Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells

Abstract

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP‐2–induced matrix calcification was studied. BMP‐2 enhances Pit‐1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. Introduction: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP‐2–induced matrix mineralization. Materials and Methods: Confluent MC3T3‐E1 osteoblast‐like cells were exposed to BMP‐2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit‐1 and Pit‐2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). Results: BMP‐2 time‐ and dose‐dependently stimulated Na‐dependent Pi transport in MC3T3‐E1 cells by increasing the Vmax of the transport system. This effect was preceded by an increase in mRNA encoding Pit‐1 but not Pit‐2. BMP‐2 also dose‐dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit‐1. Enhanced Pi transport and matrix mineralization induced by BMP‐2 were blunted by a specific inhibitor of the c‐Jun‐N‐terminal kinase (JNK) pathway. Conclusions: Results presented in this study indicate that, in addition to its well‐known effect on several markers of the differentiation of osteoblastic cells, BMP‐2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit‐1. In MC3T3‐E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP‐2.

Regulation of the Sodium-Phosphate Cotransporter Pit-1 and its Role in Vascular Calcification

“…In addition to cause the change of VSMC to the osteoblastic phenotype, BMP-2 also enhanced the calcification induced by high [Pi] EC ; the blockage of NaPi3 by PFA blunted the effect of BMP-2 in the mineralization [60], suggesting a critical role of Pit-1 in VSMC mineralization induced by BMP-2. Similar results have been obtained in osteoblastic cells, where the expression of Pit-2 was not affected by BMP-2 [61].…”

The emerging role of phosphate in vascular calcification

“…In addition, bone morphogenetic protein 2 (BMP-2), a potent osteogenic protein, has been shown to promote vascular calcification [34, 74, 75] and BMP-2 increased phosphate uptake in a time-and dose-dependent manner [76]. Interestingly, BMP-2 also promoted mineralization and upregulated Pit-1 expression in osteoblasts, and BMP2-enhanced mineralization in these cells was abrogated by Pit-1 siRNA [77]. Finally, transglutaminase (TG) appears to be an important regulator of endogenous expression, since Pit1 levels were observed in TG+/+ SMCs but absent in TG −/− SMCs in the presence or absence of elevated phosphate [78].…”

Sodium-Dependent Phosphate Cotransporters: Lessons from Gene Knockout and Mutation Studies

“…59,6062,63 PiT1 plays important roles in the regulation of Pi homeostasis in bone and cartilage in vitro, [64][65][66][67] and extracellular Pi, epinephrine, insulin-like growth factor 1, and bone morphogenic protein 2 are known to regulate PiT1 levels in osteoblast-like cells. 61,68 Recent reports have demonstrated that NaPi cotransport activity through PiT1 is associated with osteoblast differentiation. 5 Extracellular Pi affects the differentiation of chondrogenic cells induced by PiT1 functions.…”