IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2

Figures

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  Fig. 1. Upregulation of PARP12 in the damaged area of articular cartilage in patients with knee osteoarthritis (OA) and characterization of PARP12 in primary human chondrocytes (PHCs). a Heatmap of all differentially expressed genes between undamaged (U) and damaged (D) cartilage tissues of patients with OA. b Volcano plots of all differentially expressed genes, including the PARP8, PARP12, PARP14, and PARP16 members of the PARP superfamily, between U and D cartilage tissues. c, d Quantitative PCR analysis of PARP8, PARP12, PARP14, and PARP16 between U and D cartilage tissues and in PHCs treated with different concentrations of IL-1β (0, 1, 5, or 10 ng/mL) for 24 h. n = 6 per group. e Plain radiographs of patients with knee OA. f Representative image of intraoperative specimens and alcian blue, safranin O, and toluidine blue staining of U and D cartilage tissues. Scale bar: 250 µm. g Quantitative PCR analysis of PARP12 in U and D cartilage tissues. n = 30 per group. h Western blot analysis of PARP12, COL2A1, MMP13, p62, and LC3B between U and D cartilage tissues. i Heatmap of relative protein level of PARP12 in U and D cartilage tissues of different patients with OA. n = 30 per group. j…

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  Fig. 2. PARP12 is upregulated in rat OA chondrocytes and rat articular cartilage. a Experimental diagram of the OA rat model induced by the injection of 40 μL saline with or without 1 mg MIA into the intraarticular cavity of 8-week-old Sprague–Dawley rats for 1, 2, or 3 weeks. n = 6 per group. b 3D reconstruction images of micro-CT scanning. n = 5 per group. c–f Analysis of bone volume (BV)/trabecular volume (TV) ratio, bone surface (BS)/TV ratio, trabecular thickness, and trabecular numbers. n = 5 per group. g Western blot analysis of PARP12, COL2A1, MMP13, p62, and LC3B in chondrocytes of Sprague–Dawley rats injected with saline control (con) or MIA for 1, 2, or 3 weeks. h Protein quantification of (g) using ImageJ. n = 3 per group. i Representative images of Safranin O and immunohistochemistry (IHC) staining of PARP12, COL2A1, and MMP13 in chondrocytes of Sprague–Dawley rats. Scale bars: 250 µm (first row) and 50 µm (second row). j Quantification of OARSI score based on staining results in (i). n = 3 per group. k–m Quantification of PARP12, COL2A1, and MMP13 positive chondrocytes based on staining results in (i). n = 3 per group. n Colocalization of PARP12 and p62 in chondrocytes of…

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  Fig. 3. Targeting the expression of PARP12 affects mitophagy and OA-related degeneration in primary human chondrocytes (PHCs). a Western blot analysis of COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B and p62 in PHCs infected with PARP12-overexpressing (OE) or OE-NC adenoviruses. b Protein quantification of (a) using ImageJ. n = 3 per group. c Western blot analysis of COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B and p62 in PHCs infected with PARP12 KD-03 shRNA or KD-NC shRNA. d Protein quantification of (c) using ImageJ. n = 3 per group. e Immunofluorescence of PARP12, COL2A1, MMP13, LC3B and p62 in PHCs following either knockdown or overexpression of PARP12. n = 3 per group. Scale bars: 20 µm. f Chondrocytes apoptosis was assayed by flow cytometry. g Transmission electron microscopy (TEM) of autophagosomes (APs) and autolysosomes (ALs) that phagocytic mitochondria in PHCs following either knockdown or overexpression of PARP12. Blue arrows show APs and ALs that phagocytic mitochondria. Scale bars: 1 µm. h Quantification of APs and ALs that phagocytic mitochondria of (g). n = 3 per group. i PHCs were transfected with mito-Keima, incubated with knockdown or overexpression of PARP12 for…

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  Fig. 4. PARP12 interacted with ISG15. a Immunoprecipitation-mass spectrometry (IP-MS) analyses of PARP12 interaction proteins. b The string database of interaction network diagram between PARP12 and other proteins. c Colocalization of PARP12, ISG15, and MFN1/2 in primary human chondrocytes (PHCs). Scale bars: 20 µm. n = 3 per group. d Western blot analysis of ISG15, MFN1/2, PINK1 and Parkin in PHCs following either knockdown or overexpression of PARP12. e Protein quantification of (d) using ImageJ. n = 3 per group. f Representative TEM images of mitochondria in PHCs following either knockdown or overexpression of PARP12. Red arrows show mitochondria fusion. Orange arrows show mitochondria. Blue lines represent the schematic measurement of mitochondrial length and width. Scale bars: 1 µm. g Quantification of numbers, length-to-width ratio, and percentage of mitochondria fusion of (f). n = 3 per group. h Western blot analysis of OPA1 and DRP1 in PHCs following either knockdown or overexpression of PARP12. i Protein quantification of (h) using ImageJ. n = 3 per group. j Western blot analysis of NLRP3 inflammasome activity in PHCs following either knockdown or overexpression of PARP12. k…

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  Fig. 5. ISG15 upregulated by PAPR12, increasing ISGylation of MFN1/2 and then attenuating the ubiquitylation and SUMOylation to inhibit PINK1/Parkin-dependent mitophagy. a Immunoblotting (IB) analysis of ubiquitylation and ISG15 in PHCs following transfection with KD-ISG15 or KD-NC. b Protein quantification of (a) using ImageJ. n = 3 per group. c IB analysis of ubiquitylation of mitochondrial proteins. Mitochondria were isolated from KD-NC and KD-ISG15 PHCs transfected with a HA-ubiquitin construct. d Protein quantification of (d) using ImageJ. n = 3 per group. e IB analysis of SUMO2/3 and ubiquitin with SUMO2/3-conjugated proteins in KD-NC and KD-ISG15 PHCs immunoprecipitated using SUMO2/3 affinity beads. f IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3 and ISG15 in KD-NC and KD-ISG15 PHCs treated with or without MG-132 after immunoprecipitation of MFN1/2. The molecular marker of IB plots of ubiquitin, SUMO2/3, and ISG15 conjugates with MFN1/2 is 70 kD and above when considering the molecular weight of MFN1/2 at 85 kD. g Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (f) using ImageJ after immunoprecipitation of MFN1/2. n = 3 per group. h IB analysis of MFN1/2, MFN1,…

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  Fig. 6. Inflammatory cytokines-induced IRF1 activation promoted PARP12 transcription. a The JASPAR database was used to predict potential transcription factors (TFs) of PARP12 (H. sapiens). b Schematic of the predictive binding site of IRF1 at the PARP12 promoter region 2 000-bp upstream of the transcription start site (TSS), with the TSS being designated as 100. A 2 100–3 100 bp fragment was used as the negative control (NC). c Quantification of immunoprecipitated DNA by PCR following a chromatin immunoprecipitation (ChIP) assay in PHCs using normal rabbit IgG as the NC. d Quantitative PCR analysis of IRF1 in U and D cartilage tissues. n = 30 per group. e Western blot analysis of IRF1 between U and D cartilage tissues. f Relationship between age, sex, obesity gradation, affected side, disease duration, Kellgren–Lawrence gradation, and expression of IRF1 in the damaged area represented by the Sankey diagram. g Linear regression analysis of the expression of IRF1 and PARP12 in U and D areas in patients with knee OA (n = 30). h Western blot analysis of IRF1, PARP12, COL2A1, aggrecan, MMP13, RUNX2, Bcl2, Bax, LC3B, p62, MFN1, MFN2, PINK1, Parkin and NLRP3 inflammasome activity in PHCs…

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  Fig. 7. XAV-939 promoted PINK1/Parkin-mediated mitophagy and suppressed cartilage degradation via targeting PARP12. a Chemical formula structure of XAV-939. b Western blot analysis of PARP12 in primary human chondrocytes (PHCs) treated with different concentrations of XAV-939. c Protein quantification of (b) using ImageJ. n = 3 per group. d Western blot analysis of PARP12, COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B, p62, MFN1, MFN2, PINK1, Parkin and NLRP3 inflammasome activity in PHCs treated with XAV-939. e Western blot analysis of LC3B, p62, PINK1 and Parkin in PHCs treated with XAV-939 or DMSO and transfected with PARP12 KD-03 shRNA or NC. f Protein quantification of (e) using ImageJ. n = 3 per group. g Western blot analysis of LC3B, p62, PINK1 and Parkin in PHCs treated with XAV-939 or DMSO and transfected with PARP12-OE adenovirus or NC. h Protein quantification of (g) using ImageJ. n = 3 per group. i Pretreatment with RAPA (1 μmol/L) for 1 h significantly changed the expression of LC3B, p62, PINK1 and Parkin treated by XAV-939 in PHCs. j Protein quantification of (i) using ImageJ. n = 3 per group. k–m Western blot analysis, fluorescence analysis of MitoTracker Red and…

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  Fig. 8. PARP12 modulates osteoarthritis (OA) pathogenesis in monosodium iodoacetate (MIA)-treated rats. a Experimental diagram of the MIA OA rat model treated with XAV-939 or PARP12 overexpression (OE) adenovirus. Rats were evaluated at age of 10 weeks. n = 6 per group. b 3D reconstruction images of micro-CT scanning of the knees of rats treated with XAV-939 or PARP12-OE adenovirus. n = 5 per group. c–f Analysis of BV/TV, BS/TV, trabecular thickness, and trabecular numbers. n = 5 per group. g Western blot analysis of PARP12, COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B, p62, MFN1, MFN2, PINK1, Parkin and NLRP3 inflammasome activity in chondrocytes of rats treated with XAV-939 or PARP12 overexpression (OE) adenovirus. n = 3 per group. h Representative images of Safranin O and IHC staining of PARP12, COL2A1 and MMP13. Scale bars: 250 µm (first row) and 50 µm (second row). i Quantification of macroscopic score based on staining results in (h). n = 3 per group. j–l Quantification of PARP12, COL2A1, and MMP13 positive chondrocytes based on staining results in (h). n = 3 per group. m–o ROS staining, ATP level and JC-1 staining in chondrocytes of rats treated with XAV-939 or PARP12 OE…