRTF-A transcriptional activity and enhanced its nuclear translocation. Unexpectedly, MRTF-A was hyperactivated when the Arp2/3-recruiting CA region of JMY was deleted or mutated, suggesting an autoinhibitory mechanism for full-length JMY. Moreover, isolated WH2/V domains which are unable to nucleate actin were sufficient for nuclear accumulation and SRF activation. Recombinant WH2/V regions of JMY biochemically competed with MRTF-A for actin binding. Activation of MRTF-A by JMY was unaffected by Arp3 knockdown, by an Arp2/3 inhibitor, and by latrunculin which disassembles cellular F-actin. Restriction of JMY to the nucleus abrogated its MRTF-A activation. Finally, JMY RNAi reduced basal and stimulated transcriptional activation via MRTF-A.ConclusionsOur results suggest that JMY activates MRTF-SRF independently of F-actin via WH2/V-mediated competition with the RPEL region for G-actin binding in the cytoplasm. Furthermore, the C-terminal region facilitates an autoinhibitory effect on full-length JMY, possibly by intramolecular folding.

MRTFA augments megakaryocyte maturation by enhancing the SRF regulatory axis

AbstractSerum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]6GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFAOE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])–induced HEL cells, with and without MRTFAOE. We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFAOE, resulting in an upregulation of 25% of genes. MRTFAOE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFAOE lack ETS binding motifs, and MRTFAOE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP–polymerase chain reaction using HEL cells and primary human CD34+ cell–derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.

Abstract 1106: A synthetic pre-metastatic niche mimic alters the primary tumor and tumor microenvironment

Abstract Background: Immune cells at the primary tumor (PT) and pre-metastatic niche (PMN) sites are critical to metastasis progression. Recently, synthetic biomaterial scaffolds used as PMN mimics were shown to capture both immune and metastatic tumor cells in vivo (1-3). The redirection of tumor cells toward the implant also reduces tumor burden and provides a survival benefit in orthotopic breast cancer mouse models (4). Given the scaffold reduces tumor burden, we hypothesized the scaffold modulates PT and immune cell phenotypes to generate an invasion-suppressive tumor microenvironment (TME). Methods: Female NOD scid gamma (NSG) mice inoculated with tdTomato+ MDA-MB-231BR cells in the mammary fat were used as orthotopic human breast cancer models. Poly(lactide-co-glycolide) scaffolds were implanted in the intraperitoneal fat pad 7 days post-tumor inoculation (3). Mock surgery mice did not receive an implant. After 28 days, mock vs. scaffold PTs were harvested, digested, and live-cell sorted to obtain tdTomato+ tumor cells and CD45+ immune cells. The PT cell transcriptome was analyzed using RNAseq and GO analysis was performed in Metascape. CD45+ immune cells were cultured to obtain conditioned media (CM) for secretomics analysis, in vitro tumor cell invasion assays, and transcription factor activity arrays (2). Immune cells, specifically tumor associated macrophages (TAMs) (5), were characterized using flow cytometry and RT-PCR. Results: RNAseq analysis identified 892 differentially expressed genes in tumor cells in response to the scaffold implant, and Metascape GO analysis revealed signaling pathways relevant to invasion. The scaffold immune-CM decreased tumor cell invasion more than two-fold relative to mock CM. Secretomics analysis showed an increase in the pan-metastasis inhibitor decorin and a decrease in invasion-promoting CCL2 in the scaffold immune cell CM relative to mock CM. Reduced NFkB, SRF, and RAR transcription factor activity in tumor cells treated with scaffold CM relative to mock CM indicate immune secreted factors contribute to the invasion-suppressive scaffold-influenced TME. Finally, analysis of PT immune cells identified a recruited TAM population whose transcriptomic profile may contribute to the invasion-suppressive TME in scaffold bearing mice. Discussion: We demonstrate that implanted scaffolds can distally modulate secretomic and transcriptional profiles of immune cells in the TME and may reduce PT cell invasion. Our work suggests scaffolds have an active role in modulating tumor burden and may provide a foundation for developing an effective implantable therapeutic tool.

MicroRNA miR-301a is a novel cardiac regulator of Cofilin-2.

Calsarcin-1 deficient mice develop dilated cardiomyopathy (DCM) phenotype in pure C57BL/6 genetic background (Cs1-ko) despite severe contractile dysfunction and robust activation of fetal gene program. Here we performed a microRNA microarray to identify the molecular causes of this cardiac phenotype that revealed the dysregulation of several microRNAs including miR-301a, which was highly downregulated in Cs1-ko mice compared to the wild-type littermates. Cofilin-2 (Cfl2) was identified as one of the potential targets of miR-301a using prediction databases, which we validated by luciferase assay and mutation of predicted binding sites. Furthermore, expression of miR-301a contrastingly regulated Cfl2 expression levels in neonatal rat ventricular cardiomyocytes (NRVCM). Along these lines, Cfl2 was significantly upregulated in Cs1-ko mice, indicating the physiological association between miR-301a and Cfl2 in vivo. Mechanistically, we found that Cfl2 activated serum response factor response element (SRF-RE) driven luciferase activity in neonatal rat cardiomyocytes and in C2C12 cells. Similarly, knockdown of miR301a activated, whereas, its overexpression inhibited the SRF-RE driven luciferase activity, further strengthening physiological interaction between miR-301a and Cfl2. Interestingly, the expression of SRF and its target genes was strikingly increased in Cs1-ko suggesting a possible in vivo correlation between expression levels of Cfl2/miR-301a and SRF activation, which needs to be independently validated. In summary, our data demonstrates that miR-301a regulates Cofilin-2 in vitro in NRVCM, and in vivo in Cs1-ko mice. Our findings provide an additional and important layer of Cfl2 regulation, which we believe has an extended role in cardiac signal transduction and dilated cardiomyopathy presumably due to the reported involvement of Cfl2 in these mechanisms.

Abstract 19449: Upregulation of Cofilin-2 in Mouse Models Of DCM - Implications for SRF Signaling

The dilated cardiomyopathy (DCM) is a major cause for heart failure. A causative genetic background is suspected in up to 50% of all DCM cases. However, the underlying molecular pathways remain incompletely understood. Here we analyze a calsarcin-1 deficient (CS1ko) mouse line as a model for DCM. These mice showed strong upregulation of fetal genes (e.g., Nppa, Nppb ) and the calcineurin-dependent gene Rcan1.4. Interestingly, we could not detect any signes of hypertrophy. In addition, there was no increase of fibrosis in CS1ko mice compared to wildtype controls. A screening experiment revealed upregulation of Cofilin2 (Cfl2), an actin depolymerisation factor and member of the ADF/Cofilin family, in Cs1ko mice (2.6-fold, p<0.002), whereas its inactive, phosphorylated form (Ser3) remained unchanged. We also found increased amounts of Cfl2 in MLP-/- (1,8-fold p<0.05) and Calcineurin TG mice (2.7-fold p<0.01) suggesting a general role for Cfl2 in DCM. As Cfl2 is a known regulator of actin dynamics we investigated the role of Cfl2 in SRF signaling. Using an SRF-responsive luciferase reporter, overexpression of Cfl2 together with RhoA as activator of SRF signaling lead to an increase of SRF activity in C2C12 myoblasts (14-fold vs. 5.8-fold with RhoA alone, p<0.001). SiRNA-mediated knockdown of Cfl2 yielded opposite effects (0.96-fold vs. 4.6-fold, p<0.001). In the heart Cfl2 is predominantly expressed in cardiac myocytes as compared to fibroblasts. Overexpression of Cfl2 in cardiomyocytes (NRVCM) revealed an increase of Nppa and Nppb , which could be further increased by co-stimulation with phenylephrine (PE). In contrast to these findings, cell size analysis revealed a Cfl2-mediated decrease in cell size with and without PE, while knockdown of Cfl2 did not influence cellular hypertrophy. Taken together, our data imply a role for Cfl2 in the pathogenesis of DCM. Cs-1ko mice show a dilated phenotype, strong upregulation of the fetal gene program without a change in cell size. This correlates with the findings of overexpression of Cfl2 in NRVCM. Furthermore, increased Cfl2 levels induce SRF activity with potential influence on cardiomyocyte function. Further analyses are required to delineate the involved molecular pathways.

Formin-like2 regulates Rho/ROCK pathway to promote actin assembly and cell invasion of colorectal cancer.

Formin-like2 (FMNL2) is a member of the diaphanous-related formins family, which act as effectors and upstream modulators of Rho GTPases signaling and control the actin-dependent processes, such as cell motility or invasion. FMNL2 has been identified as promoting the motility and metastasis in colorectal carcinoma (CRC). However, whether FMNL2 regulates Rho signaling to promote cancer cell invasion remains unclear. In this study, we demonstrated an essential role for FMNL2 in the activations of Rho/ROCK pathway, SRF transcription or actin assembly, and subsequent CRC cell invasion. FMNL2 could activate Rho/ROCK pathway, and required ROCK to promote CRC cell invasion. Moreover, FMNL2 promoted the formation of filopodia and stress fiber, and activated the SRF transcription in a Rho-dependent manner. We also demonstrated that FMNL2 was necessary for LPA-induced invasion, RhoA/ROCK activation, actin assembly and SRF activation. FMNL2 was an essential component of LPA signal transduction toward RhoA by directly interacting with LARG. LARG silence inhibited RhoA/ROCK pathway and CRC cell invasion. Collectively, these data indicate that FMNL2, acting as upstream of RhoA by interacting with LARG, can promote actin assembly and CRC cell invasion through a Rho/ROCK-dependent mechanism.

Intravital imaging of SRF and Notch signalling identifies a key role for EZH2 in invasive melanoma cells.

The acquisition of cell motility is an early step in melanoma metastasis. Here we use intravital imaging of signalling reporter cell-lines combined with genome-wide transcriptional analysis to define signalling pathways and genes associated with melanoma metastasis. Intravital imaging revealed heterogeneous cell behaviour in vivo: less than 10% of cells were motile and both singly moving cells and streams of cells were observed. Motile melanoma cells had increased Notch- and SRF-dependent transcription. Subsequent genome-wide analysis identified an overlapping set of genes associated with high Notch and SRF activity. We identified EZH2, a histone methyltransferase in the Polycomb Repressor Complex 2, as a regulator of these genes. Heterogeneity of EZH2 levels is observed in melanoma models and co-ordinated up-regulation of genes positively regulated by EZH2 is associated with melanoma metastasis. EZH2 was also identified as regulating the amelanotic phenotype of motile cells in vivo by suppressing expression of the P-glycoprotein Oca2. Analysis of patient samples confirmed an inverse relationship between EZH2 levels and pigment. EZH2 targeting with siRNA and chemical inhibition reduced invasion in mouse and human melanoma cell lines. The EZH2 regulated SRF target genes KIF2C and KIF22 are required for melanoma cell invasion and important for lung colonisation. We propose that heterogeneity in EZH2 levels leads to heterogeneous expression of a cohort of genes associated with motile behaviour including KIF2C and KIF22. EZH2 dependent increased expression of these genes promotes melanoma cell motility and early steps in metastasis.

Co-Activation of Nuclear Factor-κB and Myocardin/Serum Response Factor Conveys the Hypertrophy Signal of High Insulin Levels in Cardiac Myoblasts

Hyperinsulinemia contributes to cardiac hypertrophy and heart failure in patients with the metabolic syndrome and type 2 diabetes. Here, high circulating levels of tumor necrosis factor (TNF)-α may synergize with insulin in signaling inflammation and cardiac hypertrophy. We tested whether high insulin affects activation of TNF-α-induced NF-κB and myocardin/serum response factor (SRF) to convey hypertrophy signaling in cardiac myoblasts. In canine cardiac myoblasts, treatment with high insulin (10(-8) to 10(-7) m) for 0-24 h increased insulin receptor substrate (IRS)-1 phosphorylation at Ser-307, decreased protein levels of chaperone-associated ubiquitin (Ub) E3 ligase C terminus of heat shock protein 70-interacting protein (CHIP), increased SRF activity, as well as β-myosin heavy chain (MHC) and myocardin expressions. Here siRNAs to myocardin or NF-κB, as well as CHIP overexpression prevented (while siRNA-mediated CHIP disruption potentiated) high insulin-induced SR element (SRE) activation and β-MHC expression. Insulin markedly potentiated TNF-α-induced NF-κB activation. Compared with insulin alone, insulin+TNF-α increased SRF/SRE binding and β-MHC expression, which was reversed by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and by NF-κB silencing. In the hearts of db/db diabetic mice, in which Akt phosphorylation was decreased, p38MAPK, Akt1, and IRS-1 phosphorylation at Ser-307 were increased, together with myocardin expression as well as SRE and NF-κB activities. In response to high insulin, cardiac myoblasts increase the expression or the promyogenic transcription factors myocardin/SRF in a CHIP-dependent manner. Insulin potentiates TNF-α in inducing NF-κB and SRF/SRE activities. In hyperinsulinemic states, myocardin may act as a nuclear effector of insulin, promoting cardiac hypertrophy.

Nuclear actin and myocardin-related transcription factors control disuse muscle atrophy through regulation of Srf activity.

Skeletal muscle atrophy is a debilitating process that is associated with a wide variety of conditions including inactivity, disease and aging. Here, we demonstrate that the actin, myocardin-related transcription factors and serum response factor (actin-Mrtf-Srf) pathway is specifically downregulated in the muscle atrophy that is induced through disuse in mice. We show in vivo that the abolition of mechanical signals leads to the rapid accumulation of G-actin in myonuclei and the export of the Srf coactivator Mrtf-A, resulting in a decrease of Mrtf-Srf-dependent transcription that contributes to atrophy. We demonstrate that inhibition of the actin-Mrtf-Srf axis through overexpression of nuclear non-polymerizable actin, through pharmacological inhibition of Mrtf-Srf and through muscle-specific Srf deletion worsens denervation-induced atrophy. Conversely, maintenance of high levels of activity of Srf or Mrtfs in denervated muscle, through overexpression of constitutively active derivatives, counteracts atrophy. Altogether, our data provide new mechanistic insights into the control of muscle mass upon disuse atrophy by the actin-Mrtf-Srf pathway, highlighting Srf as a key mediator of mechanotransduction in muscle.

Abstract 315: Mice With Cardiac-restricted Overexpression Of Myozap Are Sensitized To Biomechanical Stress And Develop A Protein-aggregate Associated Cardiomyopathy

The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes ensuring their electro-mechanical coupling. In previous work, we could identify and characterize Myozap, a novel cardiac-enriched ID protein, which interacts with several other ID proteins. We could now show in cardiomyocytes that overexpression of Myozap activates the Rho-dependent SRF pathway. Consistently, RNAi-mediated Myozap knockdown blocked Rho-mediated SRF activation linking the ID to gene regulation and actin dynamics. For further analyses of Myozap’s functions in vivo, we generated a mouse model with cardiac overexpression of Myozap cDNA (αMHC promoter). 1 year old mice developed cardiomyopathy with significant hypertrophy (heart weight/tibia length +43%, p<0.01, n=8-10) as well as LV dilation (LVEDD +20%, p<0.01) and a trend towards LV dysfunction (fractional shortening 50% vs. 42%, n=10, p=0.07). Consistently, these mice displayed upregulation of the hypertrophy-associated gene program (nppa 4.3-fold, p<0.001, and nppb 2.2-fold, p<0.01, n=7-9). On the ultrastructural level, we could detect bulky aggregates containing Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the changes in the hearts of patients suffering from desminopathies. Interestingly, desmin was not detectable in the aggregates in MyozapTG mice, but appeared to be displaced from the ID. Further molecular analyses in the aged mice also revealed dysregulation of typical members of the unfolded protein response (UPR) associated with upregulation of UPR-related apoptosis (cleaved caspase 12). Functionally, pressure overload via transverse aortic constriction (TAC) caused exaggerated cardiac hypertrophy (heart/body weight 7.9 mg/g in Myozap TG-TAC vs. 5.6 mg/g in WT-TAC, n=11 each group, p=0.002) and loss of contractility (fractional shortening of 15.3% in Myozap TG-TAC vs. 33.0% in WT-TAC, n=11 each group, p<0.001). Similarly, a physiological stimulus (voluntary running) led to a less dramatic but significant premature LV dysfunction. In conclusion, Myozap overexpression offers new insights in the pathogenesis of protein aggregate-associated cardiomyopathies.

Lamin A/C and emerin regulate MKL1–SRF activity by modulating actin dynamics

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

209 TRANSCRIPTOME ANALYSIS IDENTIFIES A NOVEL MECHANISM OF CAMP-MEDIATED GROWTH ARREST IN VSMC: PKA AND EPAC SYNERGISE TO SUPPRESS EGR1 EXPRESSION

Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. We recently demonstrated that cAMP-sensitive PKA and Epac signalling pathways synergise to inhibit VSMC proliferation but the underlying mechanisms are not understood. Using PKA and Epac-selective agonists and microarray analysis we tested the hypothesis that PKA and Epac block VSMC proliferation via coordinate regulation of immediate early genes required for cell-cycle progression. Epac agonist significantly enhanced PKA-dependent inhibition of EGR1 mRNA (4.6±0.38 fold for PKA vs 13.8±1.09 fold for PKA+EPAC, p cAMP-dependent inhibition (3.9±0.8 fold, p In summary, our results demonstrate that PKA and Epac synergise to inhibit VSMC proliferation by rapidly inhibiting EGR1 expression via a mechanism that probably involves inhibition of SRF activity. This also provides a mechanistic explanation for the CREB and MAPK-independent anti-mitogenic effects of cAMP in VSMC.

Abstract P341: MRTF-A, a Rho-Dependent Coactivator of SRF, Plays a Critical Role in Vascular Remodeling

Objective: Rho-ROCK pathway is involved in the progression of pathological vascular remodeling through the regulation of cell proliferation and migration by controlling gene transcription. While a transcriptional co-activator Myocardin-related transcription factor(MRTF)-A transduces Rho-actin signaling to SRF activation in the nucleus, roles of MRTF-A in the process underlying vascular diseases remain unknown. Methods and Results: We found that MRTF-A mRNA and protein expression in femoral arteries 2weeks after wire injury was significantly higher than that in sham-operated arteries of mice. In contrast, the expression of myocardin or MRTF-B was significantly decreased in injured arteries. We then evaluated the role played by MRTF-A in pathological vascular remodeling by creating several vascular disease models of mice lacking MRTF-A (MRTF-A -/- ). Neointima formation induced by ligation in carotid arteries of MRTF-A -/- was significantly smaller than that of MRTF-A +/- . Neointima formation induced by wire injury in femoral arteries of MRTFA -/- was significantly smaller than that of MRTF-A +/+ . Atherosclerotic lesions of MRTF-A -/- ;ApoE -/- was also significantly smaller than those of MRTF-A +/+ ;ApoE -/ . The expression of SRF-target genes involved in cellular migration, such as MMP9 and vinculin,within injured arteries of MRTF-A -/- was significantly attenuated compared to that of MRTF-A +/+ . In cultured, de-differentiated rat aortic vascular smooth muscle cells (RAVSMC), the expression of these genes was decreased by knocking down MRTF-A. Indeed, the promoter activity of vinculin gene was controlled by MRTF-A in RAVSMC. In addition, knocking down MRTF-A in RAVSMC resulted in a significant impairment in migration capacity. Furthermore, treatment with a small molecule CCG1423 inhibiting MRTF-A significantly reduced neointima formation induced by wire injury in femoral arteries of mice. Conclusions: Our study revealed that in de-differentiated vascular smooth muscle cells, in which myocardin and MRTTF-B expression is decreased, increased expression of MRTF-A contributes to the acquisition of migration capacity by maintaining SRF-target genes transcription, thereby promoting stress-induced vascular remodeling.

Role of Geminin in Mediating Phenotype Transformation of Vascular Smooth Muscle Cells in Rats

ObjectiveTo investigate the effect of Geminin on the phenotype transformation of VSMCs in rats.Methods(1) Both phenotypes were obtained after serum stimulation, and different biological function were detected induced by acetylcholine....

Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons

During corticogenesis, pyramidal neurons (~80% of cortical neurons) arise from the ventricular zone (VZ) pass through a multipolar stage to become bipolar and attach to radial glia1, 3, and then migrate to their proper position within the cortex1, 2. As pyramidal neurons migrate radially, they remain attached to their glial substrate as they pass through the subventricular (SVZ) and intermediate (IZ) zones, regions rich in tangentially migrating interneurons and axon fiber tracts. We examined the role of Lamellipodin (Lpd), a homolog of a key regulator of neuronal migration and polarization in C. elegans, in corticogenesis. Lpd depletion caused bipolar pyramidal neurons to adopt a tangential, rather than radial-glial, migration mode without affecting cell fate. Mechanistically, Lpd depletion reduced the activity of SRF, a transcription factor regulated by changes in the ratio of polymerized to unpolymerized actin. Therefore, Lpd depletion exposes a role for SRF in directing pyramidal neurons to select a radial migration pathway along glia rather than a tangential migration mode.