T L Cameron1, I L Gresshoff2, K M Bell3, K A Piróg4, L Sampurno5, C L Hartley6, E M Sanford7, R Wilson8, J Ermann9, R P Boot-Handford10, L H Glimcher11, M D Briggs12, J F Bateman13. 1. Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: trevor.cameron@mcri.edu.au. 2. Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: irma.gresshoff@unimelb.edu.au. 3. Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: katrina.bell@mcri.edu.au. 4. Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK. Electronic address: katarzyna.pirog@newcastle.ac.uk. 5. Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: lisa.sampurno@mcri.edu.au. 6. Central Manchester University Hospitals NHS Foundation Trust, St Mary's Hospital, Manchester, UK. Electronic address: claire.burgoyne@cmft.nhs.uk. 7. Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: emma.sanford@mcri.edu.au. 8. Murdoch Childrens Research Institute, Parkville, Victoria, Australia. Electronic address: richard.wilson@utas.edu.au. 9. 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. 10. 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. 11. Weill Cornell Medical College, Cornell University, New York, NY, USA. Electronic address: lglimche@med.cornell.edu. 12. Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK. Electronic address: michael.briggs@newcastle.ac.uk. 13. 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.
Abstract
OBJECTIVE: To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage. DESIGN: 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. CONCLUSION: 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.
OBJECTIVE: To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage. DESIGN: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. CONCLUSION: 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: Cheng Wang; Zhijia Tan; Ben Niu; Kwok Yeung Tsang; Andrew Tai; Wilson C W Chan; Rebecca L K Lo; Keith K H Leung; Nelson W F Dung; Nobuyuki Itoh; Michael Q Zhang; Danny Chan; Kathryn Song Eng Cheah Journal: Elife Date: 2018-07-19 Impact factor: 8.140
Authors: Trevor L Cameron; Katrina M Bell; Irma L Gresshoff; Lisa Sampurno; Lorna Mullan; Joerg Ermann; Laurie H Glimcher; Raymond P Boot-Handford; John F Bateman Journal: PLoS Genet Date: 2015-09-15 Impact factor: 5.917