title: Cartilage-specific ablation of XBP1 signaling in mouse results in a chondrodysplasia characterized by reduced chondrocyte proliferation and delayed cartilage maturation and mineralization.
abstract: OBJECTIVE
  To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage.
  
  
  METHODS
  Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation.
  
  
  RESULTS
  Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR.
  
  
  CONCLUSIONS
  Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease.
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: trevor.cameron@mcri.edu.au.
authors.forename: T L
authors.initials: TL
authors.lastname: Cameron
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: irma.gresshoff@unimelb.edu.au.
authors.forename: I L
authors.initials: IL
authors.lastname: Gresshoff
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: katrina.bell@mcri.edu.au.
authors.forename: K M
authors.initials: KM
authors.lastname: Bell
authors.affiliation: Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK. Electronic address: katarzyna.pirog@newcastle.ac.uk.
authors.forename: K A
authors.initials: KA
authors.lastname: Piróg
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: lisa.sampurno@mcri.edu.au.
authors.forename: L
authors.initials: L
authors.lastname: Sampurno
authors.affiliation: Central Manchester University Hospitals NHS Foundation Trust, St Mary's Hospital, Manchester, UK. Electronic address: claire.burgoyne@cmft.nhs.uk.
authors.forename: C L
authors.initials: CL
authors.lastname: Hartley
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: emma.sanford@mcri.edu.au.
authors.forename: E M
authors.initials: EM
authors.lastname: Sanford
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: richard.wilson@utas.edu.au.
authors.forename: R
authors.initials: R
authors.lastname: Wilson
authors.affiliation: Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA. Electronic address: jermann@partners.org.
authors.forename: J
authors.initials: J
authors.lastname: Ermann
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, The University of Manchester, Manchester, UK. Electronic address: ray.boot-handford@manchester.ac.uk.
authors.forename: R P
authors.initials: RP
authors.lastname: Boot-Handford
authors.affiliation: Weill Cornell Medical College, Cornell University, New York, NY, USA. Electronic address: lglimche@med.cornell.edu.
authors.forename: L H
authors.initials: LH
authors.lastname: Glimcher
authors.affiliation: Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK. Electronic address: michael.briggs@newcastle.ac.uk.
authors.forename: M D
authors.initials: MD
authors.lastname: Briggs
authors.affiliation: Murdoch Childrens Research Institute, Parkville, Victoria, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia. Electronic address: john.bateman@mcri.edu.au.
authors.forename: J F
authors.initials: JF
authors.lastname: Bateman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002113:Calcification, Physiologic / Q000502:physiology*; D002358:Cartilage, Articular / Q000473:pathology* / Q000503:physiopathology; D049109:Cell Proliferation / Q000502:physiology; D019902:Chondrocytes / Q000473:pathology*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D004195:Disease Models, Animal; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D017353:Gene Deletion; D006132:Growth Plate / Q000473:pathology / Q000503:physiopathology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D010009:Osteochondrodysplasias / Q000473:pathology* / Q000503:physiopathology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology; D000071701:Regulatory Factor X Transcription Factors; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D000071717:X-Box Binding Protein 1
chemical_list: D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605544:Xbp1 protein, mouse; C486556:Ern2 protein, mouse; D017346:Protein Serine-Threonine Kinases
keywords: Chondrodysplasia; ER stress; Endochondral ossification; Growth plate; XBP1