Disruption of Transforming Growth Factor β Signaling and Profibrotic Responses in Normal Skin Fibroblasts by Peroxisome Proliferator-Activated Receptor γ

Asish K. Ghosh, Swati Bhattacharyya, Gabriella Lakos, Shu Jen Chen, Yasuji Mori, John Varga*

*Corresponding author for this work

Research output: Contribution to journalArticle

160 Citations (Scopus)

Abstract

Objective. In fibroblasts, transforming growth factor β (TGFβ) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGFβ-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGFβ/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) in normal skin fibroblasts and its effect on TGFβ-induced cellular responses. Methods. The expression and activity of PPARγ in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPARγ activation by naturally occurring and synthetic ligands on collagen synthesis and α-smooth muscle actin (α-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPARγ expression constructs. Results. The PPARγ receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPARγ resulted in modest suppression of basal collagen gene expression, it abrogated TGFβ-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPARγ in fibroblasts, and was blocked by a selective antagonist of PPARγ signaling or by transfection of fibroblasts with dominant-negative PPARγ constructs. Furthermore, PPARγ ligands abrogated TGFβ-induced expression of α-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGFβ was suppressed by PPARγ despite the absence of a consensus PPARγ-response element in the targeted promoters. Ligand-induced activation of fibroblast PPARγ had no effect on protein expression of cellular Smad3 or Smad7. Conclusion. By abrogating of TGFβ-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPARγ may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPARγ activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

Original languageEnglish (US)
Pages (from-to)1305-1318
Number of pages14
JournalArthritis and Rheumatism
Volume50
Issue number4
DOIs
StatePublished - Apr 1 2004

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Peroxisome Proliferator-Activated Receptors
Transforming Growth Factors
Fibroblasts
Skin
Myofibroblasts
Collagen
Ligands
Transfection
Fibrosis
Gene Expression
Fibroblast Growth Factors
Response Elements
Cytoplasmic and Nuclear Receptors
Northern Blotting

ASJC Scopus subject areas

  • Immunology and Allergy
  • Rheumatology
  • Immunology
  • Pharmacology (medical)

Cite this

@article{d8847ae1a282493ea64d5ff1fc1d6c12,
title = "Disruption of Transforming Growth Factor β Signaling and Profibrotic Responses in Normal Skin Fibroblasts by Peroxisome Proliferator-Activated Receptor γ",
abstract = "Objective. In fibroblasts, transforming growth factor β (TGFβ) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGFβ-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGFβ/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) in normal skin fibroblasts and its effect on TGFβ-induced cellular responses. Methods. The expression and activity of PPARγ in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPARγ activation by naturally occurring and synthetic ligands on collagen synthesis and α-smooth muscle actin (α-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPARγ expression constructs. Results. The PPARγ receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPARγ resulted in modest suppression of basal collagen gene expression, it abrogated TGFβ-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPARγ in fibroblasts, and was blocked by a selective antagonist of PPARγ signaling or by transfection of fibroblasts with dominant-negative PPARγ constructs. Furthermore, PPARγ ligands abrogated TGFβ-induced expression of α-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGFβ was suppressed by PPARγ despite the absence of a consensus PPARγ-response element in the targeted promoters. Ligand-induced activation of fibroblast PPARγ had no effect on protein expression of cellular Smad3 or Smad7. Conclusion. By abrogating of TGFβ-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPARγ may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPARγ activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.",
author = "Ghosh, {Asish K.} and Swati Bhattacharyya and Gabriella Lakos and Chen, {Shu Jen} and Yasuji Mori and John Varga",
year = "2004",
month = "4",
day = "1",
doi = "10.1002/art.20104",
language = "English (US)",
volume = "50",
pages = "1305--1318",
journal = "Arthritis and Rheumatology",
issn = "2326-5191",
publisher = "John Wiley and Sons Ltd",
number = "4",

}

TY - JOUR

T1 - Disruption of Transforming Growth Factor β Signaling and Profibrotic Responses in Normal Skin Fibroblasts by Peroxisome Proliferator-Activated Receptor γ

AU - Ghosh, Asish K.

AU - Bhattacharyya, Swati

AU - Lakos, Gabriella

AU - Chen, Shu Jen

AU - Mori, Yasuji

AU - Varga, John

PY - 2004/4/1

Y1 - 2004/4/1

N2 - Objective. In fibroblasts, transforming growth factor β (TGFβ) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGFβ-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGFβ/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) in normal skin fibroblasts and its effect on TGFβ-induced cellular responses. Methods. The expression and activity of PPARγ in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPARγ activation by naturally occurring and synthetic ligands on collagen synthesis and α-smooth muscle actin (α-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPARγ expression constructs. Results. The PPARγ receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPARγ resulted in modest suppression of basal collagen gene expression, it abrogated TGFβ-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPARγ in fibroblasts, and was blocked by a selective antagonist of PPARγ signaling or by transfection of fibroblasts with dominant-negative PPARγ constructs. Furthermore, PPARγ ligands abrogated TGFβ-induced expression of α-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGFβ was suppressed by PPARγ despite the absence of a consensus PPARγ-response element in the targeted promoters. Ligand-induced activation of fibroblast PPARγ had no effect on protein expression of cellular Smad3 or Smad7. Conclusion. By abrogating of TGFβ-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPARγ may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPARγ activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

AB - Objective. In fibroblasts, transforming growth factor β (TGFβ) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGFβ-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGFβ/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) in normal skin fibroblasts and its effect on TGFβ-induced cellular responses. Methods. The expression and activity of PPARγ in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPARγ activation by naturally occurring and synthetic ligands on collagen synthesis and α-smooth muscle actin (α-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPARγ expression constructs. Results. The PPARγ receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPARγ resulted in modest suppression of basal collagen gene expression, it abrogated TGFβ-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPARγ in fibroblasts, and was blocked by a selective antagonist of PPARγ signaling or by transfection of fibroblasts with dominant-negative PPARγ constructs. Furthermore, PPARγ ligands abrogated TGFβ-induced expression of α-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGFβ was suppressed by PPARγ despite the absence of a consensus PPARγ-response element in the targeted promoters. Ligand-induced activation of fibroblast PPARγ had no effect on protein expression of cellular Smad3 or Smad7. Conclusion. By abrogating of TGFβ-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPARγ may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPARγ activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

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