Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (mCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-b activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.
These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.
Factors affecting the cross-linking of milk proteins by transglutaminase (TGase) were studied. Crosslinking of caseins in bovine skim milk was optimal over a very wide pH range. The role of micellar calcium phosphate (MCP) in maintaining the integrity of TGase-treated casein micelles was studied by incubating skim milk with 0.01% (w/v) TGase at 30°C for 1-24 h, followed by removal of MCP from untreated or TGase-treated milk by acidification and dialysis. The protein content and profile of the samples were determined by Kjeldahl and SDS-PAGE, respectively. Whey proteins in unheated milk were not susceptible to TGase-induced cross-linking. The higher level of sedimentable protein in MCP-free TGase-treated milk than in MCP-free control milk indicated that TGase treatment partially prevented disintegration of the micelle on removal of MCP, probably due to extensive intramicellar TGase-induced cross-linking of casein molecules which led to the formation of sedimentable covalently bonded casein aggregates.
Background The association between diabetes mellitus (DM) and bone diseases is acknowledged. However, the mechanistic pathways leading to the alveolar bone (AB) destruction remain unclear. This study aims to elucidate the mechanical forces (MF)‐induced AB destruction in DM and its underlying mechanism. Methods In vivo periodontal tissue responses to MF were evaluated in rats with diabetes. In vitro human periodontal ligament (PDL) cells were either treated with advanced glycation end products (AGEs) alone or with AGEs and MF. Results In vivo, the transcription of VEGF‐A, colony stimulating factor‐1 (CSF‐1), and Ager was upregulated in diabetes, whereas changes in DDOST and Glo1 mRNAs were negligible. DM induced VEGF‐A protein in the vascular cells of the PDL and subsequent angiogenesis, but DM itself did not induce osteoclastogenesis. MF‐induced AB resorption was augmented in DM, and such augmentation was morphologically substantiated by the occasional undermining resorption as well as the frontal resorption of the AB by osteoclasts. The mRNA levels of CSF‐1 and vascular endothelial growth factor (VEGF) during MF application were highly elevated in diabetes, compared with those of the normal counterparts. In vitro, AGEs treatment elevated Glut‐1 and CSF‐1 mRNA levels via the p38 and JNK pathways, whereas OGT and VEGF levels remained unchanged. Compressive MF especially caused upregulation of VEGF, CSF‐1, and Glut‐1 levels, and such upregulation was further enhanced by AGEs treatment. Conclusions Overloaded MF and AGEs metabolites may synergistically aggravate AB destruction by upregulating CSF‐1 and VEGF. Therefore, regulating the compressive overloading of teeth, as well as the levels of diabetic AGEs, may prove to be an effective therapeutic modality for managing DM‐induced AB destruction.
Fusion and apoptosis share a breakdown of the membrane phospholipids asymmetry, modes of which are largely unknown in osteoclastogenesis. Here, we investigated the externalization of phosphatidylserine (PS) and its receptors, and their biological functions in osteoclastogenesis. Strong immunoreactivities in vivo for the PS receptors TIM4, BAI1, and STAB2 were observed in the TRAP-positive multinucleated cells in the alveolar bone that was being remodeled around the developing dental follicles in rats. These receptors were significantly upregulated during M-CSF/RANKLinduced in vitro osteoclastogenesis using mouse bone marrow-derived cells. PS externalization in preosteoclasts was increased by the M-CSF/RANKL treatment. Multinucleation of preosteoclasts was markedly inhibited by antibodies against PS and its receptors. Among the investigated lipid transporter proteins, floppases (Abcb4, Abcc5, and Abcg1) were upregulated, whereas flippases (Atp11c and Atp8a1) downregulated during osteoclastogenesis. Preosteoclast fusion was markedly blocked by the ATPase inhibitor Na 3 VO 4 and siRNAs against Abcc5 and Abcg1, revealing the importance of these lipid transporters in PS externalization. Further, the levels of Cd47 and Cd31, don't-eat-me signal inducers, were increased or sustained in the early phase of osteoclastogenesis, whereas those of AnnexinI and Mfg-e8, eat-me signals inducers, were increased in the late apoptotic phase. In addition, Z-VAD-FMK, a pan caspase inhibitor, had no effect on preosteoclast fusion in the early phase of osteoclastogenesis, whereas Abs against PS, TIM4, and BAI1 decreased osteoclast apoptosis during the late phase. These results suggest that PS externalization is essential for the whole process of osteoclastogenesis and share PS receptors and transporters in the early stage fusion and late stage apoptosis. Therefore, modulation of PS and its receptors could be a useful strategy to develop anti-bone resorptive agents.
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