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


BACKGROUND
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.


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.
Suzuki
Atsushi
A
Division of Endocrinology, Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Japan.
Ghayor
Chafik
C
Guicheux
Jérome
J
Magne
David
D
Quillard
Sophie
S
Kakita
Ayako
A
Ono
Yasunaga
Y
Miura
Yoshitaka
Y
Oiso
Yutaka
Y
Itoh
Mitsuyasu
M
Caverzasio
Joseph
J
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D002113:Calcification, Physiologic / Q000502:physiology*; D017931:DNA Primers; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D050817:Transcription Factor Pit-1 / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D050817:Transcription Factor Pit-1; D016212:Transforming Growth Factor beta