ABSTRACT
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 (µCT). 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-ß 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.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cell Differentiation/drug effects , Osteoblasts/cytology , Osteogenesis/drug effects , Relaxin/pharmacology , Animals , Calcification, Physiologic/drug effects , Cell Line , Core Binding Factor Alpha 1 Subunit/metabolism , Humans , MAP Kinase Kinase Kinases/metabolism , Mice, Inbred C57BL , Osteoblasts/drug effects , Osteoblasts/enzymology , Phosphorylation/drug effects , Protein Binding/drug effects , Receptors, G-Protein-Coupled/metabolism , Smad Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
A complex and intricate cascade of gene expression is essential for late stage tooth development. This study was performed to detect molecules involved in dental hard tissue formation and tooth eruption by comparing gene expression in cap stage molar germs (before eruptive movement and dental hard tissue formation) with that in root formation stage molar germs (after eruptive movement and dental hard tissue formation). DD-PCR revealed that cyclophilin A (Cyp-A), a potent chemoattractant for monocytes as well as a ligand for extracellular matrix metalloproteinase inducer (EMMPRIN) was expressed differentially in the two stages molar germs. The levels of Cyp-A and EMMPRIN mRNA were significantly higher at the root formation stage than at the cap and crown stages of the molar germs. Immunofluorescence showed that Cyp-A and EMMPRIN were expressed strongly in the follicular cells overlaying the occlusal region of the molar germs at the root formation stage. In contrast, their immunoreactivity was weak in the follicular tissues and was not region-specific in molar germs at the cap stage. In addition, the MCP-1 and CSF-1 mRNA levels increased in parallel to that of Cyp-A mRNA and the increased number of osteoclasts at the occlusal region. Immunoreactivity against Cyp-A and EMMPRIN was also observed in the fully differentiated ameloblasts and odontoblasts. This study suggests that Cyp-A and EMMPRIN play roles in the maturation of dental hard tissue and the formation of an eruption pathway.
Subject(s)
Basigin/genetics , Cyclophilin A/genetics , Gene Expression Regulation, Developmental/genetics , Molar/growth & development , Molar/metabolism , Tooth Eruption/physiology , Ameloblasts/metabolism , Animals , Animals, Newborn , Basigin/metabolism , Cyclophilin A/metabolism , Odontoblasts/metabolism , Osteoclasts/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Slow diffusion of Cu(ClO4)2 with p-C6H4(SiMe2(4-Py))2 yields a supramolecular framework consisting of two kinds of channels via self-assembly of a 1D double-stranded chain in a prismatic fashion. The channel has a 11 x 11 A2 cross section (Cu...C = 11.13 A) with a 6 x 6 A2 square pore. The channels are infinitely arranged, resulting in another kind of channel with similar pores. Thus, two different kinds of channels with different solvate molecules coexist in a 1:2 ratio. Weak C-H...pi interaction may be one of the driving forces in the assembly of the prismatic channel structure.
