Hsp90 regulation of fibroblast activation in pulmonary fibrosis
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
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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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 2

Hsp90 and its ATPase activity elevated in fibrotic lungs.

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Hsp90 and its ATPase activity elevated in fibrotic lungs. (A) Idiopathic...
(A) Idiopathic pulmonary fibrosis (IPF, n = 4) and non-IPF (n = 6) lung tissue sections immunostained with antibodies against Hsp90 (brown) and vimentin (purple). All images obtained at low (×5; scale bars = 400 μm) and high (×40; scale bars = 50 μm) magnification. Dashed boxes indicate the area of the lung shown in high-magnification images. Arrows indicate Hsp90 staining in the cytoplasm and nucleus of vimentin-positive lung cells. Hsp90 localization was observed in epithelial cells and vimentin-positive cells of fibrotic foci. (B) The lung lysates immunoblotted with antibodies against Hsp90 to measure increases in Hsp90 in the fibrotic lungs of TGF-α–transgenic (TGF-α–Tg) mice compared with control mice on doxycycline (Dox) for 6 weeks. (C) Quantification of Hsp90 levels using Phosphor Imager software, and the amount of Hsp90 normalized with GAPDH levels in the lung lysates of TGF-α–Tg mice compared with control mice on Dox for 6 weeks (n = 2/group). (D) Hsp90 ATPase activity measured in the lung lysates of control or TGF-α–Tg mice on Dox for 6 weeks (n = 3/group). (E) Hsp90 ATPase activity measured in the lysates of fibroblasts isolated from IPF and non-IPF lungs (n = 4/group). (F) Hsp90 binding affinity towards 17-AAG was evaluated in a competitive binding assay using a biotinylated geldanamycin (biotin-GM) probe and increasing concentrations of 17-AAG in the lung lysates of control mice or TGF-α–Tg mice on Dox for 6 weeks. (G) The percentage inhibition of biotin-GM binding to Hsp90 with increasing doses of 17-AAG in lysates isolated from fibrotic lungs compared with normal lungs. The above results are representative of 2 to 3 independent experiments and reported as mean ± SEM. An unpaired 2-tailed Student’s t test was performed to measure the significance. *P < 0.05, **P < 0.005, and ***P < 0.0005.