The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress

Abstract

Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1-positive granules than wild-type in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCPR155H-knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2α unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration.

Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Characterization of arsenite-induced stress granules by immunoblot in C2C12 cells. A: Analysis of the cytoplasmic fraction shows an increment in the protein levels of p-eIF2α immediately after 200 μmol/L arsenite exposure that diminishes 1 hour after treatment (arsenite and recovery). B: Total levels of eIF2α are unchanged. C and D: G3BP (C) and ubiquitin protein (D) levels increase after arsenite treatment in the cytosol. E: LC3-II protein levels are up-regulated 1 hour after arsenite treatment. F and G: Cytoplasmic levels of TDP-43 (F) and VCP (G) are similar in all conditions. H: Representative images of the blots quantified in panels A–G. n = 6 to 7 (from 4 independent experiments). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus no arsenite. eIF2α, eukaryotic translation initiation factor 2α; G3BP, RasGAP SH3-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC3, microtubule-associated protein 1A/1B-light chain 3; p, phosphorylated; TDP-43, transactive response DNA-binding protein 43; VCP, valosin-containing protein.

Figure 2

Autophagy and lysosome inhibitors delay stress granule resolution after arsenite exposure. A: Cells treated with autophagy (12 μmol/L MHY1485) or lysosome (20 μmol/L leupeptin) inhibitors present more stress granules than untreated or 0.3% DMSO-treated cells after left to recover for 1 hour from arsenite exposure. Arrowheads indicate stress granules. B: Quantification of panel A. C: MHY1485 and leupeptin treatment does not show stress granules in the absence of arsenite exposure. D: Similarly, cells analyzed by immunoblot 1 hour after exposure with 200 μmol/L arsenite present augmented levels of the stress granule marker G3BP and polyubiquitylated proteins. E and F: Quantification of panel D. n = 100 cells per well (3 wells per condition) (C). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus untreated; †P < 0.05, †††P < 0.001 versus 0.3% DMSO. Scale bars = 50 μm (A and C). DMSO, dimethyl sulfoxide; FK2, mono- and polyubiquitylated proteins antibody; G3BP, RasGAP SH3-binding protein; TiA-1, T-intracellular antigen-1.

Figure 3

IBMPFD-relevant VCP mutants delay stress granule resolution after arsenite exposure in C2C12 cells. A: Cells expressing VCP(R155H) or VCP(A232E) show significantly more TiA-1+ stress granules than nontransfected or wt VCP-transfected cells 1 hour after arsenite exposure (right). No differences were observed on untreated cells or immediately after arsenite exposure. B–D: Similarly, more G3BP (B), pTDP-43– (C), and TDP-43+ (D) granules are observed on cells transfected with mutant VCP than nontransfected and wt-transfected cells, 1 hour after arsenite treatment (arsenite and recovery). E–H: Representative images from the arsenite and recovery condition are presented for TiA-1 (E), G3BP (F), pTDP-43 (G), and TDP-43 (H). The dsRED tag was fused to the different VCP constructs to allow identification of transfected cells. Arrowheads indicate stress granules. n = 100 cells per well (6 wells per condition). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus noT. ††P < 0.01 versus VCP (wt). Scale bar = 50 μm. G3BP, RasGAP SH3-binding protein; IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; noT, nontransfected; p, phosphorylated; TDP-43, transactive response DNA-binding protein 43; TiA-1, T-intracellular antigen-1; VCP, valosin-containing protein; wt, wild-type.

Figure 4

IBMPFD-relevant VCP mutants delay stress granule resolution in differentiated C2C12 cells. A: Cells express myosin protein after 5 days of incubation in horse serum 2% and show dsRED+ expression when transfected. B: Differentiated C2C12 cells that expressed VCP(R155H) or VCP(A232E) show significantly more TiA-1+ stress granules than nontransfected or wt VCP-transfected cells when left to recover for 1 hour after arsenite exposure. C: Representative images from the data graphed on panel B. The dsRED tag was fused to the different VCP constructs to allow identification of transfected cells. Arrowheads indicate stress granules. n = 50 cells per well (6 wells per condition). ∗P < 0.05 versus noT; †P < 0.05 versus wt VCP. Scale bars: 50 μm (A); 25 μm (C). IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; noT, nontransfected; TiA-1, T-intracellular antigen-1; VCP, valosin-containing protein; wt, wild-type.

Figure 5

IBMPFD-relevant VCP mutants do not delay stress granule resolution after heat shock treatment in C2C12 cells. A: Cells expressing VCP(R155H) or VCP(A232E) show similar TiA-1+ stress granules compared with wt VCP-expressing cells on all conditions: no treatment, immediately after heat shock, or 30 minutes after heat shock (right). B: Representative images of the heat shock and recovery condition. The dsRED tag was fused to the different VCP constructs to allow identification of transfected cells. Arrowheads indicate stress granules. n = 100 cells per well (7 to 8 wells per condition). Scale bar = 50 μm. IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; TiA-1, T-intracellular antigen-1; VCP, valosin-containing protein; wt, wild-type.

Figure 6

Oxidative stress but not stress granule markers are increased in the IBMPFD model: the VCPR155H-KI mouse. A: Immunoblot analysis of skeletal muscle lysates shows augmented ubiquitinylated proteins on 15- to 18-month-old VCPR155H-KI mice compared with wt skeletal muscle lysates of the same age. B: Similarly, the oxyblot assay reveals increments on oxidated proteins on skeletal muscle of VCPR155H-KI mice compared with wt mice. C–F: However, G3BP (C), p-eIF2α (D), total eIF2α (E), and PABP1 (F) protein levels are similar between genotypes. G: Representative blots for the different proteins analyzed. H: TiA-1 and TDP-43 present major nuclear localization when analyzed by immunofluorescence. I: Hematoxylin and eosin staining shows no major histologic differences between the wt and VCPR155H-KI skeletal muscle. J–L: Similarly, no differences are detected when skeletal muscle was stained for G3BP (J), TiA-1 (K), and TDP-43 (L). Arrowheads indicate nuclear localization of TiA-1 and TDP-43 proteins. n = 8 wt and 7 VCPR155H-KI (R155H) mice (G). ∗P < 0.05. Scale bars = 50 μm. eIF2α, eukaryotic translation initiation factor 2α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G3BP, RasGAP SH3-binding protein; IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; KI, knockin; p, phosphorylated; PABP1, poly(A)-binding protein 1; TDP-43, transactive response DNA-binding protein 43; TiA-1, T-intracellular antigen-1; VCP, valosin-containing protein; wt, wild-type.