The Experts below are selected from a list of 6210 Experts worldwide ranked by ideXlab platform
Nathan Sandbo - One of the best experts on this subject based on the ideXlab platform.
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The Novel mTOR Complex 1/2 Inhibitor P529 Inhibits Human Lung Myofibroblast Differentiation.
Journal of Cellular Biochemistry, 2017Co-Authors: Keith T. Ferguson, Elizabeth E. Torr, Ksenija Bernau, Jonathan P. Leet, David Sherris, Nathan Sandbo, Nathan SandboAbstract:Idiopathic pulmonary fibrosis is a progressive and deadly disorder with very few therapeutic options. Palomid 529 (8-(1-hydroxyethyl)-2-methoxy-3-(4-methoxybenzyloxy)-benzo[c]chromen-6-one; P529) is a novel dual inhibitor of mechanistic target of rapamycin complex 1/2 (mTORC1/2). In these studies, we investigated the effect of P529 on TGF-β-dependent signaling and Myofibroblast Differentiation. TGF-β-induced phosphorylation of the mTORC1 targets, p70 S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), were both dose dependently inhibited by P529 in human lung fibroblasts with maximal inhibition occurring between 10 and 20 μM. mTORC2-mediated phosphorylation of Akt at the S473 site was partially inhibited with a similar dose dependency, as was TGF-β-induced Myofibroblast Differentiation. Protein levels of TGF-β-induced fibronectin and collagen were similarly decreased by P529. At this dose, there was also inhibition of mRNA transcript levels for Col1 and α-SMA, suggesting inhibition of transcriptional activation. However, there was no effect of P529 on canonical TGF-β-induced Smad signaling, as assessed by receptor-associated Smad2/3 phosphorylation, Smad2/3/4 translocation, or Smad-driven gene expression, as assessed by Smad-binding element driven luciferase. Conversely, activation of mTORC1/2 signaling was dependent on TGF-β type I receptor (ALK5) signaling and on Smad2/3 expression. P529 treatment disrupted TGF-β-induced actin stress fiber formation during Myofibroblast Differentiation, the deposition of new extracellular fibronectin matrix, and linear wound closure by fibroblasts. Likewise, mTOR knockdown inhibited TGF-β-induced Myofibroblast Differentiation. In conclusion, P529 inhibits TGF-β-induced Myofibroblast Differentiation, actin stress fiber formation, and matrix protein expression and deposition. Inhibition of mTORC1/2 by P529 may be a promising approach to inhibit in vivo fibrosis. J. Cell. Biochem. 118: 2241–2249, 2017. © 2017 Wiley Periodicals, Inc.
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tensin 1 is essential for Myofibroblast Differentiation and extracellular matrix formation
American Journal of Respiratory Cell and Molecular Biology, 2017Co-Authors: Ksenija Bernau, Elizabeth E. Torr, Michael D Evans, Jason K Aoki, Caitlyn Ngam, Nathan Sandbo, Nathan SandboAbstract:Myofibroblasts, the primary effector cells that mediate matrix remodeling during pulmonary fibrosis, rapidly assemble an extracellular fibronectin matrix. Tensin (TNS) 1 is a key component of specialized cellular adhesions (fibrillar adhesions) that bind to extracellular fibronectin fibrils. We hypothesized that TNS1 may play a role in modulating Myofibroblast-mediated matrix formation. We found that TNS1 expression is increased in fibroblastic foci from lungs with idiopathic pulmonary fibrosis. Transforming growth factor (TGF)-β profoundly up-regulates TNS1 expression with kinetics that parallel the expression of the Myofibroblast marker, smooth muscle α-actin. TGF-β–induced TNS1 expression is dependent on signaling through the TGF-β receptor 1 and is Rho coiled-coiled kinase/actin/megakaryoblastic leukemia-1/serum response factor dependent. Small interfering RNA–mediated knockdown of TNS1 disrupted TGF-β–induced Myofibroblast Differentiation, without affecting TGF-β/Smad signaling. In contrast, loss of ...
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control of Myofibroblast Differentiation by microtubule dynamics through a regulated localization of mdia2
Journal of Biological Chemistry, 2013Co-Authors: Nathan Sandbo, Elizabeth E. Torr, Caitlyn Ngam, Steve Kregel, Jacob Kach, Nickolai O DulinAbstract: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.
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delayed stress fiber formation mediates pulmonary Myofibroblast Differentiation in response to tgf β
American Journal of Physiology-lung Cellular and Molecular Physiology, 2011Co-Authors: Nathan Sandbo, Caitlyn Ngam, Jacob Kach, Andrew Lau, Douglas M Yau, Nickolai O DulinAbstract:Myofibroblast Differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary Myofibroblast Differentiation. TGF-β stimulated a profound, but delayed (18-24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for Myofibroblast Differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further Myofibroblast Differentiation.
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delayed stress fiber formation mediates pulmonary Myofibroblast Differentiation in response to tgf β
American Journal of Physiology-lung Cellular and Molecular Physiology, 2011Co-Authors: Nathan Sandbo, Caitlyn Ngam, Jacob Kach, Andrew Lau, Douglas M Yau, Nickolai O DulinAbstract:Myofibroblast Differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and i...
Nickolai O Dulin - One of the best experts on this subject based on the ideXlab platform.
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sirt3 blocks Myofibroblast Differentiation and pulmonary fibrosis by preventing mitochondrial dna damage
American Journal of Physiology-lung Cellular and Molecular Physiology, 2017Co-Authors: Samik Bindu, Gökhan M. Mutlu, Nickolai O Dulin, Vinodkumar B Pillai, Abhinav Kanwal, Sadhana Samant, Eric Verdin, Mahesh P GuptaAbstract:Myofibroblast Differentiation is a key process in the pathogenesis of fibrotic diseases. Transforming growth factor-β1 (TGF-β1) is a powerful inducer of Myofibroblast Differentiation and is implicated in pathogenesis of tissue fibrosis. This study was undertaken to determine the role of mitochondrial deacetylase SIRT3 in TGF-β1-induced Myofibroblast Differentiation in vitro and lung fibrosis in vivo. Treatment of human lung fibroblasts with TGF-β1 resulted in increased expression of fibrosis markers, smooth muscle α-actin (α-SMA), collagen-1, and fibronectin. TGF-β1 treatment also caused depletion of endogenous SIRT3, which paralleled with increased production of reactive oxygen species (ROS), DNA damage, and subsequent reduction in levels of 8-oxoguanine DNA glycosylase (OGG1), an enzyme that hydrolyzes oxidized guanine (8-oxo-dG) and thus protects DNA from oxidative damage. Overexpression of SIRT3 by adenovirus-mediated transduction reversed the effects of TGF-β1 on ROS production and mitochondrial DNA damage and inhibited TGF-β1-induced Myofibroblast Differentiation. To determine the antifibrotic role of SIRT3 in vivo, we used the bleomycin-induced mouse model of pulmonary fibrosis. Compared with wild-type controls, Sirt3-knockout mice showed exacerbated fibrosis after intratracheal instillation of bleomycin. Increased lung fibrosis was associated with decreased levels of OGG1 and concomitant accumulation of 8-oxo-dG and increased mitochondrial DNA damage. In contrast, the transgenic mice with whole body Sirt3 overexpression were protected from bleomycin-induced mtDNA damage and development of lung fibrosis. These data demonstrate a critical role of SIRT3 in the control of Myofibroblast Differentiation and lung fibrosis.
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regulation of Myofibroblast Differentiation by cardiac glycosides
American Journal of Physiology-lung Cellular and Molecular Physiology, 2016Co-Authors: Eleanor B. Reed, Svetlana V. Koltsova, Olga A. Akimova, Robert B. Hamanaka, Gökhan M. Mutlu, Sergei N. Orlov, Nickolai O DulinAbstract: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.
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Regulation of Myofibroblast Differentiation by cardiac glycosides
American journal of physiology. Lung cellular and molecular physiology, 2016Co-Authors: Eleanor B. Reed, Svetlana V. Koltsova, Olga A. Akimova, Robert B. Hamanaka, Gökhan M. Mutlu, Sergei N. Orlov, Nickolai O. Dulin, Nickolai O DulinAbstract: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+] ...
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control of Myofibroblast Differentiation by microtubule dynamics through a regulated localization of mdia2
Journal of Biological Chemistry, 2013Co-Authors: Nathan Sandbo, Elizabeth E. Torr, Caitlyn Ngam, Steve Kregel, Jacob Kach, Nickolai O DulinAbstract: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.
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delayed stress fiber formation mediates pulmonary Myofibroblast Differentiation in response to tgf β
American Journal of Physiology-lung Cellular and Molecular Physiology, 2011Co-Authors: Nathan Sandbo, Caitlyn Ngam, Jacob Kach, Andrew Lau, Douglas M Yau, Nickolai O DulinAbstract:Myofibroblast Differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary Myofibroblast Differentiation. TGF-β stimulated a profound, but delayed (18-24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for Myofibroblast Differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further Myofibroblast Differentiation.
Victor J Thannickal - One of the best experts on this subject based on the ideXlab platform.
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glutaminolysis is required for transforming growth factor β1 induced Myofibroblast Differentiation and activation
Journal of Biological Chemistry, 2017Co-Authors: Karen Bernard, Naomi J Logsdon, Gloria A Benavides, Yan Y Sanders, Jianhua Zhang, Victor M Darleyusmar, Victor J ThannickalAbstract:Myofibroblasts participate in physiological wound healing and pathological fibrosis. Myofibroblast Differentiation is characterized by the expression of α-smooth muscle actin and extracellular matrix proteins and is dependent on metabolic reprogramming. In this study, we explored the role of glutaminolysis and metabolites of TCA in supporting Myofibroblast Differentiation. Glutaminolysis converts Gln into α-ketoglutarate (α-KG), a critical intermediate in the TCA cycle. Increases in the steady-state concentrations of TCA cycle metabolites including α-KG, succinate, fumarate, malate, and citrate were observed in TGF-β1-differentiated Myofibroblasts. The concentration of glutamate was also increased in TGF-β1-differentiated Myofibroblasts compared with controls, whereas glutamine levels were decreased, suggesting enhanced glutaminolysis. This was associated with TGF-β1-induced expression of the glutaminase (GLS) isoform, GLS1, which converts Gln into glutamate, at both the mRNA and protein levels. The stimulation of GLS1 expression by TGF-β1 was dependent on both SMAD3 and p38 mitogen-activated protein kinase activation. Depletion of extracellular Gln prevented TGF-β1-induced Myofibroblast Differentiation. The removal of extracellular Gln postMyofibroblast Differentiation decreased the expression of the profibrotic markers fibronectin and hypoxia-inducible factor-1α and reversed TGF-β1-induced metabolic reprogramming. Silencing of GLS1 expression, in the presence of Gln, abrogated TGF-β1-induced expression of profibrotic markers. Treatment of GLS1-deficient Myofibroblasts with exogenous glutamate or α-KG restored TGF-β1-induced expression of profibrotic markers in GLS1-deficient Myofibroblasts. Together, these data demonstrate that glutaminolysis is a critical component of Myofibroblast metabolic reprogramming that regulates Myofibroblast Differentiation.
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glycolytic reprogramming in Myofibroblast Differentiation and lung fibrosis
American Journal of Respiratory and Critical Care Medicine, 2015Co-Authors: Na Xie, Karen Bernard, Victor J Thannickal, Huachun Cui, Sami Banerjee, Ruiming Liu, Zheng Tan, Gang LiuAbstract:Rationale: Dysregulation of cellular metabolism has been shown to participate in several pathologic processes. However, the role of metabolic reprogramming is not well appreciated in the pathogenesis of organ fibrosis.Objectives: To determine if glycolytic reprogramming participates in the pathogenesis of lung fibrosis and assess the therapeutic potential of glycolytic inhibition in treating lung fibrosis.Methods: A cell metabolism assay was performed to determine glycolytic flux and mitochondrial respiration. Lactate levels were measured to assess glycolysis in fibroblasts and lungs. Glycolytic inhibition by genetic and pharmacologic approaches was used to demonstrate the critical role of glycolysis in lung fibrosis.Measurements and Main Results: Augmentation of glycolysis is an early and sustained event during Myofibroblast Differentiation, which is dependent on the increased expression of critical glycolytic enzymes, in particular, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Augment...
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mir 145 regulates Myofibroblast Differentiation and lung fibrosis
The FASEB Journal, 2013Co-Authors: Shanzhong Yang, Victor J Thannickal, Huachun Cui, Na Xie, Mert Icyuz, Sami Banerjee, Veena B Antony, Edward Abraham, Gang LiuAbstract:The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to Myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung Myofibroblast Differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung Myofibroblast Differentiation and pulmonary fibrosis. Wild-type and miR-145−/− mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-β1-reated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal hum...
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mir 145 regulates Myofibroblast Differentiation and lung fibrosis
The FASEB Journal, 2013Co-Authors: Shanzhong Yang, Victor J Thannickal, Huachun Cui, Na Xie, Mert Icyuz, Sami Banerjee, Veena B Antony, Edward Abraham, Gang LiuAbstract:The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to Myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung Myofibroblast Differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung Myofibroblast Differentiation and pulmonary fibrosis. Wild-type and miR-145(-/-) mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-β1-treated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal human lungs. Overexpression of miR-145 in lung fibroblasts increased SMA-α expression, enhanced contractility, and promoted formation of focal and fibrillar adhesions. In contrast, miR-145 deficiency diminished TGF-β1 induced SMA-α expression. miR-145 did not affect the activity of TGF-β1, but promoted the activation of latent TGF-β1. miR-145 targets KLF4, a known negative regulator of SMA-α expression. Finally, we found that miR-145(-/-) mice are protected from bleomycin-induced pulmonary fibrosis. miR-145 plays an important role in the Differentiation of lung Myofibroblasts. miR-145 deficiency is protective against bleomycin-induced lung fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic disorders.
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matrix stiffness induced Myofibroblast Differentiation is mediated by intrinsic mechanotransduction
American Journal of Respiratory Cell and Molecular Biology, 2012Co-Authors: Xiangwei Huang, Victor J Thannickal, Naiheng Yang, Vincent F. Fiore, Thomas H. Barker, Yi Sun, Stephan W. Morris, Qiang Ding, Yong ZhouAbstract:The mechanical properties of the extracellular matrix have recently been shown to promote Myofibroblast Differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates Myofibroblast Differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced Myofibroblast Differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for Myofibroblast Differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and Myofibroblast Differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes Myofibroblast Differentiation, suggesting an intrinsic mechanotransduction mechanism.
Yong Zhou - One of the best experts on this subject based on the ideXlab platform.
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therapeutic targeting of src kinase in Myofibroblast Differentiation and pulmonary fibrosis
Journal of Pharmacology and Experimental Therapeutics, 2014Co-Authors: Pulin Che, Yong Zhou, Veena B Antony, Gang Liu, Xiaosi Han, Guoqiang Cai, Tracy Luckhardt, Gene P Siegal, Ruiming Liu, Leena P DesaiAbstract:Myofibroblasts are effector cells in fibrotic disorders that synthesize and remodel the extracellular matrix (ECM). This study investigated the role of the Src kinase pathway in Myofibroblast activation in vitro and fibrogenesis in vivo. The profibrotic cytokine, transforming growth factor β1 (TGF-β1), induced rapid activation of Src kinase, which led to Myofibroblast Differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-β1–induced Src kinase activation in a dose-dependent manner. Inhibition of Src kinase significantly reduced α-smooth muscle actin (α-SMA) expression, a marker of Myofibroblast Differentiation, in TGF-β1–treated lung fibroblasts. In addition, the induced expression of collagen and fibronectin and three-dimensional collagen gel contraction were also significantly inhibited in AZD0530-treated fibroblasts. The therapeutic efficiency of Src kinase inhibition in vivo was tested in the bleomycin murine lung fibrosis model. Src kinase activation and collagen accumulation were significantly reduced in the lungs of AZD0530-treated mice when compared with controls. Furthermore, the total fibrotic area and expression of α-SMA and ECM proteins were significantly decreased in lungs of AZD0530-treated mice. These results indicate that Src kinase promotes Myofibroblast Differentiation and activation of lung fibroblasts. Additionally, these studies provide proof-of-concept for targeting the noncanonical TGF-β signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.
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Myofibroblast Differentiation and enhanced tgf b signaling in cystic fibrosis lung disease
PLOS ONE, 2013Co-Authors: William T Harris, James S Hagood, Yong Zhou, David R Kelly, Dezhi Wang, Mark Macewen, John P Clancy, Namasivayam Ambalavanan, Eric J SorscherAbstract:Rationale TGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined. Objective To investigate Myofibroblast Differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis. Methods Lung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β1, TGF-β signaling (Smad2 phosphorylation), Myofibroblast Differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain). Results TGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of Myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both Myofibroblast Differentiation and tissue fibrosis are significantly augmented (p<0.005). Conclusions These studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent Myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.
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matrix stiffness induced Myofibroblast Differentiation is mediated by intrinsic mechanotransduction
American Journal of Respiratory Cell and Molecular Biology, 2012Co-Authors: Xiangwei Huang, Victor J Thannickal, Naiheng Yang, Vincent F. Fiore, Thomas H. Barker, Yi Sun, Stephan W. Morris, Qiang Ding, Yong ZhouAbstract:The mechanical properties of the extracellular matrix have recently been shown to promote Myofibroblast Differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates Myofibroblast Differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced Myofibroblast Differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for Myofibroblast Differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and Myofibroblast Differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes Myofibroblast Differentiation, suggesting an intrinsic mechanotransduction mechanism.
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matrix stiffness induced Myofibroblast Differentiation is mediated by intrinsic mechanotransduction
American Journal of Respiratory Cell and Molecular Biology, 2012Co-Authors: Xiangwei Huang, Victor J Thannickal, Naiheng Yang, Vincent F. Fiore, Thomas H. Barker, Stephan W. Morris, Qiang Ding, Yong ZhouAbstract:The mechanical properties of the extracellular matrix have recently been shown to promote Myofibroblast Differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates Myofibroblast Differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness–induced Myofibroblast Differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a centr...
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neuronal wiskott aldrich syndrome protein n wasp is critical for formation of α smooth muscle actin filaments during Myofibroblast Differentiation
American Journal of Physiology-lung Cellular and Molecular Physiology, 2012Co-Authors: Guoqiang Cai, Victor J Thannickal, Yong Zhou, Tracy Luckhardt, Chufang Chou, Anni Zheng, Louis F Reichardt, Junlin Guan, Haotian Fang, Qiang DingAbstract:Myofibroblasts are implicated in pathological stromal responses associated with lung fibrosis. One prominent phenotypic marker of fully differentiated Myofibroblasts is the polymerized, thick cytoplasmic filaments containing newly synthesized α-smooth muscle actin (α-SMA). These α-SMA-containing cytoplasmic filaments are important for Myofibroblast contractility during tissue remodeling. However, the molecular mechanisms regulating the formation and maturation of α-SMA-containing filaments have not been defined. This study demonstrates a critical role for neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating the formation of α-SMA-containing cytoplasmic filaments during Myofibroblast Differentiation and in Myofibroblast contractility. Focal adhesion kinase (FAK) is activated by transforming growth factor-β1 (TGF-β1) and is required for phosphorylation of tyrosine residue 256 (Y256) of N-WASP. Phosphorylation of Y256 of N-WASP is essential for TGF-β1-induced formation of α-SMA-containing cytoplasmic filaments in primary human lung fibroblasts. In addition, we demonstrate that actin-related protein (Arp) 2/3 complex is downstream of N-WASP and mediates the maturation of α-SMA-containing cytoplasmic filaments. Together, this study supports a critical role of N-WASP in integrating FAK and Arp2/3 signaling to mediate formation of α-SMA-containing cytoplasmic filaments during Myofibroblast Differentiation and maturation.
Dina Behmen - One of the best experts on this subject based on the ideXlab platform.
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syndecan 4 signaling via nfat regulates extracellular matrix production and cardiac Myofibroblast Differentiation in response to mechanical stress
Journal of Molecular and Cellular Cardiology, 2013Co-Authors: Kate M Herum, Ida G Lunde, Biljana Skrbic, Geir Florholmen, Dina BehmenAbstract:Pressure overload activates cardiac fibroblasts leading to excessive production of extracellular matrix which may contribute to compromised heart function. The activated fibroblast acquires smooth muscle-like features such as expression of smooth muscle alpha-actin (SMA) and SM22 and is therefore referred to as Myofibroblast. The molecular mechanisms underlying mechanical stress-induced Myofibroblast Differentiation are poorly defined. The objective of this study was to examine the potential roles of the transmembrane proteoglycan syndecan-4 and the calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) in Myofibroblast Differentiation. Aortic banding resulted in elevated collagen land III, fibronectin, SMA and SM22 mRNA in the left ventricles of wild-type mice, whereas this response was markedly reduced in syndecan-4(-/-) mice. Myofibroblast Differentiation in vitro was associated with increased SMA, collagen I and III expression and NFAT-luciferase activity, all of which were reduced in fibroblasts from syndecan-4(-/-) mice or after treatment with calcineurin/NFAT blockers. Following cyclic stretch, NFATc4 was activated in cardiac fibroblasts in a syndecan-4- and calcineurin-dependent manner. Syndecan-4 and calcineurin co-localized and mechanical stress resulted in dephosphorylation of serine179 of syndecan-4, an intracellular residue critical for calcineurin interaction. Over-expression of NFATc4 up-regulated collagen III, MRTF-A (a transcriptional regulator of SMA) and the NFAT-target regulator of calcineurin 1.4 (RCAN1.4). Our data demonstrate that syndecan-4 is important for the Differentiation of cardiac fibroblasts into Myofibroblasts in the pressure-overloaded heart and that the calcineurin/NFAT pathway is engaged upon mechanical stress in a syndecan-4-dependent manner, playing an active role in Myofibroblast Differentiation and extracellular matrix production. This article is part of a Special Issue entitled 'Possible Editorial'. (c) 2012 Elsevier Ltd. All rights reserved.
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syndecan 4 signaling via nfat regulates extracellular matrix production and cardiac Myofibroblast Differentiation in response to mechanical stress
Journal of Molecular and Cellular Cardiology, 2013Co-Authors: Kate M Herum, Ida G Lunde, Biljana Skrbic, Geir Florholmen, Dina BehmenAbstract:Pressure overload activates cardiac fibroblasts leading to excessive production of extracellular matrix which may contribute to compromised heart function. The activated fibroblast acquires smooth muscle-like features such as expression of smooth muscle α-actin (SMA) and SM22 and is therefore referred to as Myofibroblast. The molecular mechanisms underlying mechanical stress-induced Myofibroblast Differentiation are poorly defined. The objective of this study was to examine the potential roles of the transmembrane proteoglycan syndecan-4 and the calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) in Myofibroblast Differentiation. Aortic banding resulted in elevated collagen I and III, fibronectin, SMA and SM22 mRNA in the left ventricles of wild-type mice, whereas this response was markedly reduced in syndecan-4(-/-) mice. Myofibroblast Differentiation in vitro was associated with increased SMA, collagen I and III expression and NFAT-luciferase activity, all of which were reduced in fibroblasts from syndecan-4(-/-) mice or after treatment with calcineurin/NFAT blockers. Following cyclic stretch, NFATc4 was activated in cardiac fibroblasts in a syndecan-4- and calcineurin-dependent manner. Syndecan-4 and calcineurin co-localized and mechanical stress resulted in dephosphorylation of serine179 of syndecan-4, an intracellular residue critical for calcineurin interaction. Over-expression of NFATc4 up-regulated collagen III, MRTF-A (a transcriptional regulator of SMA) and the NFAT-target regulator of calcineurin 1.4 (RCAN1.4). Our data demonstrate that syndecan-4 is important for the Differentiation of cardiac fibroblasts into Myofibroblasts in the pressure-overloaded heart and that the calcineurin/NFAT pathway is engaged upon mechanical stress in a syndecan-4-dependent manner, playing an active role in Myofibroblast Differentiation and extracellular matrix production. This article is part of a Special Issue entitled 'Possible Editorial'.
