Inorganic pyrophosphatase induces type I collagen in osteoblasts

Abstract

Introduction 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 (PPi) and generates phosphate (Pi). Humans and mice deficient in the PPi-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 iPPiase), which generates Pi via PPi hydrolysis with neutral pH optimum, remains unknown. We assessed iPPiase in Enpp1−/− and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPPiase regulates collagen I expression. Methods We treated mouse calvarial osteoblasts with ascorbate and β-glycerol phosphate to promote calcification, and we assessed cytosolic Pi and PPi levels, sodium-dependent Pi uptake, Pit-1 Pi co-transporter expression, and iPPiase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts. Results 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 PPi in early culture, Enpp1−/− osteoblasts maintained cytosolic Pi levels comparable to WT osteoblasts, in association with increased iPPiase, enhanced sodium-dependent Pi 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 Pi and decreased cytosolic but not extracellular PPi, and induced both collagen I synthesis and calcification. Conclusions Increased iPPiase is associated with “Pi hunger” and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPPiase 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 PPi.

Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) Protein Regulates Osteoblast Differentiation

“…3, H and I). This result is consistent with a previous report that demonstrated that ENPP1 expression in calvarial osteoblastic cells influenced TNAP enzyme activity to a greater extent than TNAP mRNA expression upon treatment of cells with ascorbate (38).…”

“…These results indicate that the increased levels of osteoblastic gene expression seen in pPGS/ Enpp1 cells upon ascorbate treatment are not due to increased collagen expression in the pPGS/Enpp1 cells. That Col1a1 mRNA expression is increased in Enpp1 shRNA-expressing cells and decreased in Enpp1 cDNA-expressing cells is consistent with the previous report that type I collagen expression and synthesis is increased in calvarial osteoblasts isolated from Enpp1 Ϫ/Ϫ mice (38). In support of our findings that Enpp1 enhances osteoblast differentiation, pPGS control and ENPP1-overexpressing cells were generated on two separate occasions, and results were similar with both sets of cells (data not shown).…”

Bioinorganics and biomaterials: Bone repair

“…However, this seems to be a simplification, as there are at least two more phosphatases known to be active at the site of mineralization. PHOSPHO 1 generates Pi from phospholipids [47] and inorganic pyrophosphatase generates Pi from cytosolic PPi in osteoblasts [48]. This latter pyrophosphatase was also found to significantly regulate collagen expression.…”

Dual Role of the Trps1 Transcription Factor in Dentin Mineralization

“…5D). It has been reported that Opn is up-regulated upon TNAP deficiency and by increased PP i levels (14,50); therefore, we analyzed expression of a PP i transporter Ank but detected no significant difference in Ank expression between Trps1-KD cells and controls (Fig. 5E).…”