BoneKEy-Osteovision | Commentary
Pyrophosphate, an old actor in two new roles
DOI:10.1138/2001032
Age cannot wither her, nor custom stale Her infinite variety. Antony and Cleopatra. Act ii. Sc. 2.
Inorganic pyrophosphate (PPi) was recognized about 40 years ago to be a potent inhibitor of calcium phosphate formation and dissolution (). The fact that PPi was present in various biological fluids and could inhibit ectopic mineralization in vivo led to the hypothesis that it could act as a regulator of calcification, being destroyed at calcifying sites by enzymes with pyrophosphatase activity, such as alkaline phosphatase (). Its effect on crystal dissolution opened the possibility that PPi was also involved in bone resorption, the negative results in vivo being due to its too rapid destruction (). The mineralization hypothesis was supported by the finding that in hereditary hypophosphatasia, where alkaline phosphatase is inactive, PPi is increased in urine and plasma, possibly explaining the occurrence of osteomalacia in this disease. (The alternative although unlikely interpretation, is that ALP produces high local concentrations of Pi, which exceed the solubility product of calcium phosphate). These results prompted a look for analogs of pyrophosphate which were not destroyed by hydrolysis, and this resulted in the discovery of the biological action of bisphosphonates. They turn out to share the effect of PPi on mineralization but to have a probably distinct mechanism of inhibition of the osteoclast (). Pyrophosphate now reappears on stage in a command performance: Three recent papers have identified new roles for PPi, one in a murine form of hereditary arthritis () and the others in a human bone disorder, autosomal dominant craniometaphyseal dysplasia (OMIM 123000) ().
David Kingsley's group has identified the locus of the progressive arthritis (ank) mutation in the mouse (). The autosomal recessive ank mutation causes a severe, progressive form of arthritis in which hydroxyapatite crystals appear in joint fluid and synovial cartilage, heralding destructive arthritis, osteophyte formation, ankylosis and death due to immobility. Positional cloning of genes in an interval on mouse chromosome 15 identified a nonsense mutation in a 492 amino acid multipass integral membrane protein expressed on the cell surface (Fig 1). Based on previous evidence that PPi levels are low in joint fluid of ank/ank mice, Ho et al. () studied the involvement of the ANK protein in PPi transport. In skin fibroblasts from ank/ank mice intracellular PPi levels were increased and extracellular PPi levels reciprocally reduced; this defect was corrected by expression of wt ANK in mutant cells. Conversely, overexpression of ANK in COS cells reduced intracellular PPi levels to undetectable. Finally, the fall in intracellular PPi levels induced by expression of ANK was blocked by probenecid, a general inhibitor of organic anion transport.
Thus ANK appears to function as a PPi transporter. Loss of both copies of the gene for this transporter produces a severe form of crystal-induced arthritis, presumably because low PPi levels fail to inhibit hydroxyapatite crystal formation in joint fluid and cartilage. Heterozygotes for the ank mutation are normal, and neither heterozygotes nor homozygotes were noted to have a bone phenotype other than osteophyte formation. Interestingly, mutations in an ectoenzyme that produces PPi also produce ectopic mineralization of joints and ligaments in the mouse trait tiptoe walking ().
Enter autosomal dominant craniometaphyseal dysplasia (OMIM 123000). This rare disorder was originally described by Jackson in collaboration with Fuller Albright in 1954. Affected individuals have marked osteosclerosis of the craniofacial bones, often with neurological deficits from cranial nerve compression. The metaphysis of the long bones is flared (Erlenmeyer flask abnormality), but the extracranial skeleton and joints are otherwise unaffected.
Two groups have recently identified mutations in the human ortholog of the mouse ank gene on chromosome 5p as responsible for autosomal dominant craniometaphyseal dysplasia. In contrast to the mouse ank mutation, which introduces a premature stop codon, all the changes found to date in the human gene are point mutations that cluster in the intracellular domains (Fig 1). Together, these encompass seven mutations in 11 families and three sporadic cases, with several mutations occurring more than once.
The mouse and human mutations in the ank gene produce nonoverlapping phenotypes. The mouse ank mutation is inherited recessively and apparently causes a loss of transporter function. The clustered point mutations in humans produce a disorder with dominant inheritance which is unlikely to be explained simply by haploinsufficiency (loss of one functional allele), because they are exclusively point mutations, they are highly clustered and because haploinsufficiency in the mouse (or the human cri-du-chat syndrome, in which this locus is sometimes deleted) does not give rise to an abnormal phenotype. One possibility is that the mutations have a dominant negative effect, perhaps by competing for another protein or a small molecule regulator of PPi transport. If so, the cellular environment or regulation of the transporter in bone may be different from cartilage, which is unaffected in craniometaphyseal dysplasia. Another possibility is that human mutations induce a gain of function, increasing “leakiness” of PPi through the channel.
Could the phenotype of grossly thickened bones be explained solely by increased mineralization? It appears likely that PPi transport regulates cellular functions in bone, rather than simply affecting mineralization — but are these cellcular functions of the osteoblast or the osteoclast? Reichenberger et al. report that the transporter is expressed by PCR in osteoblasts, but not cells isolated from an osteoclastoma (). However, it was previously reported that osteoclast function in vitro was abnormal in cells isolated from a patient with craniometaphyseal dysplasia (). This raises the possibility of a cell-autonomous defect in osteoclast function, as might occur the transporter is expressed in mature, active osteoclasts and if PPi accumulated in mutant cells because of a loss of PPi transporter function. Regardless, the identification of the ANK transporter as the actor in craniometaphyseal dysplasia puts pyrophosphate on the stage again in bone.
The comments of Herbert Fleisch and Gideon Rodan are gratefully acknowledged.
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