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All-trans retinoic acid and bone morphogenetic protein 2 (BMP2) synergistically induced an alkaline phosphatase (ALP) activity, one of the osteoblastic differentiation markers, and promoted the extracellular matrix calcification in a myoblastic C2C12 cell culture system. The induced ALP mRNA was not suppressed in the presence of a protein synthesis inhibitor, suggesting that the de novo protein synthesis does not influence this induction. There are three isotypes for the retinoic acid receptor (RARα, RARβ, RARγ). Both the ALP activity and the extracellular matrix calcification were inhibited by the addition of the specific siRNA for RARγ, but not by that for RARα or RARβ. When the effects of the RAR subtype-specific agonists on the ALP activity in the presence of BMP2 were examined, the RARγ-specific agonist was the most effective. The ALP activity induced by any RAR subtype-specific agonist was inhibited by the addition of the specific siRNA for RARγ, but not by that for RARα or RARβ. These results suggest that a RARγ-dependent functional crosstalk is present between the retinoic acid and BMP2 signaling to induce osteogenic transdifferentiation in myoblastic C2C12 cells. [ABSTRACT FROM AUTHOR]

Background: Enamel extracts are biologically active and capable of inducing osteogenesis and cementogenesis, but the specific molecules carrying these activities have not been ascertained. The purpose of this study was to identify osteogenic factors in porcine enamel extracts. Methods: Enamel proteins were separated by size-exclusion chromatography into four fractions, which were tested for their osteogenic activity on osteoblast-like cells (ST2) and human periodontal ligament (HPDL) cells. Results: Fraction 3 (Fr.3) and a transforming growth factor-beta 1 (TGF-β1) control reduced alkaline phosphatase (ALP) activity in ST2 but enhanced ALP activity in HPDL cells. The enhanced ALP activity was blocked by anti-TGF-β antibodies. Furthermore, using a dual-luciferase reporter assay, we demonstrated that Fr.3 can induce the promoter activity of the plasminogen activator inhibitor type 1 (PAI-1) gene. Conclusion: These results show that the osteoinductive activity of enamel extracts on HPDL cells is mediated by TGF-β1. [ABSTRACT FROM AUTHOR]

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation. [ABSTRACT FROM AUTHOR]

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins. [ABSTRACT FROM AUTHOR]