Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFβ-induced Nox4 expression and myofibroblast transdifferentiation.
Transdifferentiation of quiescent dermal fibroblasts to secretory myofibroblasts has a central role in wound healing and pathological scar formation. This myofibroblast transdifferentiation process involves TGFβ-induced de novo synthesis of alpha smooth muscle cell actin (αSMA)+ fibers that enhance contractility as well as increased expression of extracellular matrix (ECM) proteins, including collagen and fibronectin. These processes are mediated upstream by the reactive oxygen species (ROS)-producing enzyme Nox4, whose induction by TGFβ is incompletely understood. In this study, we demonstrate that Nox4 is involved in αSMA+ fiber formation and collagen production in primary human dermal fibroblasts (hDFs) using a small-molecule inhibitor and siRNA-mediated silencing. Furthermore, TGFβ-induced signaling via Smad3 is required for myofibroblast transformation and Nox4 upregulation. Immunoprecipitation-selected reaction monitoring (IP-SRM) assays of the activated Smad3 complex suggest that it couples with the epigenetic reader and transcription co-activator bromodomain and extraterminal (BET) domain containing protein 4 (BRD4) to promote Nox4 transcription. In addition, cyclin-dependent kinase 9 (CDK9), a component of positive transcription elongation factor, binds to BRD4 after TGFβ stimulation and is also required for RNA polymerase II phosphorylation and Nox4 transcription regulation. Surprisingly, BRD4 depletion decreases myofibroblast differentiation but does not affect collagen or fibronectin expression in primary skin fibroblasts, whereas knockdown of CDK9 decreases all myofibroblast genes. We observe enhanced numbers and persistence of myofibroblast formation and TGFβ signaling in hypertrophic scars. BRD4 inhibition reverses hypertrophic skin fibroblast transdifferentiation to myofibroblasts. Our data indicate that BRD4 and CDK9 have independent, coordinated roles in promoting the myofibroblast transition and suggest that inhibition of the Smad3-BRD4 pathway may be a useful strategy to limit hypertrophic scar formation after burn injury.
Department of Biochemistry and Molecuilar Biology, University of Texas Medical Branch, Galveston, TX, USA.|MD-PhD Program, University of Texas Medical Branch, Galveston, TX, USA.
Talha
T
Ijaz
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Internal Medicine - Division of Endocrinology, University of Texas Medical Branch, Galveston, TX, USA.
Mohammad
M
Jamaluddin
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Internal Medicine - Division of Endocrinology, University of Texas Medical Branch, Galveston, TX, USA.|Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX, USA.
Yingxin
Y
Zhao
Internal Medicine - Division of Endocrinology, University of Texas Medical Branch, Galveston, TX, USA.
Yueqing
Y
Zhang
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Shriner's Hospital for Children, University of Texas Medical Branch, Galveston, TX, USA.
Jayson
J
Jay
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Shriner's Hospital for Children, University of Texas Medical Branch, Galveston, TX, USA.|Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA.
Celeste C
CC
Finnerty
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Shriner's Hospital for Children, University of Texas Medical Branch, Galveston, TX, USA.|Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA.
David N
DN
Herndon
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Internal Medicine - Division of Endocrinology, University of Texas Medical Branch, Galveston, TX, USA.|Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX, USA.
Ronald G
RG
Tilton
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA.|Internal Medicine - Division of Endocrinology, University of Texas Medical Branch, Galveston, TX, USA.|Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX, USA.
Allan R
0000-0002-5012-4090
AR
Brasier
D000293:Adolescent; D018797:Cell Cycle Proteins; D054338:Cell Transdifferentiation / Q000502:physiology*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003094:Collagen / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D058628:Myofibroblasts / Q000378:metabolism* / Q000502:physiology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein; D014157:Transcription Factors / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology; D014945:Wound Healing / Q000502:physiology; D000092468:Bromodomain Containing Proteins
D018797:Cell Cycle Proteins; D003094:Collagen; D042863:Cyclin-Dependent Kinase 9; D005353:Fibronectins; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; D009687:Nuclear Proteins; D051900:Smad3 Protein; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D016212:Transforming Growth Factor beta; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C498910:NOX4 protein, human; C467969:CDK9 protein, human