title: Disruption of biomineralization pathways in spinal tissues of a mouse model of diffuse idiopathic skeletal hyperostosis.
abstract: Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1(-/-)) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1(-/-) mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1(-/-) mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1(-/-) mice at 2months of age (prior to disc mineralization), 4, and 6months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [(3)H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1(-/-) and wild-type mice at 2 or 4months of age. However, at 6months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1(-/-) mice. To assess whether changes detected in ENT1(-/-) mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1(-/-) IVDs at 2 or 6months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1(-/-) mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1(-/-) mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1(-/-) mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1(-/-) mice.
authors.affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada; Department of Oral Health School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.
authors.forename: Hisataka
authors.initials: H
authors.lastname: Ii
authors.affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Sumeeta
authors.initials: S
authors.lastname: Warraich
authors.affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Neil
authors.initials: N
authors.lastname: Tenn
authors.affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Diana
authors.initials: D
authors.lastname: Quinonez
authors.affiliation: Imaging Research Laboratories, Robarts Research Institute, Department of Surgery, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada.
authors.forename: David W
authors.initials: DW
authors.lastname: Holdsworth
authors.affiliation: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: James R
authors.initials: JR
authors.lastname: Hammond
authors.affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada.
authors.forename: S Jeffrey
authors.initials: SJ
authors.lastname: Dixon
authors.affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada. Electronic address: cheryle.seguin@schulich.uwo.ca.
authors.forename: Cheryle A
authors.initials: CA
authors.lastname: Séguin
mesh_terms: D015762:2-Chloroadenosine / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000070616:Annulus Fibrosus; D002113:Calcification, Physiologic / Q000235:genetics; D002114:Calcinosis / Q000235:genetics / Q000473:pathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D004057:Hyperostosis, Diffuse Idiopathic Skeletal / Q000000981:diagnostic imaging / Q000473:pathology* / Q000503:physiopathology*; D007403:Intervertebral Disc / Q000000981:diagnostic imaging / Q000473:pathology / Q000503:physiopathology; D008297:Male; D008810:Mice, Inbred C57BL; D012727:Sex Characteristics; D013131:Spine / Q000000981:diagnostic imaging / Q000473:pathology* / Q000503:physiopathology*; D055114:X-Ray Microtomography
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D015762:2-Chloroadenosine; D000469:Alkaline Phosphatase
keywords: Alkaline phosphatase; Annulus fibrosus; DISH; Diffuse idiopathic skeletal hyperostosis; ENT1; Equilibrative nucleoside transporter 1; Intervertebral disc; Mineralization