Therapeutic induction of Bcl2-associated athanogene 3-mediated autophagy in idiopathic pulmonary fibrosis.

BACKGROUND: Exaggerated fibroblast proliferation is a well-known feature in idiopathic pulmonary fibrosis (IPF) which may be - in part - due to insufficient autophagy, a lysosome dependent cellular surveillance pathway. Bcl2-associated athanogene 3 (BAG3) is a pivotal co-chaperone of the autophagy pathway. Here, we studied whether therapeutic modulation of BAG3-mediated autophagy can rescue insufficient autophagy and impact IPF fibroblast proliferation. METHODS: Primary interstitial fibroblasts or precision cut lung slices (PCLS) of IPF lungs were treated with (1) the antifibrotic drug pirfenidone (Pirf), (2) the demethylating agent 5-azacytidine (Aza), (3) the BAG3 modulator cantharidin (Ctd). Autophagy flux was measured following pretreatment with the autophagy inhibitors or by GFP-RFP-LC3B transfection followed by drug treatments. Proliferation was measured by 5-bromo-2'-deoxyuridine assay. BAG3, filamin C (FLNC), proliferating-cell-nuclear-antigen (PCNA), collagen1A1 (COL1A1) and autophagy proteins were assessed by immunoblotting or immunofluorescence. Loss of function experiments were performed by siRNA mediated knockdown of BAG3. RESULTS: In comparison with healthy donors, increased BAG3 protein was observed in IPF lung homogenates and IPF fibroblasts. In addition, the substrate of BAG3-mediated autophagy, FLNC, was increased in IPF fibroblasts, implying insufficient activation of BAG3-dependent autophagy. Therapeutic modulation of this pathway using Aza and Ctd alone or in combination with the IPF therapy drug Pirf rescued the insufficient BAG3-mediated autophagy and decreased fibroblast proliferation. Such effects were observed upon therapeutic modulation of BAG3 but not upon knock down of BAG3 per se in IPF fibroblasts. Similarly, PCLS of IPF patients showed a significant decrease in collagen deposition in response to these drugs, either alone or in a more potent form in combination with Pirf. CONCLUSIONS: Our study reveals that repurposing drugs that modulate autophagy regulating proteins render therapeutic benefits in IPF. Fine tuning of this pathway may hence signify a promising therapeutic strategy to ameliorate antifibrotic properties and augment the efficacy of current IPF therapy.

PACS2-TRPV1 axis is required for ER-mitochondrial tethering during ER stress and lung fibrosis.

Endoplasmic reticulum (ER) and mitochondria (mito) play a vital role in alveolar type II cell (AEC2) homeostasis and are both stressed in patients with idiopathic pulmonary fibrosis (IPF). Up to now, no data are available with regard to ER-mito cross talk and tethering under conditions of IPF. We here demonstrate that ER-mitochondrial tethering is reduced upon experimental ER stress in vitro and in the IPF AECII ex vivo, and this is-at least in part-due to decreased phosphofurin acidic cluster sorting protein 2 (PACS-2, also called PACS2) protein levels. PACS2 levels are influenced by its interaction with the transient receptor potential cation channel subfamily V member 1 (TRPV1) and can be experimentally modified by the TRPV1-modulating drug capsaicin (CPS). Employing alveolar epithelial cells with overexpression of the terminal ER stress signaling factor Chop or the IPF-associated surfactant protein C mutation (SPCΔexon4) in vitro, we observed a restoration of PACS2 levels upon treatment with CPS. Similarly, treatment of precision cut lung slices from IPF patients with CPS ex vivo forwarded similar effects. Importantly, in all models such kind of intervention also greatly reduced the extent of alveolar epithelial apoptosis. We therefore conclude that therapeutic targeting of the PACS2-TRPV1 axis represents an interesting novel, epithelial-protective approach in IPF.