Mineral Formation in Joints Caused by Complete or Joint-Specific Loss of ANK Function

Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Journal of Bone and Mineral Research (Impact Factor: 6.83). 09/2006; 21(8):1238-47. DOI: 10.1359/jbmr.060515
Source: PubMed


To reveal the ANK complete loss of function phenotype in mice, we generated conditional and null alleles. Mice homozygous for the null allele exhibited widespread joint mineralization, similar in severity to animals harboring the original ank allele. A delayed yet similar phenotype was observed in mice with joint-specific loss of ANK function.
The ANK pyrophosphate regulator was originally identified and proposed to play a key role in articular cartilage maintenance based on a single spontaneous mouse mutation (ank) that causes severe generalized arthritis. A number of human mutations have subsequently been reported in the human ortholog (ANKH), some of which produce skull and long bone defects with no apparent defects in joints or articular cartilage. None of the currently known mouse or human mutations clearly eliminate the function of the endogenous gene.
Two new Ank alleles were generated using homologous recombination in mouse embryonic stem (ES) cells. Joint range of motion assays and muCT studies were used to quantitatively assess phenotypic severity in wildtype, heterozygous, and homozygous mice carrying either the null (Anknull) or original (Ankank) allele. A Gdf5-Cre expressing line was crossed to mice harboring the conditional (Ankfloxp) allele to eliminate ANK function specifically in the joints. Histological stains and beta-galactosidase (LACZ) activity were used to determine the correlation between local loss of ANK function and defective joint phenotypes.
Anknull/Anknull mice develop severe ectopic postnatal crystal deposition in almost every joint of the body, leading to eventual joint fusion and loss of mobility. The severity of phenotype in these mice is indistinguishable from that of Ankank/Ankank mice. In addition, despite the widespread expression of Ank in many tissues, the specific deletion of Ank in joints also produces joint mineralization and ankylosis.
These studies show that ANK function is required locally in joints to inhibit mineral formation and that the Ank gene plays a key role in postnatal maintenance of joint mobility and function.

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    • "Surprisingly, although the disc contains both fibrous proteins and a hydrated extracellular matrix, there are no calcified deposits in the normal NP tissue. The local control of dystrophic mineralization in the NP is necessary to prevent dystrophic mineralization of the disc.62 "
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    ABSTRACT: Intervertebral disc (IVD) degeneration is implicated as a major cause of low back pain. The alternated phenotypes, reduced cell survival, decreased metabolic activity, loss of matrix production and dystrophic mineralization of nucleus pulposus (NP) cells may be key contributors to progressive IVD degeneration. IVD is the largest avascular structure in the body, characterized by low oxygen tension in vivo. Hypoxia-inducible factor (HIF) is a master transcription factor that is induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. This review summarizes relevant studies concerning the involvement of HIF in the regulation of biological behaviors of NP cells. We describe current data on the expression of HIF in NP cells and further discuss the various roles that HIF plays in the regulation of the phenotype, survival, metabolism, matrix production and dystrophic mineralization of NP cells. Here, we conclude that HIF may be a promising target for the prevention and treatment of IVD degeneration.
    Yonsei medical journal 07/2013; 54(4):807-812. DOI:10.3349/ymj.2013.54.4.807 · 1.29 Impact Factor
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    • "Preparation and genotyping of mouse models was previously described for Ank−/− [26], [71], Alpl−/− (previously known as Akp2−/−), and Enpp1−/− [10], [14], [16], [54]. Ank and Alpl mice were maintained on a mixed background of 129S1/SvImJ and C57BL/6 strains, and Enpp1 mice were maintained on a mixed background of C57BL/6×129/SvTerJ strains. "
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    ABSTRACT: Inorganic pyrophosphate (PP(i)) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PP(i) are controlled by antagonistic functions of factors that decrease PP(i) and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PP(i) and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PP(i) in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PP(i) dysregulation. Excess PP(i) in the Alpl(-/-) mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PP(i) in both Ank and Enpp1(-/-) mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PP(i) regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PP(i) output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PP(i) mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PP(i) inhibited mineralization and associated increases in Ank and Enpp1 mRNA. Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PP(i), directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.
    PLoS ONE 06/2012; 7(6):e38393. DOI:10.1371/journal.pone.0038393 · 3.23 Impact Factor
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    • ". Heterozygote breeding pairs were employed to prepare homozygote Ank KO mice and age-matched WT controls at specific ages during tooth development from 24 to 79 days post coitum (dpc), where birth usually falls on 19 dpc. In order to conditionally ablate Ank in joints, including the periodontal gomphosis, Gdf5-Cre mice were crossed with mice harboring a floxed Ank allele, Ank floxP , as previously described [Gurley et al., 2006a]. Gdf5-Cre / Ank floxP were prepared for 47–60 dpc. "
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    ABSTRACT: Tooth root cementum is sensitive to modulation of inorganic pyrophosphate (PP(i)), an inhibitor of hydroxyapatite precipitation. Factors increasing PP(i) include progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) while tissue nonspecific alkaline phosphatase hydrolyzes PP(i). Studies here aimed to define the role of ANK in root and cementum by analyzing tooth development in Ank knock-out (KO) mice versus wild type. Periodontal development in KO versus control mice was analyzed by histology, histomorphometry, immunohistochemistry, in situ hybridization, electron microscopy, and nanoindentation. Cementoblast cultures were used in vitro to provide mechanistic underpinnings for PP(i) modulation of cell function. Over the course of root development, Ank KO cervical cementum became 8- to 12-fold thicker than control cervical cementum. Periodontal ligament width was maintained and other dentoalveolar tissues, including apical cementum, were unaltered. Cervical cementum uncharacteristically included numerous cells, from rapid cementogenesis. Ank KO increased osteopontin and dentin matrix protein 1 gene and protein expression, and markedly increased NPP1 protein expression in cementoblasts but not in other cell types. Conditional ablation of Ank in joints and periodontia confirmed a local role for ANK in cementogenesis. In vitro studies employing cementoblasts indicated that Ank and Enpp1 mRNA levels increased in step with mineral nodule formation, supporting a role for these factors in regulation of cementum matrix mineralization. ANK, by modulating local PP(i), controls cervical cementum apposition and extracellular matrix. Loss of ANK created a local environment conducive to rapid cementogenesis; therefore, approaches modulating PP(i) in periodontal tissues have potential to promote cementum regeneration.
    Cells Tissues Organs 03/2011; 194(5):382-405. DOI:10.1159/000323457 · 2.14 Impact Factor
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