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 4

Hsp90 regulates the proliferation of fibroblasts in fibrotic lesions.

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Hsp90 regulates the proliferation of fibroblasts in fibrotic lesions. (A...
(A) Primary lung fibroblasts (CD45–Col1+) isolated from lung fibroblast cultures of human idiopathic pulmonary fibrosis (IPF) lungs by negative selection with anti-CD45 magnetic beads. Fibroblasts were treated with vehicle or 17-AAG (0.5 μM) for 24 hours and immunostained with antibodies against proliferating cell nuclear antigen (PCNA). Scale bars: 40 μm. (B) The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software (n = 3–4/group). Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. Results are cumulative of 2 independent experiments (n = 3–4/group). (C) Quantification of proliferation using the BrdU incorporation assay in lung resident fibroblasts isolated from lung cell cultures of TGF-α–transgenic (TGF-α–Tg) mice on doxycycline (Dox) for 4 weeks. Fibroblasts treated with indicated doses of 17-AAG for 48 hours from beginning of the treatment relative to vehicle is plotted. One-way ANOVA with Sidak’s multiple comparisons test was used to measure significant difference. (D) Extent of proliferation was measured using BrdU incorporation assay in lung fibroblasts isolated from lung cell cultures of TGF-α–Tg mice on Dox for 4 weeks and treated with control, Hsp90AA-specific, or Hsp90AB-specific siRNA for 72 hours. Results are cumulative of 2 independent experiments (n = 4/group). One-way ANOVA with Sidak’s multiple comparisons test was used to measure significant difference. (E) Quantification of Cdk4, Igf1, Mycn, and Sphk1 gene transcripts relative to Hprt in the lung-resident fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with vehicle or 17-AAG (0.2 μM) for 24 hours. Results are representative of 2 independent experiments with similar results (n = 4/group). (F) Quantification of Cdk4, Igf1, Mycn, and Sphk1 gene transcripts relative to Hprt in the lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with control, Hsp90AA-specific, or Hsp90AB-specific siRNA for 72 hours. Results are representative of 2 independent experiments (n = 3–4/group). Data shown are mean ± SEM. An unpaired 2-tailed Student’s t test was performed to measure the significance. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005.