Linked deficiencies in extracellular PPi and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression

Osteopontin and PPi both suppress hydroxyapatite deposition. Extracellular PPi deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PPi deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPPI) function partly to generate PPI, a physiologic calcification inhibitor. PPi transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in anklank mice. To assess how PC-1, ANK, and PPi regulate both calcification and cell differentiation, we studied cultured PC-1(-/-) and anklank mouse calvarial osteoblasts. PC-1(-/-) osteoblasts demonstrated similar to50% depressed NPP activity and markedly lowered extracellular PPi associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1(-/-) and anklank cultured osteoblasts demonstrated not only comparable extracellular PPi depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PPi and OPN), and OPN itself (greater than or equal to15 pg/ml), corrected hypercalcification by PC-1(-/-) and anklank osteoblasts. Thus, linked regulatory effects on extracellular PPi and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.