Transmission of ER stress response by ATF6 promotes endochondral bone growth

Journal of Orthopaedic Surgery and Research

Feng-Jin Guo²  Xiaofeng Han²  Peng Zhang²  Rong Jiang¹  Zhangyuan Xiong² 

[1] Laboratory of Stem Cells and Tissue Engineering, Chongqing Medical University, Chongqing, China;Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing 400016, China

关键词: Bone growth;    Runx2;    Chondrocyte differentiation;    UPR;    BMP2;    ATF6;   

Others  :  1227815
DOI  :  10.1186/s13018-015-0284-7

 received in 2015-07-15, accepted in 2015-08-31,  发布年份 2015

【 摘 要 】

Background

We reported earlier that X-box binding protein1 spliced (XBP1S), a key regulator of the unfolded protein response (UPR), as a bone morphogenetic protein 2 (BMP2)-inducible transcription factor, positively regulates endochondral bone formation by activating granulin-epithelin precursor (GEP) chondrogenic growth factor. Under the stress of misfolded or unfolded proteins in the endoplasmic reticulum (ER), the cells can be protected by the mammalian UPR. However, the influence of activating transcription factor 6 (ATF6), another transcriptional arm of UPR, in BMP2-induced chondrocyte differentiation has not yet been elucidated. In the current study, we investigate and explore the role of ATF6 in endochondral bone formation, focus on associated molecules of hypertrophic chondrocyte differentiation, as well as the molecular events underlying this process.

Methods

High-cell-density micromass cultures were used to induce ATDC5 and C3H10T1/2 cell differentiation into chondrocytes. Quantitative real-time PCR, immunoblotting analysis, and immunohistochemistry were performed to examine (1) the expression of ATF6, ATF6α, collagen II, collagen X, and matrix metalloproteinase-13 (MMP13) and (2) whether ATF6 stimulates chondrogenesis and whether ATF6 enhances runt-related transcription factor 2 (Runx2)-mediated chondrocyte hypertrophy. Culture of fetal mouse bone explants was to detect whether ATF6 stimulates chondrocyte hypertrophy, mineralization, and endochondral bone growth. Coimmunoprecipitation was employed to determine whether ATF6 associates with Runx2 in chondrocyte differentiation.

Results

ATF6 is differentially expressed in the course of BMP2-triggered chondrocyte differentiation. Overexpression of ATF6 accelerates chondrocyte differentiation, and the ex vivo studies reveal that ATF6 is a potent stimulator of chondrocyte hypertrophy, mineralization, and endochondral bone growth. Knockdown of ATF6 via a siRNA approach inhibits chondrogenesis. Furthermore, ATF6 associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. And, the stimulation effect of ATF6 is reduced during inhibition of Runx2 via a siRNA approach, suggesting that the promoting effect is required for Runx2.

Conclusions

Our observations demonstrate that ATF6 positively regulates chondrocyte hypertrophy and endochondral bone formation through activating Runx2-mediated hypertrophic chondrocyte differentiation.

【 授权许可】

   
2015 Xiong et al.

【 图 表 】

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【 参考文献 】

• [1]Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem. 2006; 97:33-44.
• [2]Colnot C. Cellular and molecular interactions regulating skeletogenesis. J Cell Biochem. 2005; 95:688-97.
• [3]Franz-Odendaal TA, Vickaryous MK. Skeletal elements in the vertebrate eye and adnexa: morphological and developmental perspectives. Dev Dyn. 2006; 235:1244-55.
• [4]Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors. 2004; 22:233-41.
• [5]Yu L, Han M, Yan M, Lee J, Muneoka K. BMP2 induces segment-specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center. Dev Biol. 2012; 372:263-73.
• [6]Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, Sekiya H, Tsumagari K, Ochiai K, Yoshinaga K, Saitoh M, Nishimura R, Yoneda T, Kou I, Furuichi T, Ikegawa S, Ikawa M, Okabe M, Wanaka A, Imaizumi K. Signaling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nat Cell Biol. 2009; 11:1205-11.
• [7]Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012; 13(2):89-102.
• [8]Zhang K, Kaufman RJ. Signaling the unfolded protein response from the endoplasmic reticulum. J Biol Chem. 2004; 279:25935-8.
• [9]Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007; 8(7):519-29.
• [10]Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest. 2002; 110(10):1389-98.
• [11]Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 1999; 13:1211-33.
• [12]Keiko N, Tatsuhiko S, Nobuhiro M. Endoplasmic reticulum stress signaling transmitted by ATF6 mediates apoptosis during muscle development. J Cell Biol. 2005; 169(4):555-60.
• [13]Guo FJ, Xiong Z, Han X, Liu C, Liu Y, Jiang R, Zhang P. XBP1S, a BMP2-inducible transcription factor, accelerates endochondral bone growth by activating GEP growth factor. J Cell Mol Med. 2014; 18(6):1157-71.
• [14]Jang WG, Kim EJ, Kim DK, Ryoo HM, Lee KB, Kim SH, Choi HS, Koh JT. BMP2 protein regulates osteocalcin expression via RUNX2-mediated ATF6 gene transcription. J Biol Chem. 2012; 287:905-15.
• [15]Han X, Zhang P, Jiang R, Xia F, Li M, Guo FJ. Explore on the effect of ATF6 on cell growth and apoptosis in cartilage development. Histochem Cell Biol. 2014; 142(5):497-509.
• [16]Guo FJ, Jiang R, Xiong Z, Xia F, Li M, Chen L, Liu CJ. IRE1a constitutes a negative feedback loop with BMP2 and acts as a novel mediator in modulating osteogenic differentiation. Cell Death Dis. 2014; 5:e1239.
• [17]Zhang Y, Kong L, Carlson CS, Liu CJ. Cbfa1-dependent expression of an interferon-inducible p204 protein is required for chondrocyte differentiation. Cell Death Differ. 2008; 15:1760-71.
• [18]Feng JQ, Guo FJ, Jiang BC, Zhang Y, Frenkel S, Wang DW, Tang W, Xie Y, Liu CJ. Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis. FASEB J. 2010; 24(6):1879-92.
• [19]Saito A, Ochiai K, Kondo S, Tsumagari K, Murakami T, Cavener DR, Imaizumi K. Endoplasmic reticulum stress response mediated by the PERK-eIF2(alpha)-ATF4 pathway is involved in osteoblast differentiation induced by BMP2. J Biol Chem. 2011; 286(6):4809-18.
• [20]Saito A, Hino S, Murakami T, Kanemoto S, Kondo S, Saitoh M, Nishimura R, Yoneda T, Furuichi T, Ikegawa S, Ikawa M, Okabe M, Imaizumi K. Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis. Nat Cell Biol. 2009; 11(10):1197-204.
• [21]Tohmonda T, Miyauchi Y, Ghosh R, Yoda M, Uchikawa S, Takito J, Morioka H, Nakamura M, Iwawaki T, Chiba K, Toyama Y, Urano F, Horiuchi K. The IRE1α-XBP1 pathway is essential for osteoblast differentiation through promoting transcription of Osterix. EMBO Rep. 2011; 12:451-7.
• [22]Liu CJ, Prazak L, Fajardo M, Yu S, Tyagi N, Di Cesare PE. Leukemia/lymphoma-related factor a POZ domain-containing transcriptional repressor, interacts with histone deacetylase-1 and inhibits cartilage oligomeric matrix protein gene expression and chondrogenesis. J Biol Chem. 2004; 279:47081-91.
• [23]Atkinson BL, Fantle KS, Benedict JJ, Huffer WE, Gutierrez-Hartmann A. Combination of osteoinductive bone proteins differentiates mesenchymal C3H/10T1/2 cells specifically to the cartilage lineage. J Cell Biochem. 1997; 65:325-39.
• [24]Canalis E, Economides AN, Gazzerro E. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 2003; 24:218-35.
• [25]Welch RD, Jones AL, Bucholz RW, Reinert CM, Tjia JS, Pierce WA, Wozney JM, Li XJ. Effect of recombinant human bone morphogenetic protein-2 on fracture healing in a goat tibial fracture model. J Bone Miner Res. 1998; 13:1483-90.
• [26]Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B. Type X collagen gene regulation by RUNX2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo. J Cell Biol. 2003; 162:833-42.
• [27]Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem. 2006;97:33–44.
• [28]Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron D. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000; 287:664-6.
• [29]Liu CY, Schröder M, Kaufman RJ. Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum. J Biol Chem. 2000; 275(32):24881-5.
• [30]Korennykh AV, Egea PF, Korostelev AA, Finer-Moore J, Zhang C, Shokat KM, Stroud RM, Walter P. The unfolded protein response signals through high-order assembly of IRE1. Nature. 2009; 457:687-93.
• [31]Lin JH, Li H, Yasumura D, Cohen HR, Zhang C, Panning B, Shokat KM, Lavail MM, Walter P. IRE1 signaling affects cell fate during the unfolded protein response. Science. 2007; 318:944-9.
• [32]Kim JW, Choi H, Jeong BC, Oh SH, Hur SW, Lee BN, Kim SH, Nör JE, Koh JT, Hwang YC. Transcriptional factor ATF6 is involved in odontoblastic differentiation. J Dent Res. 2014; 93(5):483-9.
• [33]Vekich JA, Belmont PJ, Thuerauf DJ, Glembotski CC. Protein disulfide isomerase-associated 6 is an ATF6-inducible ER stress response protein that protects cardiac myocytes from ischemia/reperfusion-mediated cell death. J Mol Cell Cardiol. 2012; 53(2):259-67.
• [34]Glembotski CC. Roles for ATF6 and the sarco/endoplasmic reticulum protein quality control system in the heart. J Mol Cell Cardiol. 2014; 71:11-5.
• [35]Howarth DL, Lindtner C, Vacaru AM, Sachidanandam R, Tsedensodnom O, Vasilkova T, Buettner C, Sadler KC. Activating transcription factor 6 is necessary and sufficient for alcoholic fatty liver disease in zebrafish. PLoS Genet. 2014; 10(5):e1004335.
• [36]Wu J, Ruas JL, Estall JL, Rasbach KA, Choi JH, Ye L, Boström P, Tyra HM, Crawford RW, Campbell KP, Rutkowski DT, Kaufman RJ, Spiegelman BM. The unfolded protein response mediates adaptation to exercise in skeletal muscle through a PGC-1α/ATF6α complex. Cell Metab. 2011; 13(2):160-9.
• [37]Li M, Baumeister P, Roy B, Phan T, Foti D, Luo S, Lee AS. ATF6 as a transcription activator of the endoplasmic reticulum stress element: thapsigargin stress-induced changes and synergistic interactions with NF-Y and YY1. Mol Cell Biol USA. 2000; 20(14):5096-106.
• [38]Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K, Yamana K, Zanma A, Takada K, Ito Y, Komori T. RUNX2 and Runx3 are essential for chondrocyte maturation, and RUNX2 regulates limb growth through induction of Indian hedgehog. Genes Dev. 2004; 18:952-63.
• [39]Drissi MH, Li X, Sheu TJ, Zuscik MJ, Schwarz EM, Puzas JE, Rosier RN, O’Keefe RJ. RUNX2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes. J Cell Biochem. 2003; 90:1287-98.
• [40]Tsang KY, Chan D, Cheslett D, Chan WC, So CL, Melhado IG, Chan TW, Kwan KM, Hunziker EB, Yamada Y, et al. Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function. PLoS Biol. 2007;5. doi:10.1371/journal.pbio.0050044.
• [41]Gutierrez S, Javed A, Tennant DK, van Rees M, Montecino M, Stein GS, Stein JL, Lian JB. CCAAT/enhancer-binding proteins (C/EBP) beta and delta activate osteocalcin gene transcription and synergize with RUNX2 at the C/EBP element to regulate bone-specific expression. J Biol Chem. 2002; 277:1316-23.
• [42]Schroeder TM, Kahler RA, Li X, Westendorf JJ. Histone deacetylase 3 interacts with Runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation. J Biol Chem. 2004; 279:41998-2007.
• [43]Yoshida CA, Furuichi T, Fujita T, Fukuyama R, Kanatani N, Kobayashi S, Satake M, Takada K, Komori T. Core-binding factor beta interacts with Runx2 and is required for skeletal development. Nat Genet. 2002; 32:633-8.