Research ArticleOsteoarthritis

ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone

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Science Translational Medicine  12 Sep 2018:
Vol. 10, Issue 458, eaar8426
DOI: 10.1126/scitranslmed.aar8426
  • Fig. 1 Loss of ANP32A increases osteoarthritis severity and susceptibility.

    (A and B) Real-time polymerase chain reaction (PCR) (A) and immunoblot (B) analysis of ANP32A mRNA and protein (with actin as loading control) in human articular chondrocytes from hip osteoarthritis (OA) and fracture patients (non-OA) (n = 4 per group; *t6 = 3.79, P = 0.009, t test). (C) Immunohistochemical staining for ANP32A in OA and non-OA human cartilage (n = 4 per group; scale bar, 400 μm). (D) ANP32A expression by RNA sequencing in paired preserved and damaged cartilage from hips (circles) and knees (triangles) from osteoarthritis patients [log2-fold change (Log2FC): damaged versus preserved] (n = 21, P < 0.001, Benjamini-Hochberg–adjusted paired t test). (E and F) Immunohistochemical staining for ANP32A in male wild-type (WT) mice 12 weeks after induction of osteoarthritis by DMM (Surgery) compared to sham-operated mice (Sham) (E) and in 8-week-old male or 12-month-old female mice (F) [n = 5 (E) and 3 (F) per group]. (G to J) Hematoxylin–safranin-O–stained sections (G and I) and quantification by Osteoarthritis Research Society International (OARSI) severity grade (H and J) of cartilage damage in knee joints after DMM and after 12-month aging of mice [n = 8 and 10 male mice, *t34 = 5.16, P < 0.001, Bonferroni-corrected for six tests in one-way analysis of variance (ANOVA) (H); n = 10 female mice per group, *t18 = 3.05, P = 0.007, t test (J)]. (E to G and I) Scale bars, 200 μm. Error bars indicate mean ± SD. IgG, immunoglobulin G.

  • Fig. 2 ANP32A deficiency reduces ATM and triggers oxidative stress in cartilage.

    (A and B) Volcano plot (A) and PANTHER pathway analysis (B) of microarray data comparing articular cartilage of 8-week-old male Anp32a-deficient to wild-type mice (n = 4 per group). (C and D) Real-time PCR (C) and immunoblot analysis (D) of ATM mRNA and protein (with actin as loading control) in mouse articular chondrocytes. (E to G) Immunohistochemical staining for ATM (E), 8-hydroxydeoxyguanosine (8-OHdG) (F), and DHE staining (G) in knees from 8-week-old male mice (n = 3). DAPI, 4′,6-diamidino-2-phenylindole. (H and I) Real-time PCR (H) and immunoblot (I) analysis of ATM mRNA and protein in articular chondrocytes from hip osteoarthritis (OA) and fracture patients (non-OA) [n = 4 per group; *t6 = 9.04, P < 0.001, t test]. (J and K) ATM expression (J) and correlation with ANP32A expression (K) by RNA sequencing in paired preserved and damaged cartilage from hips (circles) and knees (triangles) from osteoarthritis patients [log2-fold change (Log2FC) of damaged versus preserved] [n = 21, P = 0.008, Benjamini-Hochberg–adjusted paired t test (J), Pearson’s correlation = 0.536, P < 0.001 (K)]. (L to N) Real-time PCR analysis of ANP32A (L) and ATM expression (M) and 2′,7′- dichlorofluorescin diacetate (DCFDA) ROS activity staining (N) in human articular chondrocytes transfected with ANP32A (siANP) or scrambled siRNA (siSCR), treated with H2O2 and recombinant ATM protein (rATM) {0.5 μg/ml [low dose (LD)] or 1 μg/ml [high dose (HD)]} (n = 2, error bars indicate mean ± SD of three technical replicates). Scale bars, 200 μm (F and G) and 50 μm (E).

  • Fig. 3 ANP32A directly induces ATM expression to prevent oxidative stress in cartilage.

    (A) ChIP-qPCR analysis of ANP32A binding to different regions of the ATM gene promoter in non-osteoarthritic human articular chondrocytes. (B) ChIP-qPCR analysis of RNA polymerase II binding to the ATM promoter in human articular chondrocytes transfected with siANP or scrambled siRNA. (C and D) Real-time PCR (C) and immunoblot (D) analysis of ATM mRNA and protein in articular chondrocytes treated over time with H2O2 or vehicle (V) for 72 hours. (E) Immunoblot analysis of cytosolic and nuclear ATM and ANP32A protein amounts in response to H2O2 treatment for the indicated times in human articular chondrocytes. The images are representative of images from three independent experiments. (F) ChIP-qPCR analysis of ANP32A binding to the ATM promoter upon H2O2 treatment in human articular chondrocytes. (A, B, and F) Data from two biologically independent experiments. (C) Data are from two experiments with three technical replicates. Error bars indicate mean ± SD.

  • Fig. 4 Antioxidant treatment prevents osteoarthritis in Anp32a-deficient mice.

    (A and B) Hematoxylin–safranin-O–stained sections (A) and quantification by OARSI severity grade (B) of articular cartilage damage in knee joints of 20-week-old male mice 12 weeks after induction of osteoarthritis by the DMM model, treated with vehicle or NAC [n = 2 (WT-Sham NAC), 5 (WT-Surgery), 5 (WT-Surgery NAC), 2 (Anp32a−/−-Surgery), and 5 (Anp32a−/−-Surgery NAC), *t14 = 3.58, P = 0.03, Bonferroni-corrected for 10 tests in one-way ANOVA]. (C) Immunohistochemical detection of 8-OHdG to measure ROS in knees from WT and Anp32a−/− mice after induction of DMM osteoarthritis with or without NAC treatment. Scale bar, 200 μm. (D and E) Immunohistochemistry (D) and quantification by digital image analysis (E) of COLX in the articular cartilage of 16-week-old Anp32a−/− compared to WT mice with or without NAC treatment [n = 3, NAC reduced relative intensity more in knockout (KO) than in WT, interaction F1,8 = 5.78, P = 0.0429 by two-way ANOVA]. Images are representative of images from three to five different mice. Scale bars, 200 μm (A and C) and 50 μm (D). Error bars indicate mean ± SD.

  • Fig. 5 Anp32a deficiency leads to ataxia-like neurological defects, prevented by antioxidant treatment.

    (A) Real-time PCR analysis of ATM expression in adult brain from 8-week-old male WT and Anp32a-deficient mice (n = 9). (B) Immunohistochemical staining for ATM in cerebellum from 8-week-old male Anp32a-deficient mice compared to WT mice. (C) Immunohistochemical detection of 8-OHdG to measure ROS in 16-week-old male cerebellum. (D) Time course of oral treatment with NAC and CatWalk automated gait analysis in WT and Anp32a-deficient (KO) mice [n = 5 (WT no NAC), 7 (KO no NAC), 5 (WT NAC), 5 (KO NAC)]. (E) Gait analysis of 8-week-old male mice. Footprint colors were assigned manually (green, right; red, left; light print, forelimbs; dark print, hindlimbs). (F) Average stride length analyzed with ANOVA accounting for genotype (WT, KO), NAC (yes, no), paw [front paw (FP), hind paw (HP)], and all interactions (genotype/NAC interaction F1,18 = 6.587, P = 0.019; *for indicated pairwise comparisons, all P < 0.001, Bonferroni-corrected for 12 tests). (G) Regularity index of walking pattern analyzed with ANOVA accounting for genotype, NAC, and their interaction (genotype/NAC interaction F1,18 = 9.227, P = 0.007; *for indicated pairwise comparisons, all P ≤ 0.006, Bonferroni-corrected for six tests). Error bars indicate mean ± SD. Scale bars, 500 μm. Details about ANOVA in data file S1.

  • Fig. 6 Anp32a deficiency leads to osteopenia that is responsive to antioxidant treatment.

    (A) Subcapital DEXA analysis of bone mineral density (BMD), bone mineral content (BMC), and lean body mass in 12-week-old female Anp32a−/− mice compared to WT littermates (n = 11 and 9, *all t18 ≥ 2.224, all P ≤ 0.039, t test). (B) pQCT of trabecular and cortical bone parameters in femora from 12-week-old female Anp32a−/− mice and WT littermates (n = 10 and 9, *all t17 ≥ 2.215, all P ≤ 0.041, t test). (C) Hematoxylin–safranin-O staining of tibiae from 16-week-old male Anp32a−/− and WT mice with or without NAC treatment for 13 weeks. Scale bar, 250 μm. (D) In vivo μCT of tibiae from female Anp32a−/− mice with or without NAC treatment for 12 weeks from the age of 3 until 6 months [bone volume/tissue volume (BV/TV)] (n = 2). (E) Histomorphometry analysis of tibiae from male Anp32a−/− mice treated or not with NAC for 13 weeks from the age of 3 until 16 weeks (n = 5 per group, *t8 = 2.366, P = 0.046, t test). Error bars indicate mean ± SD.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/458/eaar8426/DC1

    Materials and Methods

    Fig. S1. Loss of ANP32A increases the severity of osteoarthritis in the collagenase- and papain-induced mouse models.

    Fig. S2. Expression of molecular markers associated with healthy chondrocytes in the presence or absence of ANP32A.

    Fig. S3. Transcriptome network analysis of articular cartilage of Anp32a-deficient mice.

    Fig. S4. Compensatory regulation of antioxidant systems in the articular cartilage of Anp32a-deficient mice.

    Fig. S5. Immunohistochemistry of COLX in the growth plates of 16-week-old male Anp32a−/− compared to WT mice.

    Fig. S6. Calbindin immunostaining of cerebellar Purkinje cells of 16-week-old male WT and Anp32a-deficient mice.

    Fig. S7. Late-stage antioxidant intervention in Anp32a-deficient mice ameliorates ataxia-related defects.

    Fig. S8. Model for the role of ANP32A on oxidative stress.

    Table S1. Patient characteristics.

    Table S2. Top ranked genes of transcriptome network of articular cartilage of 8-week-old Anp32a-deficient male mice compared to WT mice.

    Table S3. Gene expression of main antioxidants in transcriptome network of articular cartilage of 8-week-old male Anp32a-deficient mice compared to WT mice.

    Table S4. Gait parameters of early-stage antioxidant intervention with NAC in Anp32a-deficient mice.

    Table S5. Animal experiments: Overview, setup, and analysis details.

    Table S6. Primers used in qPCR analysis.

    Data file S1. Statistical analysis.

    References (7278)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Loss of ANP32A increases the severity of osteoarthritis in the collagenase- and papain-induced mouse models.
    • Fig. S2. Expression of molecular markers associated with healthy chondrocytes in the presence or absence of ANP32A.
    • Fig. S3. Transcriptome network analysis of articular cartilage of Anp32a-deficient mice.
    • Fig. S4. Compensatory regulation of antioxidant systems in the articular cartilage of Anp32a-deficient mice.
    • Fig. S5. Immunohistochemistry of COLX in the growth plates of 16-week-old male Anp32a−/− compared to WT mice.
    • Fig. S6. Calbindin immunostaining of cerebellar Purkinje cells of 16-week-old male WT and Anp32a-deficient mice.
    • Fig. S7. Late-stage antioxidant intervention in Anp32a-deficient mice ameliorates ataxia-related defects.
    • Fig. S8. Model for the role of ANP32A on oxidative stress.
    • Table S1. Patient characteristics.
    • Table S2. Top ranked genes of transcriptome network of articular cartilage of 8-week-old Anp32a-deficient male mice compared to WT mice.
    • Table S3. Gene expression of main antioxidants in transcriptome network of articular cartilage of 8-week-old male Anp32a-deficient mice compared to WT mice.
    • Table S4. Gait parameters of early-stage antioxidant intervention with NAC in Anp32a-deficient mice.
    • Table S5. Animal experiments: Overview, setup, and analysis details.
    • Table S6. Primers used in qPCR analysis.
    • References (7278)

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    Other Supplementary Material for this manuscript includes the following:

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