Hsp90 regulation of fibroblast activation in pulmonary fibrosis
Vishwaraj Sontake, … , Anil G. Jegga, Satish K. Madala
Vishwaraj Sontake, … , Anil G. Jegga, Satish K. Madala
Published February 23, 2017
Citation Information: JCI Insight. 2017;2(4):e91454. https://doi.org/10.1172/jci.insight.91454.
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Research Article Immunology Pulmonology

Hsp90 regulation of fibroblast activation in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-β–driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease.

Authors

Vishwaraj Sontake, Yunguan Wang, Rajesh K. Kasam, Debora Sinner, Geereddy B. Reddy, Anjaparavanda P. Naren, Francis X. McCormack, Eric S. White, Anil G. Jegga, Satish K. Madala

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Figure 6

Hsp90 inhibition attenuates TGF-β–induced myofibroblast transformation and ECM gene expression.

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Hsp90 inhibition attenuates TGF-β–induced myofibroblast transformation a...
(A) Primary lung-resident fibroblasts (CD45–Col1+) were isolated from lung cell cultures of αSMAcreERROSAmTmG mice and treated with TGF-β and 4-hydroxy tamoxifen in the presence and absence of 17-AAG for 72 hours. Immunofluorescence images were obtained at an original magnification of ×10. Scale bars: 200 μm. (B) The number of GFP-positive and total DAPI-positive (blue) cells were quantified using MetaMorph image analysis software (n = 4–5/group) and are indicated as the percentage αSMA-positive cells in total DAPI-positive cells. Data are cumulative of 2 independent experiments with similar results. (C and D) Human lung-resident fibroblasts (CD45–Col1+) were isolated from non–idiopathic pulmonary fibrosis (non-IPF) lung fibroblast cultures and treated with TGF-β in the presence and absence of 17-AAG for 24 hours. Transcripts of extracellular matrix (ECM) genes COL1α and FN1 were quantified using qRT-PCR and are shown as the fold-induced gene transcripts relative to GAPDH. Data are representative of 2 independent experiments with similar results (n = 4–5/group). (E) Lung-resident fibroblasts of αSMAcreERROSAmTmG mice were transfected with control, Hsp90AA-specific, or Hsp90AB-specific siRNA. After a 24-hour transfection, cells were treated with TGF-β and 4-hydroxy tamoxifen for 72 hours and the number of GFP-positive and total DAPI-positive cells were quantified using MetaMorph image analysis software (n = 4–5/group). Data are cumulative of 2 independent experiments with similar results. One-way ANOVA with Sidak’s multiple comparisons test was used to measure significant difference for all the experiments. All data shown are mean ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005. ns, not significant.