Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment.

Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment.

Genetic and epigenetic regulation of osteopontin by cyclic adenosine 3' 5'-monophosphate in osteoblasts.

Osteopontin (OPN) is not only a marker of osteoblasts but it is also related to cancer progression and inflammation. The expression of OPN increases in response to inflammatory cytokines, hormones, and mechanical stress. Among them, cyclic-AMP (cAMP) elevating agents stimulate OPN expression in the presence of 1, 25-OH vitamin D3 (VD3). We aimed to clarify the mechanism by which cAMP enhances OPN expression in osteoblastic cells. The OPN promoter (-2335 to +76, OPNp2335) exerted a cell type specific response to forskolin (FK) and VD3. Sequential deletion analysis of OPNp revealed that the OPNp (-833 to +76) contained essential responsive regions to respond to cAMP signaling. In particular, both Vitamin D response element (VDRE, -758 to -743) and osteoblast-specific cis- acting element 2 (OSE2, -695 to -690) were essential for cAMP-mediated OPNp activity. The expression of vitamin D receptor (VDR), but not runt-related transcription factor 2 (Runx2), a nuclear receptor for OSE2, was induced by the treatment of the cells with FK. Although, VD3-induced OPNp activity was slightly enhanced in VDR-overexpressing osteoblasts, it reached the same level as that of osteoblasts induced by both VD3 and FK in the presence of histone deacetylase (HDAC) inhibitor. Moreover, we identified histone acetylation on the OPN promoter region by FK treatment. These results strongly suggest that OPNp activity is controlled by the cAMP signaling via genetic and epigenetic regulations.

Low-intensity pulsed ultrasound reduces periodontal atrophy in occlusal hypofunctional teeth.

OBJECTIVE: To clarify whether low-intensity pulsed ultrasound (LIPUS) exposure has recovery effects on the hypofunctional periodontal ligament (PDL) and interradicular alveolar bone (IRAB). MATERIALS AND METHODS: Twelve-week-old male Sprague-Dawley rats were divided into three groups (n = 5 each): a normal occlusion (C) group, an occlusal hypofunction (H) group, and an occlusal hypofunction group subjected to LIPUS (HL) treatment. Hypofunctional occlusion of the maxillary first molar (M1) of the H and HL groups was induced by the bite-raising technique. Only the HL group was irradiated with LIPUS for 5 days. The IRAB and PDL of M1 were examined by microcomputed tomography (micro-CT) analysis. To quantify mRNA expression of cytokines involved in PDL proliferation and development, real-time reverse transcription quantitative PCR (qRT-PCR) was performed for twist family bHLH transcription factor 1 (Twist1), periostin, and connective tissue growth factor (CTGF) in the PDL samples. RESULTS: Micro-CT analysis showed that the PDL volume was decreased in the H group compared with that of the C and HL groups. Both bone volume per tissue volume (BV/TV) of IRAB was decreased in the H group compared with that in the C group. LIPUS exposure restored BV/TV in the IRAB of the HL group. qRT-PCR analysis showed that Twist1, periostin, and CTGF mRNA levels were decreased in the H group and increased in the HL group. CONCLUSION: LIPUS exposure reduced the atrophic changes of alveolar bone by inducing the upregulation of periostin and CTGF expression to promote PDL healing after induction of occlusal hypofunction.