Biochemical and Genetic Analysis of ANK in Arthritis and Bone Disease

Department of Developmental Biology , Stanford University, Stanford, California, United States
The American Journal of Human Genetics (Impact Factor: 10.93). 01/2007; 79(6):1017-29. DOI: 10.1086/509881
Source: PubMed


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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    • "ANKH encodes a multipass transmembrane protein (ANKH) in joints and other tissues and participates in the export of intracellular PPi [30,31]. PPi cannot diffuse across cell membranes passively, and ANKH is the principal way in which intracellular PPi reaches the extracellular environment. "
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    ABSTRACT: Chondrocalcinosis (CC) most commonly results from calcium pyrophosphate crystal deposition (CPPD). The objective of this study is to examine the association between candidate single nucleotide polymorphisms (SNPs) and radiographic CC. SNPs in ankylosis human (ANKH), high ferritin (HFE), tissue non-specific alkaline phosphatase (TNAP), ecto-neucleotide pyrophosphatase (ENPP1) and transferrin (TE) genes were genotyped in participants of the Genetics of Osteoarthritis (OA) and Lifestyle (GOAL) and Nottingham OA case-control studies. Adjusted genotype odds ratio (aORGENOTYPE), the OR for association between one additional minor allele and CC was calculated, and adjusted for age, gender, body mass index (BMI), and OA using binary logistic regression. Statistical significance was set at P <=0.003 after Bonferroni correction for multiple tests. The -4bpG > A polymorphism in the 5'UTR of ANKH associated with CC after Bonferroni correction. This was independent of age, gender, OA, and BMI (aORGENOTYPE (95%CI) 1.39 (1.14-1.69), P = 0.001). rs3045 and rs875525, two other SNPs in ANKH associated with CC (aORGENOTYPE (95%CI) of 1.31 (1.09-1.58), P = 0.005; and 1.18 (1.03-1.35), P = 0.015 respectively), however, this was non-significant after Bonferroni correction. This study validates the association between a functional polymorphism in the 5'UTR of ANKH and CC, and shows for the first time that this is independent of age and OA - the two key risk factors for CC. It shows that other SNPs in ANKH may also associate with CC. This supports the role of extracellular inorganic pyrophosphate in the pathogenesis of CC. The findings of this hospital-based study require replication in a community-based population.
    Full-text · Article · Jan 2014 · Arthritis research & therapy
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    • "ANK is a 492 amino acid multipass transmembrane protein originally described as the mutated protein in ank/ank mice [9]. Considerable evidence supports its role in extracellular pyrophosphate (ePPi) transport [9,10]. ePPi is a key regulator of pathologic mineralization in cartilage and other tissues. "
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    ABSTRACT: Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process. We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release. eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels. These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.
    Full-text · Article · Oct 2013 · Arthritis research & therapy
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    • "The progressive ankylosis protein (mouse Ank and the human homolog, ANKH), is a pyrophosphate transporter that regulates PPi transport across plasma membrane between intra- and extracellular compartments [26], [29]. Reduced fertility has been reported in ank mutant mice [26]. "
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    ABSTRACT: Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.
    Preview · Article · Apr 2012 · PLoS ONE
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