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 3

Mechanisms of Hsp90-driven migration and invasion in fibroblasts.

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Mechanisms of Hsp90-driven migration and invasion in fibroblasts. Primar...
Primary lung fibroblasts (CD45–Col1+) isolated from lung fibroblast cultures of human idiopathic pulmonary fibrosis (IPF) or TGF-α–transgenic (TGF-α–Tg) mice on doxycycline (Dox) for 4 weeks by negative selection with anti-CD45 magnetic beads. (A) Quantification of migration of IPF fibroblasts treated with vehicle or 17-AAG (1 μM) for 24 hours (n = 6/group). (B) Quantification of invasiveness of IPF fibroblasts treated with vehicle or 17-AAG (1 μM) for 24 hours (n = 6/group). (C) Fibroblasts of TGF-α–Tg mice on Dox for 4 weeks were transiently transfected with control, Hsp90AA-specific, or Hsp90AB-specific siRNA for 48 hours and the migration was quantified for 24 hours (n = 6/group). (D) Costaining of F-actin and Hsp90 in lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks. Hsp90 colocalized (white signal) with F-actin in cytoplasmic fibrillary adhesions or focal complexes. Images were obtained at an original magnification of ×40. Scale bars: 40 μm. (E) F-actin stained using phalloidin in lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks treated with vehicle or 17-AAG (1 μM) for 24 hours. Scale bars: 40 μm. (F) Immunoblot analysis for FAK, p-Tyr576/577FAK, ERK, p-Tyr468ERK, cell division control protein 42 homolog (Cdc42), and Ras homolog gene family, member A (RhoA) in the lysates of lung fibroblast of TGF-α–Tg mice on Dox for 4 weeks treated with vehicle or 17-AAG (0.5 μM) for 48 hours. (G) The total expression and phosphorylation of the indicated proteins were measured and normalized to GAPDH levels in the total lysates (n = 3/group). (H) Quantification of Ccl3, Ccl20, Il-10, and Ccr5 gene transcripts relative to Hprt in the lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with vehicle or 17-AAG (0.5 μM) for 24 hours. (I) Quantification of Has2, Cd44, Mmp12, and Timp1 gene transcripts relative to Hprt in the lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with vehicle or 17-AAG (0.5 μM) for 24 hours (n = 3/group). Results are representation of 3 independent experiments with similar results. 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.