Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFβ-induced Nox4 expression and myofibroblast transdifferentiation

Talha Ijaz, Mohammad Jamaluddin, Yingxin Zhao, Yueqing Zhang, Jayson Jay, Celeste Finnerty, David Herndon, Ronald Tilton, Allan R. Brasier

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)e2606
JournalCell death & disease
Volume8
Issue number2
DOIs
StatePublished - Feb 9 2017

Fingerprint

Cyclin-Dependent Kinase 9
Myofibroblasts
Fibroblasts
Hypertrophic Cicatrix
Skin
Collagen
Fibronectins
Smooth Muscle Myocytes
Actins
Peptide Elongation Factors
RNA Polymerase II
Extracellular Matrix Proteins
Immunoprecipitation
Epigenomics
Wound Healing
Small Interfering RNA
Cicatrix
Reactive Oxygen Species
Transcription Factors
Up-Regulation

ASJC Scopus subject areas

  • Immunology
  • Cellular and Molecular Neuroscience
  • Cell Biology
  • Cancer Research

Cite this

Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFβ-induced Nox4 expression and myofibroblast transdifferentiation. / Ijaz, Talha; Jamaluddin, Mohammad; Zhao, Yingxin; Zhang, Yueqing; Jay, Jayson; Finnerty, Celeste; Herndon, David; Tilton, Ronald; Brasier, Allan R.

In: Cell death & disease, Vol. 8, No. 2, 09.02.2017, p. e2606.

Research output: Contribution to journalArticle

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abstract = "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.",
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AU - Zhang, Yueqing

AU - Jay, Jayson

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AU - Herndon, David

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