Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli

Amos Dodi, Iyabode O. Ajayi, Christine Chang, Meghan Beard, Shanna L. Ashley, Steven K. Huang, Victor J. Thannickal, Daniel J. Tschumperlin, Thomas H. Sisson, Jeffrey C. Horowitz

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Methods: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Results: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Conclusions: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

Original languageEnglish (US)
Article number91
JournalRespiratory Research
Volume19
Issue number1
DOIs
StatePublished - May 10 2018
Externally publishedYes

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Fibroblasts
Apoptosis
Lung
Western Blotting
Enzyme-Linked Immunosorbent Assay
Focal Adhesion Protein-Tyrosine Kinases
Fas Ligand Protein
Elastic Modulus
Protein Kinase Inhibitors
Conditioned Culture Medium
Epigenomics
Plastics
Compliance
Real-Time Polymerase Chain Reaction
Cell Death
Cytokines
Phenotype
Messenger RNA
Wounds and Injuries
Proteins

Keywords

  • Apoptosis
  • CD-95
  • Extracellular matrix
  • Fibrosis
  • Lung injury
  • Myofibroblast
  • Wound-repair

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine

Cite this

Dodi, A., Ajayi, I. O., Chang, C., Beard, M., Ashley, S. L., Huang, S. K., ... Horowitz, J. C. (2018). Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli. Respiratory Research, 19(1), [91]. https://doi.org/10.1186/s12931-018-0801-4

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli. / Dodi, Amos; Ajayi, Iyabode O.; Chang, Christine; Beard, Meghan; Ashley, Shanna L.; Huang, Steven K.; Thannickal, Victor J.; Tschumperlin, Daniel J.; Sisson, Thomas H.; Horowitz, Jeffrey C.

In: Respiratory Research, Vol. 19, No. 1, 91, 10.05.2018.

Research output: Contribution to journalArticle

Dodi, A, Ajayi, IO, Chang, C, Beard, M, Ashley, SL, Huang, SK, Thannickal, VJ, Tschumperlin, DJ, Sisson, TH & Horowitz, JC 2018, 'Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli', Respiratory Research, vol. 19, no. 1, 91. https://doi.org/10.1186/s12931-018-0801-4
Dodi, Amos ; Ajayi, Iyabode O. ; Chang, Christine ; Beard, Meghan ; Ashley, Shanna L. ; Huang, Steven K. ; Thannickal, Victor J. ; Tschumperlin, Daniel J. ; Sisson, Thomas H. ; Horowitz, Jeffrey C. / Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli. In: Respiratory Research. 2018 ; Vol. 19, No. 1.
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abstract = "Background: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Methods: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Results: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Conclusions: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.",
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AU - Dodi, Amos

AU - Ajayi, Iyabode O.

AU - Chang, Christine

AU - Beard, Meghan

AU - Ashley, Shanna L.

AU - Huang, Steven K.

AU - Thannickal, Victor J.

AU - Tschumperlin, Daniel J.

AU - Sisson, Thomas H.

AU - Horowitz, Jeffrey C.

PY - 2018/5/10

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N2 - Background: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Methods: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Results: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Conclusions: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

AB - Background: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Methods: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Results: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Conclusions: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

KW - Apoptosis

KW - CD-95

KW - Extracellular matrix

KW - Fibrosis

KW - Lung injury

KW - Myofibroblast

KW - Wound-repair

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