Regulation of Cementoblast Gene Expression by Inorganic Phosphate In Vitro

Abstract

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (P(i)). Immortalized murine cementoblasts were treated with P(i) in vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1-10 mM) and time-course (1-48 hours) assays were performed, as well as studies including the Na-P(i) uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM P(i) upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6-24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-P(i) cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

“…Studies have shown that coculture of endothelial cells with chondrocytes induces both collagen type X and ALP expression, 75 as was also shown in the collagen type X staining presented in our previous study. 49 Finally, b-glycerophosphate is the source of phosphate needed to produce hydroxyapatite, [26][27][28] but we propose that in this coculture model, these phosphates are naturally provided by cartilage cells undergoing hypertrophy within the cartilage template, 76 or from the serum contained in the expansion medium. The results suggest that a direct coculture technique can obviate the need for osteogenic supplements to induce osteogenesis in vitro.…”

“…12,[23][24][25] It also regulates expression of genes including osteopontin and BMP-2. [26][27][28] Exposure of rat MSCs, 12,[14][15][16][17] human MSCs (hMSCs), 9,11,13 or murine osteoblasts 22,29 to dexamethasone, ascorbic acid, and b-glycerophosphate can significantly increase alkaline phosphatase (ALP) activity in vitro.…”

Non-destructive characterisation of mesenchymal stem cell differentiation using LC-MS-based metabolite footprinting

“…6,7 βGP provides inorganic phosphate (Pi) that facilitates intracellular signalling molecules. [8][9][10] Since metabolites provide the phenotypic outcome of gene expression or metabolic activities of a cell, global metabolite profiling can give a rapid snapshot of the cell physiology by monitoring concentration changes of a broad range of metabolites and provide insight into relationships between genotype and phenotype. 11,12 Therefore, metabolomics has been used widely for understanding underlying molecular processes in applications for human health and disease including biomarker and drug discovery.…”

Surface properties and early murine pre-osteoblastic cell responses of phosphoric acid modified titanium surface

“…It has previously been shown that phosphate ions promoted osteogenic differentiation of various cell types, for example osteoblasts, cementoblasts, and periodontal ligament cells. [18][19][20][21] In the present study, titanium surfaces were modified with different H 3 PO 4 concentration using low temperature treatment. No significant difference of surface roughness was noted.…”