Phosphate regulates expression of SIBLINGs and MMPs in cementoblasts

Introduction: Cementoblasts, the cells responsible for tooth root cementum formation, are especially sensitive to local phosphate and pyrophosphate during development, as evidenced by cementum phenotypes resulting from altered phosphate/pyrophosphate distribution. SIBLING family members BSP, OPN, and DMP-1 are regulated by phosphate in cementoblasts and have been shown to activate three specific matrix metalloproteinase (MMP) partners: MMP2, MMP3, and MMP9, respectively, in vitro. The aim of this study was to examine regulatory effects of phosphate on SIBLING and MMP expression in cementoblasts, in vitro. Materials & Methods: Immortalized murine cementoblasts were treated with inorganic phosphate, in vitro, and effects on gene expression (by real time RT-PCR and mouse total genome microarray) were observed. Dose-response 80.1-10 mM phosphate) and time-course (1-48hr) assays were performed. A sodium-phosphate uptake inhibitor, foscarnet, was used to better define phosphate-mediated effects on cells. Results: Three SIBLING family members were regulated by phosphate: OPN (increased over 3000f control), DMP-1 (increased over 3,00027777701060f control), and BSP (decreased). MMP3 was dramatically increased (4,00026651125000f control), paralleling regulation of its partner OPN. Both MMP2 and MMP9 were slightly down-regulated. Time-course experiments indicated a response for SIBLING and MMP genes within 24 hr. Use of foscarnet demonstrated that phosphate uptake was required for observed changes in gene expression. Discussion: These results indicate an effect of phosphate on cementoblast SIBLING and MMP expression in vitro. During cementum formation, phosphate may be an important regulator of cementoblast activity, including modulation of biomineralization, attachment, and matrix modification

Role of PHOSPHO1 in periodontal development and function

The tooth root and periodontal apparatus, including the acellular and cellular cementum, periodontal ligament (PDL), and alveolar bone, are critical for tooth function. Cementum and bone mineralization is regulated by factors including enzymes and extracellular matrix proteins that promote or inhibit hydroxyapatite crystal growth. Orphan Phosphatase 1 (Phospho1, PHOSPHO1) is a phosphatase expressed by chondrocytes, osteoblasts, and odontoblasts that functions in skeletal and dentin mineralization by initiating deposition of hydroxyapatite inside membrane-limited matrix vesicles. The role of PHOSPHO1 in periodontal formation remains unknown and we aimed to determine its functional importance in these tissues. We hypothesized that the enzyme would regulate proper mineralization of the periodontal apparatus. Spatiotemporal expression of PHOSPHO1 was mapped during periodontal development, and Phospho1(-/-) mice were analyzed using histology, immunohistochemistry, in situ hybridization, radiography, and micro–computed tomography. The Phospho1 gene and PHOSPHO1 protein were expressed by active alveolar bone osteoblasts and cementoblasts during cellular cementum formation. In Phospho1(-/-) mice, acellular cementum formation and mineralization were unaffected, whereas cellular cementum deposition increased although it displayed delayed mineralization and cementoid. Phospho1(-/-) mice featured disturbances in alveolar bone mineralization, shown by accumulation of unmineralized osteoid matrix and interglobular patterns of protein deposition. Parallel to other skeletal sites, deposition of mineral-regulating protein osteopontin (OPN) was increased in alveolar bone in Phospho1(-/-) mice. In contrast to the skeleton, genetic ablation of Spp1, the gene encoding OPN, did not ameliorate dentoalveolar defects in Phospho1(-/-) mice. Despite alveolar bone mineralization defects, periodontal attachment and function appeared undisturbed in Phospho1(-/-) mice, with normal PDL architecture and no evidence of bone loss over time. This study highlights the role of PHOSPHO1 in mineralization of alveolar bone and cellular cementum, further revealing that acellular cementum formation is not substantially regulated by PHOSPHO1 and likely does not rely on matrix vesicle–mediated initiation of mineralization

Current concepts in periodontal bioengineering

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73112/1/j.1601-6343.2005.00352.x.pd

Characterization of the Autocrine/Paracrine Function of Vitamin D in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background: We previously demonstrated that 25-hydroxyvitamin D-3, the precursor of 1 alpha,25-dihydroxyvitamin D-3, is abundant around periodontal soft tissues. Here we investigate whether 25-hydroxyvitamin D-3 is converted to 1 alpha,25-dihydroxyvitamin D-3 in periodontal soft tissue cells and explore the possibility of an autocrine/paracrine function of 1 alpha,25-dihydroxyvitamin D-3 in periodontal soft tissue cells. Methodology/Principal Findings: We established primary cultures of human gingival fibroblasts and human periodontal ligament cells from 5 individual donors. We demonstrated that 1 alpha-hydroxylase was expressed in human gingival fibroblasts and periodontal ligament cells, as was cubilin. After incubation with the 1 alpha-hydroxylase substrate 25-hydroxyvitamin D-3, human gingival fibroblasts and periodontal ligament cells generated detectable 1 alpha,25-dihydroxyvitamin D-3 that resulted in an up-regulation of CYP24A1 and RANKL mRNA. A specific knockdown of 1 alpha-hydroxylase in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 1 alpha, 25-dihydroxyvitamin D-3 production and mRNA expression of CYP24A1 and RANKL. The classical renal regulators of 1 alpha-hydroxylase (parathyroid hormone, calcium and 1 alpha,25-dihydroxyvitamin D-3) and Porphyromonas gingivalis lipopolysaccharide did not influence 1 alpha-hydroxylase expression significantly, however, interleukin-1 beta and sodium butyrate strongly induced 1 alpha-hydroxylase expression in human gingival fibroblasts and periodontal ligament cells. Conclusions/Significance: In this study, the expression, activity and functionality of 1 alpha-hydroxylase were detected in human gingival fibroblasts and periodontal ligament cells, raising the possibility that vitamin D acts in an autocrine/paracrine manner in these cells.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000305781700070&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Multidisciplinary SciencesSCI(E)PubMed13ARTICLE6e39878

Parathyroid hormone protects against periodontitis-associated bone loss

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In addition to the well-established catabolic effects (activation of bone resorption) of PTH, it is now recognized that intermittent PTH administration has anabolic effects (promotion of bone formation). The aim of this study was to investigate whether intermittent administration of PTH in rodents would block the alveolar bone loss observed in rats when a ligature model of periodontitis is used. Morphometric analysis showed that intermittent PTH administration (40 mug/kg) was able to protect the tooth site from periodontitis-induced bone resorption. In addition, there was a significant reduction in the number of inflammatory cells at the marginal gingival area in sections obtained from animals receiving PTH compared with control animals. These findings demonstrated that intermittent PTH administration was able to protect against periodontitis-associated bone loss in a rodent model.821079179

Modulation of Phosphate/Pyrophosphate Metabolism to Regenerate the Periodontium: A Novel In Vivo Approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Background: The developing periodontium is sensitive to local levels of inorganic phosphate (P(i)) and inorganic pyrophosphate (PP(i)) as demonstrated by cementum phenotypes resulting from the loss of function of protein regulators of P(i)/PP(i) homeostasis. The progressive ankylosis protein (ANK) regulates the transport of PP(i), and progressive ankylosis gene (Ank) and knock-out (KO) mice feature a rapidly forming and thick cementum. We hypothesized that, besides affecting cementum formation, decreased extracellular PP(i) levels in Ank KO mice would also impact cementum regeneration. Methods: Periodontal fenestration defects (approximate to 2 mm in length, 1 mm in width, and 0.5 mm in depth) were created on buccal aspects of mandibular molars in Ank KO and wild-type (WT) mice. Mandibles were harvested at 15 and 30 days post-surgery for histology, histomorphometry, evaluation of in vivo fluorochrome labeling, and immunohistochemistry (IHC) for proteins including bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). Results: A greater amount of new cementum was observed in Ank KO mice at 15 and 30 days post-surgery (P < 0.05), which was confirmed by fluorochrome labeling showing a higher new cementum appositional activity in defect areas in Ank KO mice versus controls. At days 15 and 30 during healing, regenerating cementum and associated cells in Ank KO samples recapitulated expression patterns mapped during development, including limited BSP and positive OPN and DMP1 in the cementum matrix as well as elevated NPP1 in cemento-blasts. Conclusions: Within the limits of the study, these findings suggest that reduced local levels of PP, could promote increased cementum regeneration. Therefore, the local modulation of P(i)/PP(i) may be a potential therapeutic approach for achieving improved cementum regeneration. J Periodontol 2011;82: 1757-1766.821217571766Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)National Institutes of Health, Bethesda, Maryland [5R03TW007590-03]National Institute of Dental and Craniofacial Research (NIDCR) [DE15109]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [08/00534-7]National Institutes of Health, Bethesda, Maryland [5R03TW007590-03]National Institute of Dental and Craniofacial Research (NIDCR) [DE15109

Cementoblast gene expression is regulated by Porphyromonas gingivalis lipopolysaccharide partially via Toll-like receptor-4/MD-2

Lipopolysaccharides are potent inflammatory mediators considered to contribute to destruction of periodontal tissues. Here, we hypothesized that Porphyromonas gingivalis lipopolysaccharide (P-LPS) treatment would regulate gene expression in murine cementoblasts through Toll-like receptor 4. Real-time (RT)-PCR and Northern blot analysis indicated that P-LPS decreased expression of transcripts for osteocalcin (OCN) and receptor activator of nuclear factor kappaB ligand (RANKL). In contrast, a dose-dependent up-regulation in mRNA levels for osteopontin (OPN) and osteoprotegerin (OPG) was observed. Similarly, ELISA demonstrated decreased RANKL and increased OPG levels. A monoclonal antibody specific for mouse TLR-4/MD-2 partially neutralized the P-LPS effect on cementoblasts. These results indicate that exposure of cementoblasts to P-LPS can alter cell function by regulating markers of osteoclastic activity (e.g., RANKL/OPG), thereby potentially affecting the inflammation-associated resorption of mineralized tissues.83860260