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Michael P. Czubryt - One of the best experts on this subject based on the ideXlab platform.
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Scleraxis regulates twist1 and snai1 expression in the epithelial to mesenchymal transition
American Journal of Physiology-heart and Circulatory Physiology, 2018Co-Authors: Danah S Alhattab, Rushita A. Bagchi, Raghu S Nagalingam, Hamza A. Safi, Matthew Taras Stecy, Michael P. CzubrytAbstract:The molecular mechanism by which the transcription factor Scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epi...
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Regulation of cardiac fibroblast MMP2 gene expression by Scleraxis.
Journal of molecular and cellular cardiology, 2018Co-Authors: Raghu S Nagalingam, Rushita A. Bagchi, Ian M C Dixon, Jeffrey T Wigle, Hamza A. Safi, Danah S. Al-hattab, Natalie M. Landry, Michael P. CzubrytAbstract:Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor Scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that Scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which Scleraxis binds. Cardiac myo-fibroblasts isolated from Scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, Scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFβ occurs via a Scleraxis-dependent mechanism: TGFβ induces recruitment of Scleraxis to the MMP2 promoter, and TGFβ was unable to up-regulate MMP2 expression in cells lacking Scleraxis due to either gene knockdown or knockout. These results reveal that Scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.
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Scleraxis a force responsive cell phenotype regulator
Current Opinion in Physiology, 2018Co-Authors: Hamza A. Safi, Raghu S Nagalingam, Michael P. CzubrytAbstract:Changes in cell phenotype underlie many of the body's responses to altered environmental conditions, stress, or damage, resulting in both physiological and pathophysiological alterations to tissue function. Transcriptional regulators play a critical role in reprogramming cell identity and behavior. Emerging evidence has implicated the transcription factor Scleraxis as a novel and powerful determiner of cell phenotype in cells that produce large quantities of extracellular matrix, including tenocytes and cardiac fibroblasts. In this review, we examine the role of Scleraxis in epithelial-to-mesenchymal transition and in altering cell phenotype, and discuss the implications of this role in tissue repair and pathology. This specialized role for Scleraxis makes it a potential target for therapies aimed at improving wound healing or attenuating tissue fibrosis.
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abstract 19631 Scleraxis regulates pressure overload induced cardiac fibrosis
Circulation, 2017Co-Authors: Raghu S Nagalingam, Nina Aroutiounova, Davinder S Jassal, David Cheung, Michael P. CzubrytAbstract:Introduction: Cardiac fibrosis is an independent risk factor for heart failure and death that currently lacks any specific treatment. Fibrosis is the uncontrolled synthesis of matrix components such as collagen and fibronectin, elevating myocardial wall stiffness, decreasing compliance, increasing arrhythmogenesis, and contributing to heart failure. Proliferation and phenotype conversion of fibroblasts to myofibroblasts contributes critically to the progression of fibrosis. Restricting the activation of myofibroblasts could thus be beneficial for restoring the function of the fibrotic heart. Our previous studies have shown that the transcription factor Scleraxis governs the production of major matrix proteins including collagen, α-smooth muscle actin and fibronectin, and regulates fibroblast to myofibroblast phenoconversion. Scleraxis knockout animals exhibit a 50% reduction in the resident cardiac fibroblast population and decreased basal extracellular matrix production, but the role of Scleraxis in card...
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role of Scleraxis in mechanical stretch mediated regulation of cardiac myofibroblast phenotype
American Journal of Physiology-cell Physiology, 2016Co-Authors: Patricia Roche, Rushita A. Bagchi, Nina Aroutiounova, Raghu S Nagalingam, Breanna M J Belisle, Jeffrey T Wigle, Michael P. CzubrytAbstract:The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-β, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor Scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as Scleraxis expression via activation of the Scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in Scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of Scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that Scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.
Eric N Olson - One of the best experts on this subject based on the ideXlab platform.
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coordinated expression of Scleraxis and sox9 genes during embryonic development of tendons and cartilage
Journal of Orthopaedic Research, 2002Co-Authors: Yoshinori Asou, Akira Nifuji, Eric N Olson, Kenichi Shinomiya, Kunikazu Tsuji, Peter Koopman, Masaki NodaAbstract:Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages, Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor. is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways, To address this point, we investigated Scleraxis and Sox9 rnRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons, In the early stage of mesenchymal tissue development at 10.5 d.p.c., Scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c.. Scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, Scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia, At 13.5 d.p.c., Scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then. at 15.5 d.p.c., Scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of Scleraxis expression during embryonic development of tendon precursor cells in close association with that of So,0 expression in chondrogenic cells in skeletal tissues. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
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dual role of the basic helix loop helix transcription factor Scleraxis in mesoderm formation and chondrogenesis during mouse embryogenesis
Development, 1999Co-Authors: Doris Brown, Daniel S Wagner, James A Richardson, Eric N OlsonAbstract:Scleraxis is a basic helix-loop-helix (bHLH) transcription factor shown previously to be expressed in developing chondrogenic cell lineages during embryogenesis. To investigate its function in embryonic development, we produced Scleraxis-null mice by gene targeting. Homozygous mutant embryos developed normally until the early egg cylinder stage (embryonic day 6.0), when they became growth-arrested and failed to gastrulate. Consistent with this early embryonic phenotype, Scleraxis was found to be expressed throughout the embryo at the time of gastrulation before becoming restricted to chondrogenic precursor cells at embryonic day 9.5. At the time of developmental arrest, Scleraxis-null embryos consisted of ectodermal and primitive endodermal cell layers, but lacked a primitive streak or recognizable mesoderm. Analysis of molecular markers of the three embryonic germ layers confirmed that Scleraxis mutant embryos were unable to form mesoderm. By generating chimeric embryos, using lacZ-marked Scleraxis-null and wild-type embryonic stem cells, we examined the ability of mutant cells to contribute to regions of the embryo beyond the time of lethality of homozygous mutants. Scleraxis-null cells were specifically excluded from the sclerotomal compartment of somites, which gives rise to the axial skeleton, and from developing ribs, but were able to contribute to most other regions of the embryo, including mesoderm-derived tissues. These results reveal an essential early role for Scleraxis in mesoderm formation, as well as a later role in formation of somite-derived chondrogenic lineages, and suggest that Scleraxis target genes mediate these processes.
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downregulation of the transcription factor Scleraxis in brain of patients with down syndrome
Journal of Neural Transmission-supplement, 1999Co-Authors: K Yeghiazaryan, Eric N Olson, D Turhanischatzmann, O Labudova, Elisabeth Schuller, Nigel J Cairns, Gert LubecAbstract:Performing gene hunting in fetal Down Syndrome (DS) brain, we found a downregulated sequence with 100% homology to the basic — helix — loop — helix transcription factor (TF) Scleraxis (Scl).
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fibroblast growth factor downregulates expression of a basic helix loop helix type transcription factor Scleraxis in a chondrocyte like cell line tc6
Journal of Cellular Biochemistry, 1998Co-Authors: Toshiyuki Kawauchi, Akira Nifuji, Eric N Olson, Nobuko Mataga, Jacky Bonaventure, Kenichi Shinomiya, Ying Liu, Masaki NodaAbstract:Scleraxis is a basic helix-loop-helix-type transcription factor that is expressed in sclerotome. Fibroblast growth factor (FGF) is one of the cytokines produced by the cells in skeletal tissues and is a potent modulator of skeletogenesis. The aim of this study was to examine the effects of FGF on the expression of Scleraxis in chondrocyte-like cells, TC6. In these cells, Scleraxis mRNA was constitutively expressed as a 1 .2 kb message at a high level in contrast to its low levels of expression in fibroblast-like cells or osteoblast-like cells. Upon treatment with FGF, Scleraxis mRNA level was decreased within 12 h. This effect was at its nadir at 24 h and the Scleraxis mRNA level returned to its base line level by 48 h. The FGF effect was maximal at 1 ng/ml. FGF effects on Scleraxis were blocked by actinomycin D but not by cycloheximide, suggesting the involvement of transcriptional events that do not require new protein synthesis. The FGF effects on Scleraxis were blocked by genistein, suggesting the involvement of tyrosine kinase in the post-receptor signaling. TGFbeta treatment of TC6 cells enhanced Scleraxis mRNA expression; however, combination of the saturation doses of FGF and TGFbeta resulted in suppression of Scleraxis mRNA level. BMP2 also suppressed Scleraxis mRNA expression in TC6 cells and no further suppression was observed in combination with FGF. These results indicate that Scleraxis is expressed in chondrocyte-like TC6 cells and it is one of the targets of FGF action in these cells.
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overexpression of a single helix loop helix type transcription factor Scleraxis enhances aggrecan gene expression in osteoblastic osteosarcoma ros17 2 8 cells
Journal of Biological Chemistry, 1997Co-Authors: Ying Liu, Akira Nifuji, Eric N Olson, Hideto Watanabe, Yoshihiko Yamada, Masaki NodaAbstract:Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of Scleraxis, we overexpressed Scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of Scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of Scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that Scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to Scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to in vitro translated Scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing Scleraxis and was supershifted by the antiserum raised against Scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to Scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to Scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, Scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.
Rushita A. Bagchi - One of the best experts on this subject based on the ideXlab platform.
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Scleraxis regulates twist1 and snai1 expression in the epithelial to mesenchymal transition
American Journal of Physiology-heart and Circulatory Physiology, 2018Co-Authors: Danah S Alhattab, Rushita A. Bagchi, Raghu S Nagalingam, Hamza A. Safi, Matthew Taras Stecy, Michael P. CzubrytAbstract:The molecular mechanism by which the transcription factor Scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epi...
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Regulation of cardiac fibroblast MMP2 gene expression by Scleraxis.
Journal of molecular and cellular cardiology, 2018Co-Authors: Raghu S Nagalingam, Rushita A. Bagchi, Ian M C Dixon, Jeffrey T Wigle, Hamza A. Safi, Danah S. Al-hattab, Natalie M. Landry, Michael P. CzubrytAbstract:Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor Scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that Scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which Scleraxis binds. Cardiac myo-fibroblasts isolated from Scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, Scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFβ occurs via a Scleraxis-dependent mechanism: TGFβ induces recruitment of Scleraxis to the MMP2 promoter, and TGFβ was unable to up-regulate MMP2 expression in cells lacking Scleraxis due to either gene knockdown or knockout. These results reveal that Scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.
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role of Scleraxis in mechanical stretch mediated regulation of cardiac myofibroblast phenotype
American Journal of Physiology-cell Physiology, 2016Co-Authors: Patricia Roche, Rushita A. Bagchi, Nina Aroutiounova, Raghu S Nagalingam, Breanna M J Belisle, Jeffrey T Wigle, Michael P. CzubrytAbstract:The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-β, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor Scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as Scleraxis expression via activation of the Scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in Scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of Scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that Scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.
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regulation of fibronectin gene expression in cardiac fibroblasts by Scleraxis
Cell and Tissue Research, 2016Co-Authors: Rushita A. Bagchi, Justin Lin, Ryan Wang, Michael P. CzubrytAbstract:The glycoprotein fibronectin is a key component of the extracellular matrix. By interacting with numerous matrix and cell surface proteins, fibronectin plays important roles in cell adhesion, migration and intracellular signaling. Up-regulation of fibronectin occurs in tissue fibrosis, and previous studies have identified the pro-fibrotic factor TGFβ as an inducer of fibronectin expression, although the mechanism responsible remains unknown. We have previously shown that a key downstream effector of TGFβ signaling in cardiac fibroblasts is the transcription factor Scleraxis, which in turn regulates the expression of a wide variety of extracellular matrix genes. We noted that fibronectin expression tracked closely with Scleraxis expression, but it was unclear whether Scleraxis directly regulated the fibronectin gene. Here, we report that Scleraxis acts via two E-box binding sites in the proximal human fibronectin promoter to govern fibronectin expression, with the second E-box being both sufficient and necessary for Scleraxis-mediated fibronectin expression to occur. A combination of electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Scleraxis interacted to a greater degree with the second E-box. Over-expression or knockdown of Scleraxis resulted in increased or decreased fibronectin expression, respectively, and Scleraxis null mice presented with dramatically decreased immunolabeling for fibronectin in cardiac tissue sections compared to wild-type controls. Furthermore, Scleraxis was required for TGFβ-induced fibronectin expression: TGFβ lost its ability to induce fibronectin expression following Scleraxis knockdown. Together, these results demonstrate a novel and required role for Scleraxis in the regulation of cardiac fibroblast fibronectin gene expression basally or in response to TGFβ.
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the transcription factor Scleraxis is a critical regulator of cardiac fibroblast phenotype
BMC Biology, 2016Co-Authors: Rushita A. Bagchi, Ronen Schweitzer, Patricia Roche, Nina Aroutiounova, Leon Espira, Bernard Abrenica, Michael P. CzubrytAbstract:Resident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis. Using explanted primary cardiac fibroblasts in gain- and loss-of-function studies, we found that Scleraxis critically controls cardiac fibroblast/myofibroblast phenotype by direct transcriptional regulation of myriad genes that effectively define these cells, including extracellular matrix components and α-smooth muscle actin. Scleraxis furthermore potentiated the TGFβ/Smad3 signaling pathway, a key regulator of myofibroblast conversion, by facilitating transcription complex formation. While Scleraxis promoted fibroblast to myofibroblast conversion, loss of Scleraxis attenuated myofibroblast function and gene expression. These results were confirmed in Scleraxis knockout mice, which were cardiac matrix-deficient and lost ~50 % of their complement of cardiac fibroblasts, with evidence of impaired epithelial-to-mesenchymal transition (EMT). Scleraxis directly transactivated several EMT marker genes, and was sufficient to induce mesenchymal/fibroblast phenotype conversion of A549 epithelial cells. Conversely, loss of Scleraxis attenuated TGFβ-induced EMT marker expression. Our results demonstrate that Scleraxis is a novel and potent regulator of cellular progression along the continuum culminating in the cardiac myofibroblast phenotype. Scleraxis was both sufficient to drive conversion, and required for full conversion to occur. Scleraxis fulfills this role by direct transcriptional regulation of key target genes, and by facilitating TGFβ/Smad signaling. Given the key role of fibroblast to myofibroblast conversion in fibrotic diseases in the heart and other tissue types, Scleraxis may be an important target for therapeutic development.
Masaki Noda - One of the best experts on this subject based on the ideXlab platform.
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coordinated expression of Scleraxis and sox9 genes during embryonic development of tendons and cartilage
Journal of Orthopaedic Research, 2002Co-Authors: Yoshinori Asou, Akira Nifuji, Eric N Olson, Kenichi Shinomiya, Kunikazu Tsuji, Peter Koopman, Masaki NodaAbstract:Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages, Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor. is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways, To address this point, we investigated Scleraxis and Sox9 rnRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons, In the early stage of mesenchymal tissue development at 10.5 d.p.c., Scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c.. Scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, Scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia, At 13.5 d.p.c., Scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then. at 15.5 d.p.c., Scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of Scleraxis expression during embryonic development of tendon precursor cells in close association with that of So,0 expression in chondrogenic cells in skeletal tissues. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
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fibroblast growth factor downregulates expression of a basic helix loop helix type transcription factor Scleraxis in a chondrocyte like cell line tc6
Journal of Cellular Biochemistry, 1998Co-Authors: Toshiyuki Kawauchi, Akira Nifuji, Eric N Olson, Nobuko Mataga, Jacky Bonaventure, Kenichi Shinomiya, Ying Liu, Masaki NodaAbstract:Scleraxis is a basic helix-loop-helix-type transcription factor that is expressed in sclerotome. Fibroblast growth factor (FGF) is one of the cytokines produced by the cells in skeletal tissues and is a potent modulator of skeletogenesis. The aim of this study was to examine the effects of FGF on the expression of Scleraxis in chondrocyte-like cells, TC6. In these cells, Scleraxis mRNA was constitutively expressed as a 1 .2 kb message at a high level in contrast to its low levels of expression in fibroblast-like cells or osteoblast-like cells. Upon treatment with FGF, Scleraxis mRNA level was decreased within 12 h. This effect was at its nadir at 24 h and the Scleraxis mRNA level returned to its base line level by 48 h. The FGF effect was maximal at 1 ng/ml. FGF effects on Scleraxis were blocked by actinomycin D but not by cycloheximide, suggesting the involvement of transcriptional events that do not require new protein synthesis. The FGF effects on Scleraxis were blocked by genistein, suggesting the involvement of tyrosine kinase in the post-receptor signaling. TGFbeta treatment of TC6 cells enhanced Scleraxis mRNA expression; however, combination of the saturation doses of FGF and TGFbeta resulted in suppression of Scleraxis mRNA level. BMP2 also suppressed Scleraxis mRNA expression in TC6 cells and no further suppression was observed in combination with FGF. These results indicate that Scleraxis is expressed in chondrocyte-like TC6 cells and it is one of the targets of FGF action in these cells.
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overexpression of a single helix loop helix type transcription factor Scleraxis enhances aggrecan gene expression in osteoblastic osteosarcoma ros17 2 8 cells
Journal of Biological Chemistry, 1997Co-Authors: Ying Liu, Akira Nifuji, Eric N Olson, Hideto Watanabe, Yoshihiko Yamada, Masaki NodaAbstract:Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of Scleraxis, we overexpressed Scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of Scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of Scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that Scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to Scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to in vitro translated Scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing Scleraxis and was supershifted by the antiserum raised against Scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to Scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to Scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, Scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.
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Scleraxis messenger ribonucleic acid is expressed in c2c12 myoblasts and its level is down regulated by bone morphogenetic protein 2 bmp2
Journal of Cellular Biochemistry, 1997Co-Authors: Ying Liu, Akira Nifuji, Eric N Olson, Masato Tamura, John M Wozney, Masaki NodaAbstract:We examined the mRNA expression of Scleraxis, a non-myogenic helix-loop-helix type transcription factor in C2C12 myogenic cells. Scleraxis mRNA has been shown to be expressed in sclerotome and perichondrium of the embryos. We found that C2C12 cells express 1.2 kb Scleraxis mRNA constitutively. Since BMP was reported to induce ectopic bone formation when implanted in muscle, we examined the effects of BMP on Scleraxis expression. Scleraxis mRNA expression in C2C12 cells was suppressed by the treatment with BMP2. This suppression was observed at 200 ng/ml but not at the lower concentrations. BMP2 treatment suppressed Scleraxis mRNA level within 24 h and lasted at least up to 48 h. Electrophoresis mobility shift assay showed that the proteins in the crude nuclear extracts prepared from C2C12 cells bound to an Scx-E-box sequence, CATGTG, which is preferentially recognized by Scleraxis. This binding was competed out by 100-fold molar excess of cold Scx-E-box sequence but not by the one with mutations in the E-box. This band was supershifted by the addition of antiserum raised against Scleraxis. BMP2 treatment suppressed the Scx-E binding activity in C2C12 cells. This suppression of the Scx-E-box binding activity was in parallel to the BMP2 suppression of the transcriptional activity of the Scx-E-CAT reporter gene transfected into C2C12 cells. These data indicated that although the default pathway for C2C12 cells is to differentiate into muscle cells, these cells do express non-myogenic transcription factor, Scleraxis, whose expression is suppressed by BMP2. J. Cell. Biochem. 67:66–74, 1997. © 1997 Wiley-Liss, Inc.
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Sclerotome-related helix-loop-helix type transcription factor (Scleraxis) mRNA is expressed in osteoblasts and its level is enhanced by type-β transforming growth factor
The Journal of endocrinology, 1996Co-Authors: Ying Liu, Akira Nifuji, Eric N Olson, P Cserjesi, Masaki NodaAbstract:Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of Scleraxis mRNA in rat osteoblastic cells and found that the Scleraxis gene was expressed as a 1.2 kb mRNA species in osteoblastic osteosarcoma ROS17/2.8 cells. The Scleraxis mRNA expression was enhanced by type-beta transforming growth factor (TGF beta) treatment. The TGF beta effect was observed in a dose-dependent manner starting at 0.2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGF beta effect was blocked by cycloheximide, while no effect on Scleraxis mRNA stability was observed. TGF beta treatment enhanced Scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2.8 cells. Furthermore, TGF beta enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGF beta treatment also enhanced Scleraxis gene expression in osteoblast-enriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helix-loop-helix type transcription factor (Scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGF beta.
Patricia Roche - One of the best experts on this subject based on the ideXlab platform.
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role of Scleraxis in mechanical stretch mediated regulation of cardiac myofibroblast phenotype
American Journal of Physiology-cell Physiology, 2016Co-Authors: Patricia Roche, Rushita A. Bagchi, Nina Aroutiounova, Raghu S Nagalingam, Breanna M J Belisle, Jeffrey T Wigle, Michael P. CzubrytAbstract:The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-β, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor Scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as Scleraxis expression via activation of the Scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in Scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of Scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that Scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.
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the transcription factor Scleraxis is a critical regulator of cardiac fibroblast phenotype
BMC Biology, 2016Co-Authors: Rushita A. Bagchi, Ronen Schweitzer, Patricia Roche, Nina Aroutiounova, Leon Espira, Bernard Abrenica, Michael P. CzubrytAbstract:Resident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis. Using explanted primary cardiac fibroblasts in gain- and loss-of-function studies, we found that Scleraxis critically controls cardiac fibroblast/myofibroblast phenotype by direct transcriptional regulation of myriad genes that effectively define these cells, including extracellular matrix components and α-smooth muscle actin. Scleraxis furthermore potentiated the TGFβ/Smad3 signaling pathway, a key regulator of myofibroblast conversion, by facilitating transcription complex formation. While Scleraxis promoted fibroblast to myofibroblast conversion, loss of Scleraxis attenuated myofibroblast function and gene expression. These results were confirmed in Scleraxis knockout mice, which were cardiac matrix-deficient and lost ~50 % of their complement of cardiac fibroblasts, with evidence of impaired epithelial-to-mesenchymal transition (EMT). Scleraxis directly transactivated several EMT marker genes, and was sufficient to induce mesenchymal/fibroblast phenotype conversion of A549 epithelial cells. Conversely, loss of Scleraxis attenuated TGFβ-induced EMT marker expression. Our results demonstrate that Scleraxis is a novel and potent regulator of cellular progression along the continuum culminating in the cardiac myofibroblast phenotype. Scleraxis was both sufficient to drive conversion, and required for full conversion to occur. Scleraxis fulfills this role by direct transcriptional regulation of key target genes, and by facilitating TGFβ/Smad signaling. Given the key role of fibroblast to myofibroblast conversion in fibrotic diseases in the heart and other tissue types, Scleraxis may be an important target for therapeutic development.
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tgfβ1 regulates Scleraxis expression in primary cardiac myofibroblasts by a smad independent mechanism
American Journal of Physiology-heart and Circulatory Physiology, 2016Co-Authors: Matthew R Zeglinski, Patricia Roche, Michael P. Czubryt, Jeffrey T Wigle, Mark Hnatowich, Davinder S Jassal, Ian M C DixonAbstract:In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.
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Additional file 4: Figure S3. of The transcription factor Scleraxis is a critical regulator of cardiac fibroblast phenotype
2016Co-Authors: Rushita Bagchi, Ronen Schweitzer, Patricia Roche, Nina Aroutiounova, Leon Espira, Bernard Abrenica, Michael P. CzubrytAbstract:Capillary density and cardiomyocyte cross-sectional area analysis of cardiac sections from WT and Scleraxis KO mice. (A) Cardiac tissue sections (6 μm) from WT and Scleraxis KO mice were stained with Rhodamine-labeled GSL I and capillaries counted to assess density. (B) Results for n = 3 independent samples per genotype (two fields/sample); mean ± SEM; *P
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abstract 12519 regulation of cardiac fibroblast phenotype by Scleraxis
Circulation, 2014Co-Authors: Rushita A. Bagchi, Ronen Schweitzer, Patricia Roche, Michael P. CzubrytAbstract:Cardiac fibroblasts constitute the primary extracellular matrix synthesis machinery in the myocardium. Activation of fibroblasts into a hyper-synthetic and contractile phenotype potentiates fibrosi...