Biochemical and Genetic Analysis of ANK in Arthritis and Bone Disease

Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
The American Journal of Human Genetics (Impact Factor: 10.99). 01/2007; 79(6):1017-29. DOI: 10.1086/509881
Source: PubMed

ABSTRACT Mutations in the progressive ankylosis gene (Ank/ANKH) cause surprisingly different skeletal phenotypes in mice and humans. In mice, recessive loss-of-function mutations cause arthritis, ectopic crystal formation, and joint fusion throughout the body. In humans, some dominant mutations cause chondrocalcinosis, an adult-onset disease characterized by the deposition of ectopic joint crystals. Other dominant mutations cause craniometaphyseal dysplasia, a childhood disease characterized by sclerosis of the skull and abnormal modeling of the long bones, with little or no joint pathology. Ank encodes a multiple-pass transmembrane protein that regulates pyrophosphate levels inside and outside tissue culture cells in vitro, but its mechanism of action is not yet clear, and conflicting models have been proposed to explain the effects of the human mutations. Here, we test wild-type and mutant forms of ANK for radiolabeled pyrophosphate-transport activity in frog oocytes. We also reconstruct two human mutations in a bacterial artificial chromosome and test them in transgenic mice for rescue of the Ank null phenotype and for induction of new skeletal phenotypes. Wild-type ANK stimulates saturable transport of pyrophosphate ions across the plasma membrane, with half maximal rates attained at physiological levels of pyrophosphate. Chondrocalcinosis mutations retain apparently wild-type transport activity and can rescue the joint-fusion phenotype of Ank null mice. Craniometaphyseal dysplasia mutations do not transport pyrophosphate and cannot rescue the defects of Ank null mice. Furthermore, microcomputed tomography revealed previously unappreciated phenotypes in Ank null mice that are reminiscent of craniometaphyseal dysplasia. The combination of biochemical and genetic analyses presented here provides insight into how mutations in ANKH cause human skeletal disease.

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    • "Loss of ANK function results in decreased extracellular PP i , and mice lacking ANK exhibit an arthritis-like phenotype characterized by ectopic calcifications in cartilage and joint tissues [Ho et al., 2000; Terkeltaub, 2001; Harmey et al., 2004; Gurley et al., 2006a]. In humans, ANKH mutations are linked to both craniometaphyseal dysplasia (CMD), characterized by overgrowth and sclerosis of craniofacial bones and abnormal modeling of long-bone metaphyses [Nurnberg et al., 2001; Reichenberger et al., 2001], and chondrocalcinosis, resulting from deposition of calcium pyrophosphate dihydrate within articular cartilage [Ryan, 2001; Pendleton et al., 2002; Netter et al., 2004; Gurley et al., 2006b]. Our group described the intriguing tooth phenotype in classical Ank mutant mice, characterized by a marked increase in cementum formation, while other tooth tissues (e.g. "
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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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    • "In contrast, Ank with CMD mutations had an opposite effect on PPi levels in cultured cells [Gurley et al., 2006b]. Our immunofluorescence labeling study suggests that the reduced extracellular PPi concentration in CMD cells may be the result of an interrupted intracellular protein trafficking leading to decreased levels of ANKH targeting the plasma membrane. "
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    ABSTRACT: Craniometaphyseal dysplasia is caused by mutations in ANKH (ankylosis, progressive homolog [mouse]) in the majority of cases, and all of the reported mutations are single amino acid changes. Genomic DNA from an affected patient, his biological parents, and a sibling was amplified and ANKH was sequenced. The affected patient had a complex heterozygous mutation in exon 7 (c.936T > C, c.938C > G, c.942_953delTGGTTGACGGAA), predicting p.Try290Gln and p.Trp292_Glu295del. We studied the effect of the predicted mutation on the subcellular distribution of ANKH protein. Immunofluorescent labeling of COS-7 cells transduced with normal or mutant Ank (murine progressive ankylosis), showed that normal Ank localized to both the plasma membrane and cytoplasm, whereas mutant Ank was detected only in the cytoplasmic compartment. We propose that this craniometaphyseal dysplasia mutation causes a loss of ANKH protein expression and activity in the plasma membrane as a result of aberrant intracellular protein trafficking.
    American Journal of Medical Genetics Part A 03/2010; 152A(3):770-6. DOI:10.1002/ajmg.a.33317 · 2.05 Impact Factor
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    • "The lack of ANK function in humans and mice results in thickened craniofacial bones, whereas the long bones show an osteopenic phenotype [Gurley et al., 2006]. The results of our study, showing that ANK is required for the differentiation of preosteoblastic cells into immature osteoblasts, are in accordance with osteopenia of the long bones in individuals with suppressed ANK function . "
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    ABSTRACT: The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu. Human mutations of ank lead to craniometaphyseal dysplasia, a disease which is characterized by the overgrowth of craniofacial bones and osteopenia in long bones, suggesting that ANK plays a regulatory role in osteoblast differentiation. To determine the role of ANK in osteoblast differentiation, we suppressed ANK expression in the osteoblastic MC3T3-E1 cell line using siRNA and determined the expression of osteoblastic marker genes and the transcription factors osterix and runx2. In addition, we determined the osteoblastic differentiation of bone marrow stromal cells isolated from the bone marrow of ank/ank mice, which express a truncated, nonfunctional ANK protein, or wild-type littermates. Suppression of ANK expression in MC3T3-E1 cells led to a decrease in bone marker gene expression, including alkaline phosphatase, bone sialoprotein, osteocalcin and type I collagen. In addition, osterix gene expression was decreased in ANK expression-suppressed MC3T3 cells, whereas runx2 expression was increased. Bone marrow stromal cells isolated from ank/ank mice cultured in the presence of ascorbate-2-phosphate for up to 35 days showed markedly reduced mineralization compared to the mineralization of bone marrow stromal cells isolated from wild-type littermates. In conclusion, these findings suggest that ANK is a positive regulator of differentiation events towards a mature osteoblastic phenotype.
    Cells Tissues Organs 09/2008; 189(1-4):158-62. DOI:10.1159/000151725 · 2.14 Impact Factor
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