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

Abstract

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.

Keywords: 5-azacytidine; BAG3; autophagy; cantharidin; fibroblasts; filamin C; idiopathic pulmonary fibrosis; pirfenidone.

FIGURE 1

BAG3‐mediated autophagy is insufficient in IPF fibroblasts. (A) Immunoblot analysis of BAG3, BAG1 or β‐actin from lung homogenates (LH) of IPF patients or healthy donors (HD) (left). The right part shows respective quantifications after normalizing their integrated density values (IDV) from n = 9 IPF patients and 5 HD. (B) Immunoblots from cell lysates of primary interstitial fibroblasts of IPF or HD for the indicated proteins. The right part shows respective IDVs that were normalized to β‐actin from n = 8 each for IPF and HD. p value summary: *p ≤0 .05, ** p ≤ 0.01, *** p ≤0.001. Abbreviation: ns, not significant.

FIGURE 2

Insufficient autophagy in IPF fibroblasts. (A) Immunoblots from cell lysates of primary interstitial fibroblasts of IPF or HD for the indicated proteins. The right part shows IDVs of LC3BII or p62 were normalized to β‐actin. Blots and quantifications were performed from fibroblasts derived from n = 8 each for IPF and HD. (B) IPF or HD interstitial fibroblasts were pretreated with bafilomycin A1 (Baf) or chloroquine (CQ) for 2 h. Cells were lysed and the lysates were subjected to western blotting of LC3B and β‐actin. Two independent duplicates of treated IPF and HD samples were loaded along with one vehicle sample (left). IDVs for LC3BII were normalized to β‐actin (right) from n = 5 each for IPF or HD. p‐Value summary: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Abbreviation: ns, not significant.

FIGURE 3

Pirfenidone (Pirf), 5‐azacytidine (Aza) and cantharidin (Ctd) increase autophagy flux in IPF fibroblasts. (A) IPF fibroblasts were pretreated with Baf, followed by treatments with vehicle (Veh), pirfenidone (Pirf), 5‐azacytidine (Aza), or cantharidin (Ctd) at the indicated concentrations for 4 h in the presence of Baf. Cells were lysed, and immunoblots were performed for LC3B and β‐actin. Relative LC3BII protein levels from n = 5 IPF patients were normalized to β‐actin. For the sake of understanding, LC3BII expression in Veh‐treated cell lysates was set as one. (B) IPF fibroblasts were transiently transfected with ptf‐LC3B (mRFP‐GFP‐LC3B) followed by treatments with Veh, Pirf, Aza or Ctd for 24 h at the indicated concentrations. Cells were then fixed and visualized by fluorescence microscopy for GFP or RFP puncta or for their co‐localization. Nuclei were stained with DAPI, scale bar = 15 µm. Puncta representing autophagosomes or autolysosomes or both were measured per cell using Image J, and their ratio was calculated for each treatment. Fibroblasts from five IPF patients were used for transfection, treatments and analysis. p Value summary: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

FIGURE 4

Pirf, Aza and Ctd activate BAG3‐mediated autophagy and decrease IPF fibroblast proliferation. (A) IPF interstitial fibroblasts were treated with Veh, Pirf, Aza or Ctd alone or Pirf in combination with Aza or Ctd as indicated for 24 h. Cells were lysed, and immunoblotting was performed for the indicated proteins (left). Relative protein amounts from five IPF patients were normalized to β‐actin and their fold change was obtained after the values in Veh‐treated controls was set as one (right). (B) Representative immunofluorescence staining for PCNA (red) in IPF fibroblasts after treating with Veh or the indicated drugs for 24 h. Nuclei were stained with DAPI (blue), scale bar = 25 µm. In the right panel, fluorescence intensity of PCNA was quantified using ImageJ. Analysis was performed from n = 5 IPF patient fibroblasts. (C) Proliferation of IPF fibroblasts (n = 5) as assessed by BrdU incorporation following indicated drug treatments for 24 h. Triplicates were measured, and absorbance values were set to one in Veh‐treated cells for the ease of understanding. (D) Analysis of BAG3 mRNA using qRT‐PCR in fibroblasts derived from IPF or HD lungs after treating them with the indicated drugs. Values were normalized to the house‐keeping gene β‐actin (ACTB). BAG3 mRNA expression in Veh‐treated IPF/HD fibroblasts was set as one. p‐Value summary: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 show significant difference as compared to Veh‐treated cells and ^§ p ≤ 0.05, ^§§ p ≤ 0.01, ^§§§ p ≤ 0.001 indicate significant difference as compared to Pirf‐treated cells

FIGURE 5

Therapeutic modulation but not siBAG3 per se affects autophagy in IPF fibroblasts. (A) IPF fibroblasts were transfected either with nontargeting siRNA (si‐NT) or with BAG3 siRNA (si‐BAG3) for 48 h followed by treatments with the indicated drugs for 24 h. Cell lysates were prepared, and western blots were performed for the indicated proteins. Bar graphs represent IDVs of BAG3, FLNC, LC3BII or α‐SMA that were normalized to β‐actin. Blots and analysis were performed from n = 3 IPF patient fibroblasts. p Value summary: *** p ≤ 0.001, significant difference between si‐NT and si‐BAG3 groups. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 show significant difference between Veh‐treated and the respective drug treated groups in cells transfected with si‐NT. ^§ p ≤ 0.05, ^§§ p ≤ 0.01, ^§§§ p ≤ 0.001 show significant difference between si‐BAG3 transfected cells with the indicated drug treatments versus their respective drug‐treated counterparts in si‐NT transfected cells

FIGURE 6

Therapeutic modulation of BAG3 decreases proliferation and COL1A1 in IPF PCLS. PCLS from explanted IPF patient lungs were treated with drugs, as indicated for 24 h. (A) PCLS lysates were prepared and tested for the indicated proteins via western blotting (top panel). Lower panels represent the quantifications of the indicated proteins after with β‐actin. Values in Veh‐treated PCLS were set as one. Treatment and representative analysis and blots from n = 3 IPF patients are shown. (B) Immunofluorescence staining for COL1A1 (red) on IPF PCLS upon Veh or drug treatments for 24 h has indicated. Nuclei were stained with DAPI (blue), scale bar = 200 µm. Lower panel indicates COL1A1 fluorescence intensity after drug treatments. Stainings and analysis were performed from n = 3 IPF PCLS per each group and about three technical replicates were performed. p Value summary: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 show significant difference as compared to Veh‐treated PCLS and ^§ p ≤ 0.05, ^§§ p ≤ 0.01, ^§§§ p ≤ 0.001 indicate significant difference as compared to Pirf‐treated PCLS

FIGURE 7

Schematic representation of this study. Restoring insufficient BAG3‐mediated autophagy in IPF fibroblasts by the indicated drugs. The slope represents the synergistic effect of the indicated drugs on BAG3 inhibition. Small font indicates decreased expression or decreased autophagy flux and vice versa. To the left, highly proliferative IPF fibroblasts (green) are depicted, enwrapped in thick extracellular matrix (brown) with increased BAG3, FLNC and COL1A1 and insufficient BAG3‐mediated autophagy. To the right, decreased IPF fibroblast proliferation, decreased FLNC, COL1A1 and BAG3 protein levels and restored autophagy upon treatments with the indicated drugs is shown