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

Figures

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  Fig. 1. ER–mitochondria tethering is impaired upon Chop overexpression. A MLE12 cells were either not induced or were induced with 1 µg/mL doxycycline for Chop expression for 12 and 24 h, followed by immunoblots for the given proteins from total cell lysates. The positions of molecular weight markers are shown. B and C Quantification of significant increase in cl.caspase 3 protein levels (C) upon Chop induction (B) obtained from (A). Relative protein amounts were normalized to Gapdh and their level in −dox cells was set as one. D Representative immunoflourescence images of MLE12 cells either non-induced or induced with dox for 24 h for Chop expression. Staining for ER (calnexin, green) and mitochondria (MitoTracker red) is shown. Nuclei were stained with DAPI (blue), scale bar = 60 µm. E Fluorescence intensity of co-localization was quantified using ImageJ, and its intensity in −dox cells was set to one. F Proximity ligation assay with antibodies against calnexin and TOM20 or calnexin and VDAC1, followed by fluorescence microscopy images are shown; scale bar = 60 µm. G and H Fluorescence intensity was quantified using ImageJ, and its intensity in uninduced cells was set to one. Blots,…

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  Fig. 2. Pacs2 is decreased upon Chop overexpression. A MLE12 cells were either not induced or were induced with 1 µg/ml doxycycline for Chop expression for 12 and 24 h, followed by immunoblots for the given proteins from total lysates. B Quantification of significant decrease in Pacs2 protein levels upon Chop induction. Relative protein amounts were normalized to Gapdh and their level in −dox cells was set as one. C Representative immunoflourescence images of Pacs2 (green) in −dox or + dox-treated cells for 24 h. Nuclei were stained with DAPI (blue), scale bar = 60 µm. D Fluorescence intensity was quantified using ImageJ, and its intensity in −dox cells was set to one. Blots and analysis were performed from n = 3 experiments and statistical significance is indicated as: **p ≤ 0.01

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  Fig. 3. Pacs2 overexpression rescues phenotype of Chop-induced cells. MLE12 cells were either mock transfected, or transfected with empty-Myc or Myc-PACS2 for 4 h, followed by no dox (− dox) or with dox (+ dox, 12 h) treatment for Chop overexpression, followed by (A) proximity ligation assay with antibodies against calnexin and VDAC1 and subsequent fluorescence microscopy, scale bar = 60 µm. B Fluorescence intensity was quantified using ImageJ, its intensity in untreated −dox cells was set to one. C Immunoblots for the indicated proteins. D Relative protein levels of Pacs2 and E cleaved caspase 3 were normalized to Gapdh and their level in −dox cells was set as one. Representative images and stainings from n of 3 independent experiments are shown, statistical significance is indicated as: *p ≤ 0.05, ***p ≤ 0.001

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  Fig. 4. Modulation of Trpv1 improves ER–mitochondrial tethering in cells overexpressing Chop. A Immunoblot analysis of Trpv1 in total cell lysates of MLE12 cells untreated or treated with dox for Chop expression for 12 and 24 h. B Relative protein level of Trpv1 were normalized to Gapdh and its level in −dox cells was set as one. C MLE12 cells were either left untreated or treated with dox for 12 h, followed by treatments with DMSO or CPS at indicated concentrations followed by immunoblots for the indicated proteins. D and E Quantification of Trpv1 (D) and Pacs2 (E) protein expression is shown. Relative protein amounts were normalized to Gapdh and their mean value in respective DMSO-treated controls was set as one. F Representative fluorescence images following proximity ligation assay with antibodies against calnexin and VDAC1, and cells treated with CPS at the indicated concentrations or DMSO upon Chop induction are shown, scale bar = 60 µm. G Fluorescence intensity was quantified using ImageJ, its intensity in −dox, DMSO-treated cells was set to one. ‘n’ of three independent experiments were performed and statistical significance is indicated as: *p ≤ 0.05, ***p ≤ 0.01, ***p ≤ 0.001

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  Fig. 5. Modulating PACS2–TRPV1 axis with CPS rescues ER–mitochondrial tethering and PACS2 protein in cells overexpressing SPCΔexon4. A MEL188 cells were stably transfected with either SPCWT or SPCΔexon4 plasmids or left un-transfected, followed by immunoblots for the given proteins. B and C Quantification of PACS2 (B) and CHOP (C) protein levels are shown. Relative protein amounts were normalized to GAPDH and their mean value in untransfected cells was set as one. D Representative fluorescence microscopy images following proximity ligation assay with antibodies against calnexin and VDAC1 in SPCWT or SPCΔexon4 overexpressing cells, scale bar = 60 µm. E Fluorescence intensity was quantified using ImageJ, and its intensity in untransfected cells was set to one. F Cells stably expressing SPCWTor SPCΔexon4 or control cells were treated with DMSO or CPS, followed by immunoblotting of PACS2 and GAPDH. G Quantification of PACS2 protein expression is shown. Relative protein amounts were normalized to GAPDH and its mean value in control cells was set as one. H Fluorescence microscopy images following proximity ligation assay with antibodies against calnexin and VDAC1 in control cells or in cells…

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  Fig. 6. PACS2 protein and ER–mitochondrial tethering are decreased in IPF AECII. A Representative immunofluorescence images for PACS2 (red) and ABCA3 (green) in IPF and Donor lung sections, and nuclei were stained with DAPI (blue) scale bar = 25 µm. B Fluorescence intensity of PACS2 was quantified using ImageJ, and its intensity in Donor sections was set as one. Lung sections from seven IPF to seven Donors were used for stainings. Statistical significance is indicated as: *p ≤ 0.05. C Representative transmission electron microscopic images from alveolar epithelial type II cells (characterized by the presence of lamellar bodies (LB)) are shown. At the ultrastructural level, the appearance of the contact zones between ER and mitochondria (Mito) in IPF differed from the healthy control. The closest contacts (arrows) appeared to be rather point shaped with the ER piston-like widened in IPF. In healthy controls, the contacts were in general elongated and more laminar so that the ER and mitochondrial membranes had a parallel run

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  Fig. 7. Modulation of TRPV1 decreases apoptosis and COLA1A level in PCLS of IPF patients. A PCLS from explanted IPF patient lungs were treated with either DMSO or CPS with the indicated concentrations for 8 h, followed by immunoblot analysis for PACS2, cleaved caspase 3 and GAPDH. B-D Quantification of PACS2 (B) and cl.caspase3 (C) protein levels is shown. Relative protein amounts were normalized to GAPDH and their mean value in DMSO-treated PCLS was set as one. D Immunofluorescence staining for cl.caspase 3 (red) in AECII (ABCA3, green) on IPF PCLS upon Veh, CPS or Nin treatment for 8 h. Nuclei were stained with DAPI (blue), scale bar = 60 µm. E Fluorescence intensity of cleaved caspase 3 in ABCA3-positive cells was quantified using ImageJ, and its intensity in DMSO-treated cells was set as one. F Immunofluorescence staining for COLA1A on IPF PCLS upon Veh, CPS or Nin treatment for 8 h. Nuclei were stained with DAPI (blue), scale bar = 60 µm. H Fluorescence intensity of COLA1A was quantified using ImageJ, and its intensity in DMSO-treated cells was set as one. Stainings and analysis were performed in PCLS performed from three IPF patients and significance is indicated as: *p ≤ 0.05, **p…

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  Fig. 8. PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis. Cartoon summarizing the results of this study: left side: upon ER stress/CHOP induction, SPCΔexon4 overexpression or in IPF AECII where persistent pro-apoptotic ER stress is observed, PACS2 protein is severely decreased and, therefore, a reduction in ER–mitochondrial tethering is observed alongside with severe AECII apoptosis. Upon treatment with CPS, a TRPV1 modulator, PACS2 protein levels are restored, ER–mitochondrial tethering is rescued together with a decrease in AECII apoptosis and COL1A1