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 V max 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.

BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells

“…Many osteotropic factors regulate phosphate uptake are associated with osteogenic differentiation and mineralization in osteoblasts. Upregulation of Pit-1 may be a common mechanism by which osteotropic factors, such as BMP-2, TGF-β and PDGF, promote biomineralization [20,21,32]. Consistent with these studies, we previously reported that phosphate uptake via Pit-1 was also required for SMC calcification, suggesting a crucial role of Pit-1 in osteogenic differentiation and mineralization of SMC [8].…”

“…Csiszar et al showed that ERK and PKC play important roles in BMP-2-induced proinflammatory phenotype in endothelial cells [19]. In addition, JNK and PI3-kinase are also involved in BMP-2-upregulated Pit-1 expression [20,21].…”

Low concentration of 3‐carene stimulates the differentiation of mouse osteoblastic MC3T3‐E1 subclone 4 cells

“…Subsequently, the activation of these pathways can regulate the induction of genes such as osteopontin and type I collagen, of which products play an important role in the process of osteoblastic mineralization (Hipskind and Bilbe, 1998). Recently, the involvement of ERK and JNK activation in the induction of ALP activity and the matrix mineralization have been reported in MC3T3-E1 cells (Hanai et al, 2006;Suzuki et al, 2006). However, considering that the ERK and JNK cascades target an overlapping but not identical set of genes (Hipskind and Bilbe, 1998), Figure 4.…”

Pulsed electromagnetic fields enhance BMP‐2 dependent osteoblastic differentiation of human mesenchymal stem cells

“…[1][2][3][4] Bone morphogenetic protein 2 (BMP-2) induces osteoblastic differentiation by acting directly on mesenchymal stem cells (MSCs), increasing expression of the osteogenic transcription factors osterix and RUNX2. 5,6 In addition, BMP-2 stimulates osteoblastlike cells to produce alkaline phosphatase (ALP) and to calcify their extracellular matrix, 7,8 and longterm exposure to BMP-2 induces apoptosis. 9 This suggests that cells must be competent to respond to differentiation signals and that the downstream effects of the signal depend on the differentiated status of the responding cell population.…”