Wolfram syndrome 2 (WFS2) is a premature aging syndrome caused by an irreversible mitochondria-mediated disorder. Cisd2, which regulates mitochondrial electron transport, has been recently identified as the causative gene of WFS2. The mouse Cisd2 knockout (KO) (Cisd2−/−) recapitulates most of the clinical manifestations of WFS2, including growth retardation, osteopenia, and lordokyphosis. However, the precise mechanisms underlying osteopenia in WFS2 and Cisd2 KO mice remain unknown. In this study, we collected embryonic fibroblasts from Cisd2-deficient embryos and reprogrammed them into induced pluripotent stem cells (iPSCs) via retroviral transduction with Oct4/Sox2/Klf4/c-Myc. Cisd2-deficient mouse iPSCs (miPSCs) exhibited structural abnormalities in their mitochondria and an impaired proliferative capability. The global gene expression profiles of Cisd2+/+, Cisd2+/−, and Cisd2−/− miPSCs revealed that Cisd2 functions as a regulator of both mitochondrial electron transport and Wnt/β-catenin signaling, which is critical for cell proliferation and osteogenic differentiation. Notably, Cisd2−/− miPSCs exhibited impaired Wnt/β-catenin signaling, with the downregulation of downstream genes, such as Tcf1, Fosl1, and Jun and the osteogenic regulator Runx2. Several differentiation markers for tridermal lineages were globally impaired in Cisd2−/− miPSCs. Alizarin red S staining and flow cytometry analysis further revealed that Cisd2−/− miPSCs failed to undergo osteogenic differentiation. Taken together, our results, as determined using an miPSC-based platform, have demonstrated that Cisd2 regulates mitochondrial function, proliferation, intracellular Ca2+ homeostasis, and Wnt pathway signaling. Cisd2 deficiency impairs the activation of Wnt/β-catenin signaling and thereby contributes to the pathogeneses of osteopenia and lordokyphosis in WFS2 patients.
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References
1.
WileySE, AndreyevAY, DivakaruniAS, KarischR, PerkinsG, WallEA, van der GeerP, ChenYF, TsaiTF, et al. (2013). Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis. EMBO Mol Med, 5:904–918.
2.
WileySE, MurphyAN, RossSA, van der GeerP and DixonJE. (2007). MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity. Proc Natl Acad Sci U S A, 104:5318–5323.
3.
TaminelliGL, SotomayorV, ValdiviesoAG, TeiberML, MarinMC and Santa-ColomaTA. (2008). CISD1 codifies a mitochondrial protein upregulated by the CFTR channel. Biochem Biophys Res Commun, 365:856–862.
4.
ChangNC, NguyenM, GermainM and ShoreGC. (2010). Antagonism of Beclin 1-dependent autophagy by BCL-2 at the endoplasmic reticulum requires NAF-1. EMBO J, 29:606–618.
5.
ChangNC, NguyenM, BourdonJ, RissePA, MartinJ, DanialouG, RizzutoR, PetrofBJ and ShoreGC. (2012). Bcl-2-associated autophagy regulator Naf-1 required for maintenance of skeletal muscle. Hum Mol Genet, 21:2277–2287.
6.
KankiT and KlionskyDJ. (2009). Mitochondrial abnormalities drive cell death in Wolfram syndrome 2. Cell Res, 19:922–923.
7.
ChenYF, KaoCH, ChenYT, WangCH, WuCY, TsaiCY, LiuFC, YangCW, WeiYH, et al. (2009). Cisd2 deficiency drives premature aging and causes mitochondria-mediated defects in mice. Genes Dev, 23:1183–1194.
8.
ChenCT, HsuSH and WeiYH. (2012). Mitochondrial bioenergetic function and metabolic plasticity in stem cell differentiation and cellular reprogramming. Biochim Biophys Acta, 1820:571–576.
9.
DiMauroS and SchonEA. (2003). Mitochondrial respiratory-chain diseases. N Engl J Med, 348:2656–2668.
10.
BukowieckiR, AdjayeJ and PrigioneA. (2014). Mitochondrial function in pluripotent stem cells and cellular reprogramming. Gerontology, 60:174–182.
11.
RuizS, PanopoulosAD, HerreriasA, BissigKD, LutzM, BerggrenWT, VermaIM and Izpisua BelmonteJC. (2011). A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity. Curr Biol, 21:45–52.
12.
WallaceDC. (1999). Mitochondrial diseases in man and mouse. Science, 283:1482–1488.
13.
MoyesCD, Mathieu-CostelloOA, TsuchiyaN, FilburnC and HansfordRG. (1997). Mitochondrial biogenesis during cellular differentiation. Am J Physiol, 272:C1345–C1351.
14.
AtmaniH, ChappardD and BasleMF. (2003). Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol. J Cell Biochem, 89:364–372.
15.
LonerganT, BrennerC and BavisterB. (2006). Differentiation-related changes in mitochondrial properties as indicators of stem cell competence. J Cell Physiol, 208:149–153.
16.
MandalS, LindgrenAG, SrivastavaAS, ClarkAT and BanerjeeU. (2011). Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells. Stem Cells, 29:486–495.
17.
Facucho-OliveiraJM, AldersonJ, SpikingsEC, EggintonS and St JohnJC. (2007). Mitochondrial DNA replication during differentiation of murine embryonic stem cells. J Cell Sci, 120:4025–4034.
18.
ChenCT, ShihYR, KuoTK, LeeOK and WeiYH. (2008). Coordinated changes of mitochondrial biogenesis and antioxidant enzymes during osteogenic differentiation of human mesenchymal stem cells. Stem Cells, 26:960–968.
19.
WangCH, ChenYF, WuCY, WuPC, HuangYL, KaoCH, LinCH, KaoLS, TsaiTF and WeiYH. (2014). Cisd2 modulates the differentiation and functioning of adipocytes by regulating intracellular Ca2+ homeostasis. Hum Mol Genet, 23:4770–4785.
20.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
21.
TakahashiK and YamanakaS. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.
22.
ChiouSH, JiangBH, YuYL, ChouSJ, TsaiPH, ChangWC, ChenLK, ChenLH, ChienY and ChiouGY. (2013). Poly(ADP-ribose) polymerase 1 regulates nuclear reprogramming and promotes iPSC generation without c-Myc. J Exp Med, 210:85–98.
23.
SungLY, GaoS, ShenH, YuH, SongY, SmithSL, ChangCC, InoueK, KuoL, et al. (2006). Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer. Nat Genet, 38:1323–1328.
24.
CossarizzaA, Baccarani-ContriM, KalashnikovaG and FranceschiC. (1993). A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun, 197:40–45.
25.
SmileyST, ReersM, Mottola-HartshornC, LinM, ChenA, SmithTW, SteeleGDJr. and ChenLB. (1991). Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci U S A, 88:3671–3675.
26.
ManciniM, SedghinasabM, KnowltonK, TamA, HockenberyD and AndersonBO. (1998). Flow cytometric measurement of mitochondrial mass and function: a novel method for assessing chemoresistance. Ann Surg Oncol, 5:287–295.
27.
KochP, OpitzT, SteinbeckJA, LadewigJ and BrustleO. (2009). A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Natl Acad Sci U S A, 106:3225–3230.
ChenYF, KaoCH, KirbyR and TsaiTF. (2009). Cisd2 mediates mitochondrial integrity and life span in mammals. Autophagy, 5:1043–1045.
31.
ConlanAR, AxelrodHL, CohenAE, AbreschEC, ZurisJ, YeeD, NechushtaiR, JenningsPA and PaddockML. (2009). Crystal structure of Miner1: The redox-active 2Fe-2S protein causative in Wolfram Syndrome 2. J Mol Biol, 392:143–153.
32.
Van GansenP and Van LerbergheN. (1988). Potential and limitations of cultivated fibroblasts in the study of senescence in animals. A review on the murine skin fibroblasts system. Arch Gerontol Geriatr, 7:31–74.
33.
VafiadakiE, ArvanitisDA, PagakisSN, PapaloukaV, SanoudouD, Kontrogianni-KonstantopoulosA and KraniasEG. (2009). The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival. Mol Biol Cell, 20:306–318.
34.
Vande VeldeC, CizeauJ, DubikD, AlimontiJ, BrownT, IsraelsS, HakemR and GreenbergAH. (2000). BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol, 20:5454–5468.
35.
AquilanoK, VigilanzaP, RotilioG and CirioloMR. (2006). Mitochondrial damage due to SOD1 deficiency in SH-SY5Y neuroblastoma cells: a rationale for the redundancy of SOD1. FASEB J, 20:1683–1685.
36.
VasevaAV and MollUM. (2009). The mitochondrial p53 pathway. Biochim Biophys Acta, 1787:414–420.
37.
MartinBL and KimelmanD. (2008). Regulation of canonical Wnt signaling by Brachyury is essential for posterior mesoderm formation. Dev Cell, 15:121–133.
38.
LindsleyRC, GillJG, KybaM, MurphyTL and MurphyKM. (2006). Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm. Development, 133:3787–3796.
39.
MacDonaldBT, TamaiK and HeX. (2009). Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell, 17:9–26.
40.
MatsuoK, OwensJM, TonkoM, ElliottC, ChambersTJ and WagnerEF. (2000). Fosl1 is a transcriptional target of c-Fos during osteoclast differentiation. Nat Genet, 24:184–187.
41.
NiidaA, HirokoT, KasaiM, FurukawaY, NakamuraY, SuzukiY, SuganoS and AkiyamaT. (2004). DKK1, a negative regulator of Wnt signaling, is a target of the beta-catenin/TCF pathway. Oncogene, 23:8520–8526.
42.
van EsJH, BarkerN and CleversH. (2003). You Wnt some, you lose some: oncogenes in the Wnt signaling pathway. Curr Opin Genet Dev, 13:28–33.
43.
GaurT, LengnerCJ, HovhannisyanH, BhatRA, BodinePV, KommBS, JavedA, van WijnenAJ, SteinJL, SteinGS and LianJB. (2005). Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem, 280:33132–33140.
44.
GregoryCA, GunnWG, PeisterA and ProckopDJ. (2004). An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem, 329:77–84.
45.
VladimirovYA, ProskurninaEV and AlekseevAV. (2013). Molecular mechanisms of apoptosis. structure of cytochrome c-cardiolipin complex. Biochemistry, 78:1086–1097.
46.
MandalS, GuptanP, Owusu-AnsahE and BanerjeeU. (2005). Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev Cell, 9:843–854.
47.
Owusu-AnsahE, YavariA, MandalS and BanerjeeU. (2008). Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint. Nat Genet, 40:356–361.
48.
NiimiT, HayashiY, FutakiS and SekiguchiK. (2004). SOX7 and SOX17 regulate the parietal endoderm-specific enhancer activity of mouse laminin alpha1 gene. J Biol Chem, 279:38055–38061.
49.
BondueA, LapougeG, PaulissenC, SemeraroC, IacovinoM, KybaM and BlanpainC. (2008). Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell, 3:69–84.
50.
NelsonTJ, ChiriacA, FaustinoRS, Crespo-DiazRJ, BehfarA and TerzicA. (2009). Lineage specification of Flk-1 + progenitors is associated with divergent Sox7 expression in cardiopoiesis. Differentiation, 77:248–255.
51.
GandilletA, SerranoAG, PearsonS, LieALM, LacaudG and KouskoffV. (2009). Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification. Blood, 114:4813–4822.
52.
MukherjeeA and RotweinP. (2009). Akt promotes BMP2-mediated osteoblast differentiation and bone development. J Cell Sci, 122:716–726.
53.
PhimphilaiM, ZhaoZ, BoulesH, RocaH and FranceschiRT. (2006). BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype. J Bone Miner Res, 21:637–646.
54.
WangYK, YuX, CohenDM, WozniakMA, YangMT, GaoL, EyckmansJ and ChenCS. (2012). Bone morphogenetic protein-2-induced signaling and osteogenesis is regulated by cell shape, RhoA/ROCK, and cytoskeletal tension. Stem Cells Dev, 21:1176–1186.
55.
MariePJ, HayE and SaidakZ. (2014). Integrin and cadherin signaling in bone: role and potential therapeutic targets. Trends Endocrinol Metab, 25:567–575.
56.
ChangSF, ChangTK, PengHH, YehYT, LeeDY, YehCR, ZhouJ, ChengCK, ChangCA and ChiuJJ. (2009). BMP-4 induction of arrest and differentiation of osteoblast-like cells via p21 CIP1 and p27 KIP1 regulation. Mol Endocrinol, 23:1827–1838.
57.
JonasonJH, XiaoG, ZhangM, XingL and ChenD. (2009). Post-translational regulation of Runx2 in bone and cartilage. J Dent Res, 88:693–703.
58.
MarcelliniS, HenriquezJP and BertinA. (2012). Control of osteogenesis by the canonical Wnt and BMP pathways in vivo: cooperation and antagonism between the canonical Wnt and BMP pathways as cells differentiate from osteochondroprogenitors to osteoblasts and osteocytes. Bioessays, 34:953–962.
59.
YaccobyS. (2010). The role of the proteasome in bone formation and osteoclastogenesis. IBMS BoneKEy, 7:147–155.
60.
ZhangX, AkechJ, BrowneG, RussellS, WixtedJJ, SteinJL, SteinGS and LianJB. (2015). Runx2-smad signaling impacts the progression of tumor-induced bone disease. Int J Cancer, 136:1321–1332.
61.
JavedA, BaeJS, AfzalF, GutierrezS, PratapJ, ZaidiSK, LouY, van WijnenAJ, SteinJL, SteinGS and LianJB. (2008). Structural coupling of Smad and Runx2 for execution of the BMP2 osteogenic signal. J Biol Chem, 283:8412–8422.
62.
BruntKR, ZhangY, MihicA, LiM, LiS-H, XueP, ZhangW, BasmajiS, TsangK, et al. (2012). Role of WNT/β-catenin signaling in rejuvenating myogenic differentiation of aged mesenchymal stem cells from cardiac patients. Am J Pathol, 181:2067–2078.
63.
KimJH, LiuX, WangJ, ChenX, ZhangH, KimSH, CuiJ, LiR, ZhangW, et al. (2013). Wnt signaling in bone formation and its therapeutic potential for bone diseases. Ther Adv Musculoskelet Dis, 5:13–31.
64.
LongoKA, WrightWS, KangS, GerinI, ChiangSH, LucasPC, OppMR and MacDougaldOA. (2004). Wnt10b inhibits development of white and brown adipose tissues. J Biol Chem, 279:35503–35509.
65.
HwangSG, YuSS, LeeSW and ChunJS. (2005). Wnt-3a regulates chondrocyte differentiation via c-Jun/AP-1 pathway. FEBS Lett, 579:4837–4842.
66.
JinEJ, LeeSY, ChoiYA, JungJC, BangOS and KangSS. (2006). BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway. Mol Cells, 22:353–359.
67.
YoonJC, NgA, KimBH, BiancoA, XavierRJ and ElledgeSJ. (2010). Wnt signaling regulates mitochondrial physiology and insulin sensitivity. Genes Dev, 24:1507–1518.
68.
SugimoriK, MatsuiK, MotomuraH, TokoroT, WangJ, HigaS, KimuraT and KitajimaI. (2005). BMP-2 prevents apoptosis of the N1511 chondrocytic cell line through PI3K/Akt-mediated NF-kappaB activation. J Bone Miner Metab, 23:411–419.
69.
HyzySL, Olivares-NavarreteR, SchwartzZ and BoyanBD. (2012). BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner. J Cell Biochem, 113:3236–3245.
70.
RaiszLG. (1999). Physiology and pathophysiology of bone remodeling. Clin Chem, 45:1353–1358.
71.
TiYF, WangR and ZhaoJN. (2014). [Mechanism of osteoclast in bone resorption]. Zhongguo Gu Shang, 27:529–532.
72.
BakerJC, BeddingtonRS and HarlandRM. (1999). Wnt signaling in Xenopus embryos inhibits bmp4 expression and activates neural development. Genes Dev, 13:3149–3159.
73.
KamiyaN, KobayashiT, MochidaY, YuPB, YamauchiM, KronenbergHM and MishinaY. (2010). Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts. J Bone Miner Res, 25:200–210.
74.
GlantschnigH, FisherJE, WesolowskiG, RodanGA and ReszkaAA. (2003). M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase. Cell Death Differ, 10:1165–1177.
75.
FaccioR, TakeshitaS, ZalloneA, RossFP and TeitelbaumSL. (2003). c-Fms and the alphavbeta3 integrin collaborate during osteoclast differentiation. J Clin Invest, 111:749–758.
76.
CapulliM, PaoneR and RucciN. (2014). Osteoblast and osteocyte: games without frontiers. Arch Biochem Biophys, 561:3–12.
77.
ChangNC, NguyenM and ShoreGC. (2012). BCL2-CISD2: An ER complex at the nexus of autophagy and calcium homeostasis?. Autophagy, 8:856–857.
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