Inorganic pyrophosphatase induces type I collagen in osteoblasts

The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PP(i)) and generates phosphate (P(i)). Humans and mice deficient in the PP(i)-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPP(i)ase), which generates P(i) via PP(i) hydrolysis with neutral pH optimum, remains unknown. We assessed iPP(i)ase in Enpp1(-/-) and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPP(i)ase regulates collagen I expression.

We treated mouse calvarial osteoblasts with ascorbate and beta-glycerol phosphate to promote calcification, and we assessed cytosolic P(i) and PP(i) levels, sodium-dependent P(i) uptake, Pit-1 P(i) co-transporter expression, and iPP(i)ase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.

Inorganic pyrophosphatase but not TNAP was elevated in Enpp1(-/-) calvariae in situ. Cultured primary Enpp1(-/-) calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PP(i) in early culture, Enpp1(-/-) osteoblasts maintained cytosolic P(i) levels comparable to WT osteoblasts, in association with increased iPP(i)ase, enhanced sodium-dependent P(i) uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1(-/-) osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic P(i) and decreased cytosolic but not extracellular PP(i), and induced both collagen I synthesis and calcification.

Increased iPP(i)ase is associated with "P(i) hunger" and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPP(i)ase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PP(i).