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--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.

Wilms’ tumor 1 drives fibroproliferation and myofibroblast transformation in severe fibrotic lung disease

“…αSMA YFP reporter mice used in the confocal analysis of WT1 expression in embryonic lung development and transcript analysis were described previously and used to efficiently label embryonic and adult smooth muscle cells (62,63). The heterozygous αSMA CreERT2/mTmG male transgenic mouse strain was generated as previously described (30). These mice express CreERT2 under the control of the promoter for mouse smooth muscle myosin, heavy polypeptide 11 and have been shown to track the genetic lineage of α-SMA-expressing cells (64).…”

“…SiRNA transfections. Primary human or mouse fibroblast cells were transfected with stealth siRNA (human WT1 siRNA [catalog HSS111390, Invitrogen], mouse WT1 siRNA [catalog MSS212628, Invitrogen], or stealth negative control siRNA [catalog 12935300, Invitrogen]) using the Lipofectamine 3000 Transfection kit (catalog L3000-015, Invitrogen) as previously described (30). Transfected cells were harvested 72 hours after transfection and used for RNA isolation and whole transcriptome analysis.…”

“…RNA sequencing (RNA-sequencing; RNA-Seq) showed that the knockdown of WT1 altered the expression of 2,425 genes, half of which were induced (1,193 genes) and half of which were repressed (1,232 genes) by WT1 (Supplemental Figure 4). To identify profibrotic gene transcripts altered by WT1 in IPF, we performed an enrichment analysis of the negatively correlated gene sets between IPF lungs and WT1-specific siRNA-treated lung fibroblasts (30). Gene expression profiles of IPF lungs from a previously published transcriptomic data set (8) (Gene Expression Omnibus [GEO] accession number GSE53845; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) were compared with that of WT1 altered genes in fibroblasts, and we identified multiple transcripts that were either upregulated (107 genes) or downregulated (63 genes) by WT1 in IPF (Supplemental Figure 5A, Supplemental Table 1).…”

The BAG3-dependent and -independent roles of cardiac small heat shock proteins

“…These multifaceted interactions enable BAG3 to assemble large multichaperone complexes (22)(23)(24) that play essential roles in cardiac protein homeostasis, cardiac structure, and cardiac function (39,40).…”

“…Binding with BAG3 promotes the deoligomerization of sHSPs, likely because BAG3 competes with the self-interactions among sHSPs, which normally stabilize large oligomers (23). Forming a complex with HSP70 through BAG3 is essential to the chaperone function of sHSPs because sHSPs lack the enzymatic activity that appears to be required for active remodeling or refolding by the other categories of chaperones (23,39,43,44). Thus, the sHSPs-BAG3-HSP70 complex is essential for denatured proteins to refold to protect against protein aggregation.…”