Amplified canonical transforming growth factor beta signaling via heat shock protein 90 in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic and usually lethal lung disease of unknown causes with limited therapeutic options. Active myofibroblasts in the intestinal and fibroblast foci are responsible for excessive tissue remodeling in response to recurrent alveolar epithelial micro-injuries. Although the underlying mechanisms of IPF are not fully understood, transforming growth factor beta 1 (TGF-beta 1) is recognized as the key player in disease progression. HSP90 is a highly conserved chaperone protein and is essential for stabilization or degradation of many signaling proteins. HSP90 is centrally involved in the pathobiology of cancer and HSP90 inhibitors have been widely investigated in preclinical and clinical studies against cancer. Recent evidence shows that HSP90 promotes epithelial-mesenchymal transition (EMT) of cancer cells and TGF-beta 1 signaling via the stabilizing of the TGF-beta receptors, however little is known about the role of HSP90 in IPF. The purpose of the present study is to discover the role of HSP90 in lung fibroblast activation, particularly with regard to TGF-beta 1-induced profibrotic phenotype, and to determine the effects of HSP90 inhibitor on bleomycin-induced pulmonary fibrosis in mice. Immunohistochemistry demonstrated intensive immunoreactivity of HSP90 in activated pulmonary fibroblasts in lungs from IPF patients. Moreover, the expression profiling using western blot showed a significant upregulation of HSP90alpha; and HSP90beta in lungs as well as primary interstitial lung fibroblasts (ILFBs) derived from IPF patients when compared with donors. Those finding directed the study at effects of HSP90 inhibition on lung fibroblast activation and fibrotic response in vitro. Both resident fibroblasts and alveolar epithelial cells are sources of lung myofibroblasts via cytokine-induced transdifferentiation. TGF-beta 1, as such a crucial cytokine promoting fibrosis, was used to induce fibroblast activation and EMT of A549 cells in the in vitro study. 17-AGG, a selective HSP90 inhibitor, blocked ILFBs activation and collagen production induced by TGF-beta 1, as demonstrated by western blot of alpha-SMA expression and Sircol Soluble Collagen assay, respectively. In addition, transwell assay showed that 17-AAG inhibited ILFBs migration induced by serum. Interestingly, 17-AAG also prevented EMT of A549 cells induced by TGF-beta 1, revealed by western blot and immunofluorescence staining of mesenchymal and epithelial markers.Furthermore, the diminished protein level of TGF-beta RI and/or TGF-beta RII was observed, along with less Smad2 phosphorylation after treatment with 17-AAG in both ILFBs and A549 cells stimulated with TGF-beta 1. In addition, co-immunoprecipitation confirmed a direct interaction between TGF-beta RII and HSP90beta in ILFBs, which implies that TGF-beta receptors are clients of HSP90 and are stabilized by HSP90. And the result with MG-132, a proteasome inhibitor, indicates that the compromised interaction of HSP90 and TGF-beta Rs leads to proteasomal degradation of TGF-beta Rs.In light of the promising in vitro effects, the in vivo effects of the highly selective HSP90 inhibitor (17-DMAG) was evaluated in attenuation of pulmonary fibrosis, using the established bleomycin-induced pulmonary fibrosis mouse model. Western blot showed increased expression of HSP90 in ILFBs from bleomycin-challenged mice as compared to sham control. Treatment with 17-DMAG (10 or 25 mg/kg) for 14 days was carried out 7 days after bleomycin challenge. 17-DMAG rescued the bodyweight loss and improved the lung compliance. Pulmonary inflammation induced by bleomycin was attenuated by 17-DMAG treatment, indicated by inflammatory cell composition from bronchoalveolar lavage fluid (BALF) and less active matrix metalloproteases (MMPs) measured by in vivo fluorescence molecular tomography (FMT) in combined with micro-computed tomography (µCT). Histological assessment including Ashcroft fibrosis scoring and collagen deposition all suggested that 17-DMAG ameliorates bleomycin-induced pulmonary fibrosis.Taken together, this study supports a central role of HSP90 in progressive pulmonary fibrosis by manipulating TGF-beta/Smad signaling and suggests a therapeutic potential of HSP90 inhibitors against IPF.