Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-β activity in MCDS chondrocytes may offer therapeutic opportunities.
References
[1]
Mackie EJ, Ahmed YA, Tatarczuch L, Chen KS, Mirams M (2008) Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol 40: 46–62. pmid:17659995 doi: 10.1016/j.biocel.2007.06.009
[2]
Asada R, Kanemoto S, Kondo S, Saito A, Imaizumi K (2011) The signalling from endoplasmic reticulum-resident bZIP transcription factors involved in diverse cellular physiology. J Biochem 149: 507–518. doi: 10.1093/jb/mvr041. pmid:21454302
[3]
Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13: 89–102. doi: 10.1038/nrm3270. pmid:22251901
[4]
Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8: 519–529. pmid:17565364 doi: 10.1038/nrm2199
[5]
Schroder M, Kaufman RJ (2005) The mammalian unfolded protein response. Annu Rev Biochem 74: 739–789. pmid:15952902 doi: 10.1146/annurev.biochem.73.011303.074134
[6]
Haze K, Yoshida H, Yanagi H, Yura T, Mori K (1999) Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 10: 3787–3799. pmid:10564271 doi: 10.1091/mbc.10.11.3787
[7]
Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397: 271–274. pmid:9930704
[8]
Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, et al. (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6: 1099–1108. pmid:11106749 doi: 10.1016/s1097-2765(00)00108-8
[9]
Lu PD, Harding HP, Ron D (2004) Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J Cell Biol 167: 27–33. pmid:15479734 doi: 10.1083/jcb.200408003
[10]
Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107: 881–891. pmid:11779464 doi: 10.1016/s0092-8674(01)00611-0
[11]
Rajpar MH, McDermott B, Kung L, Eardley R, Knowles L, et al. (2009) Targeted induction of endoplasmic reticulum stress induces cartilage pathology. PLoS Genet 5: e1000691. doi: 10.1371/journal.pgen.1000691. pmid:19834559
[12]
Cameron TL, Bell KM, Tatarczuch L, Mackie EJ, Rajpar MH, et al. (2011) Transcriptional profiling of chondrodysplasia growth plate cartilage reveals adaptive ER-stress networks that allow survival but disrupt hypertrophy. PLoS One 6: e24600. doi: 10.1371/journal.pone.0024600. pmid:21935428
[13]
He Y, Sun S, Sha H, Liu Z, Yang L, et al. (2010) Emerging roles for XBP1, a sUPeR transcription factor. Gene Expr 15: 13–25. pmid:21061914 doi: 10.3727/105221610x12819686555051
[14]
Cameron TL, Gresshoff IL, Bell KM, Pirog KA, Sampurno L, et al. (2015) Cartilage-Specific Ablation of XBP1 Signaling in Mouse Results in a Chondrodysplasia Characterized by Reduced Chondrocyte Proliferation and Delayed Cartilage Maturation and Mineralization. Osteoarthritis Cartilage 23: 661–670. doi: 10.1016/j.joca.2015.01.001. pmid:25600960
[15]
Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11: 381–389. pmid:14685163 doi: 10.1038/sj.cdd.4401373
[16]
Hirata M, Kugimiya F, Fukai A, Ohba S, Kawamura N, et al. (2009) C/EBPbeta Promotes transition from proliferation to hypertrophic differentiation of chondrocytes through transactivation of p57. PLoS One 4: e4543. doi: 10.1371/journal.pone.0004543. pmid:19229324
[17]
Hirata M, Kugimiya F, Fukai A, Saito T, Yano F, et al. (2012) C/EBPbeta and RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2alpha as the inducer in chondrocytes. Hum Mol Genet 21: 1111–1123. doi: 10.1093/hmg/ddr540. pmid:22095691
[18]
Ushijima T, Okazaki K, Tsushima H, Iwamoto Y (2014) CCAAT/enhancer-binding protein beta regulates the repression of type II collagen expression during the differentiation from proliferative to hypertrophic chondrocytes. J Biol Chem 289: 2852–2863. doi: 10.1074/jbc.M113.492843. pmid:24344131
[19]
Lee AH, Iwakoshi NN, Glimcher LH (2003) XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23: 7448–7459. pmid:14559994 doi: 10.1128/mcb.23.21.7448-7459.2003
[20]
Tsuchimochi K, Otero M, Dragomir CL, Plumb DA, Zerbini LF, et al. (2010) GADD45beta enhances Col10a1 transcription via the MTK1/MKK3/6/p38 axis and activation of C/EBPbeta-TAD4 in terminally differentiating chondrocytes. J Biol Chem 285: 8395–8407. doi: 10.1074/jbc.M109.038638. pmid:20048163
[21]
Ijiri K, Zerbini LF, Peng H, Correa RG, Lu B, et al. (2005) A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation. J Biol Chem 280: 38544–38555. pmid:16144844 doi: 10.1074/jbc.m504202200
[22]
Ron D, Habener JF (1992) CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev 6: 439–453. pmid:1547942 doi: 10.1101/gad.6.3.439
[23]
Wu J, Rutkowski DT, Dubois M, Swathirajan J, Saunders T, et al. (2007) ATF6alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress. Dev Cell 13: 351–364. pmid:17765679 doi: 10.1016/j.devcel.2007.07.005
[24]
Okada T, Yoshida H, Akazawa R, Negishi M, Mori K (2002) Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochem J 366: 585–594. pmid:12014989 doi: 10.1042/bj20020391
[25]
Maurel M, Chevet E, Tavernier J, Gerlo S (2014) Getting RIDD of RNA: IRE1 in cell fate regulation. Trends Biochem Sci 39: 245–254. doi: 10.1016/j.tibs.2014.02.008. pmid:24657016
[26]
Chen Y, Brandizzi F (2013) IRE1: ER stress sensor and cell fate executor. Trends Cell Biol 23: 547–555. doi: 10.1016/j.tcb.2013.06.005. pmid:23880584
[27]
Tsang KY, Chan D, Cheslett D, Chan WC, So CL, et al. (2007) Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function. PLoS Biol 5: e44. pmid:17298185 doi: 10.1371/journal.pbio.0050044
[28]
Ho MS, Tsang KY, Lo RL, Susic M, Makitie O, et al. (2007) COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid. Hum Mol Genet 16: 1201–1215. pmid:17403716 doi: 10.1093/hmg/ddm067
[29]
Kung LH, Rajpar MH, Briggs MD, Boot-Handford RP (2012) Hypertrophic chondrocytes have a limited capacity to cope with increases in endoplasmic reticulum stress without triggering the unfolded protein response. J Histochem Cytochem 60: 734–748. pmid:22859705 doi: 10.1369/0022155412458436
[30]
Adachi Y, Yamamoto K, Okada T, Yoshida H, Harada A, et al. (2008) ATF6 is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum. Cell Struct Funct 33: 75–89. pmid:18360008 doi: 10.1247/csf.07044
[31]
Han J, Back SH, Hur J, Lin YH, Gildersleeve R, et al. (2013) ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol 15: 481–490. doi: 10.1038/ncb2738. pmid:23624402
Chikka MR, McCabe DD, Tyra HM, Rutkowski DT (2013) C/EBP homologous protein (CHOP) contributes to suppression of metabolic genes during endoplasmic reticulum stress in the liver. J Biol Chem 288: 4405–4415. doi: 10.1074/jbc.M112.432344. pmid:23281479
[34]
Rutkowski DT, Wu J, Back SH, Callaghan MU, Ferris SP, et al. (2008) UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators. Dev Cell 15: 829–840. doi: 10.1016/j.devcel.2008.10.015. pmid:19081072
[35]
Zhang P, Liegeois NJ, Wong C, Finegold M, Hou H, et al. (1997) Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome. Nature 387: 151–158. pmid:9144284 doi: 10.1038/387151a0
[36]
Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, et al. (2004) Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci U S A 101: 17192–17197. pmid:15563592 doi: 10.1073/pnas.0407788101
[37]
Woehlbier U, Hetz C (2011) Modulating stress responses by the UPRosome: a matter of life and death. Trends Biochem Sci 36: 329–337. doi: 10.1016/j.tibs.2011.03.001. pmid:21482118
[38]
Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey V., Dudoit S., Irizarry R., Huber W., editor. Bioinformatics and Computational Biology Solutions using R and Bioconductor: Springer, New York. pp. 397–420.
[39]
Dunning M, Lynch A., Eldridge M. (2014) illuminaMousev2.db: Illumina MouseWG6v2 annotation data (chip illuminaMousev2). R package version 1.22.1.
[40]
Wu D, Lim E, Vaillant F, Asselin-Labat ML, Visvader JE, et al. (2010) ROAST: rotation gene set tests for complex microarray experiments. Bioinformatics 26: 2176–2182. doi: 10.1093/bioinformatics/btq401. pmid:20610611
