PTH, via the PTH/PTH-related protein receptor type 1 that couples to both protein kinase A (PKA) and protein kinase C (PKC) pathways, and the canonical Wnt-beta-catenin signaling pathway play important roles in bone formation. In the present study we have examined the interaction between the PTH and Wnt signaling pathways in mouse osteoblastic MC3T3-E1 cells. PTH dose- and time-dependently increased the concentrations of beta-catenin. The PKA activator, forskolin, and the PKC activator, phorbol 12-myristate-13-acetate, as well as the PTH analog, [Nle(8,18),Tyr(34)]human PTH-(3-34)amide, all increased beta-catenin levels. Both H-89, a specific PKA inhibitor, and PKC inhibitors, staurosporine and calphostin C, antagonized PTH stimulation of beta-catenin levels. TGF-beta as well as transfection of the TGF-beta-signaling molecule, Smad3, enhanced beta-catenin levels, and this was antagonized by transfection of a dominant-negative Smad3. The transcriptional activity of transfected dominant-active beta-catenin was enhanced by PTH, an effect that was antagonized by cotransfection of a dominant-negative Smad3. PTH as well as LiCl(2), which mimics the effects of the Wnt-beta-catenin pathway, rescued the dexamethasone- and etoposide-induced apoptosis of osteoblastic cells. In conclusion, the data demonstrate that PTH stimulates osteoblast beta-catenin levels via Smad3, and that both PKA and PKC pathways are involved. The canonical Wnt-beta-catenin pathway is likely to be involved in the antiapoptotic actions of PTH by acting through Smad3 in osteoblasts.

Deuterium solid-state NMR was used to study the dynamics and molecular structure of the serine (Ser) side chains in silk fibroin from Bombyx mori and from Samia cynthia ricini. Samples were selectively labeled with [3,3-2H2]Ser, and the 2H NMR powder spectra were analyzed by line shape simulation. Two types of motion could be characterized quantitatively:  one component undergoing a rapid three-site jump (25%) and a second component representing a slow exchange between sites with unequal occupancies and with a small amplitude of libration (75%). From these results, it is concluded that approximately 75% of the Ser residues contribute to the formation of hydrogen bonds in both kinds of silk fibroin from B. mori and S. cynthia ricini. Furthermore, uniaxially oriented silk fibers were used to determine the side-chain conformation and the orientational distribution of the Ser residues in the slow motional component of B. mori silk fibroin. The gauche+ conformer around N−Cα−Cβ−O was found to be dominant, suggesting that the hydroxyl groups of Ser interact with carbonyl groups on adjacent chains and thereby contribute to the intermolecular hydrogen-bonding network of the fiber.

No abstract

Mice null for menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, exhibit cranial and facial hypoplasia suggesting a role for menin in bone formation. We have shown previously that menin is required for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage in part by interacting with the bone morphogenetic protein (BMP)-2 signaling molecules Smad1/5, and the key osteoblast transcriptional regulator, Runx2 (Sowa H., Kaji, H., Hendy, G. N., Canaff, L., Komori, T., Sugimoto, T., and Chihara, K. (2004)

No abstract

Smad3, a critical component of the TGF-beta signaling pathways, plays an important role in the regulation of bone formation. However, how Smad3 affects osteoblast at the different differentiation stage remains still unknown. In the present study, we examined the effects of Smad3 on osteoblast phenotype by employing mouse bone marrow ST-2 cells and mouse osteoblastic MC3T3-E1 cells at the different differentiation stage. Smad3 overexpression significantly inhibited bone morphogenetic protein-2 (BMP-2)-induced ALP activity in ST-2 cells, indicating that Smad3 suppresses the commitment of pluripotent mesenchymal cells into osteoblastic cells. Smad3 increased the levels of COLI and ALP mRNA at 7 day cultures in MC3T3-E1 cells, and its effects on COL1 were decreased as the culture periods progress, although its effects on ALP were sustained during 21 day cultures. Smad3 overexpression enhanced the level of Runx2 and OCN mRNA at 14 day and 21 day cultures. Smad3 increased the levels of MGP and NPP-1 mRNA, although the extent of increase in MGP and NPP-1 was reduced and enhanced during the progression of culture period, respectively. Smad3 did not affect the level of ANK mRNA. On the other hand, Smad3 enhanced the level of MEPE mRNA at 14 and 21 day cultures, although Smad3 decreased it at 7 day cultures. In conclusion, Smad3 inhibits the osteoblastic commitment of ST-2 cells, while promotes the early stage of differentiation and maturation of osteoblastic committed MC3T3-E1 cells. Also, Smad3 enhanced the expression of mineralization-related genes at the maturation phase of MC3T3-E1 cells.

Statins possess pleiotropic effects in several tissues. Among them, their bone anabolic actions have been recently noted. We have proposed that Smad3, a TGF-beta-signaling molecule, is a promoter of bone formation. However, whether statins would affect TGF-beta-Smad3 pathway in osteoblasts is still unknown. The present study was performed to examine the effects of statin on Smad3 expression and cell apoptosis by employing mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Statins (pitavastatin, mevastatin, and simvastatin) as well as alendronate increased the levels of Smad3 in MC3T3-E1 cells. The effects of pitavastatin on Smad3 levels were observed from 3 hours and later. Pitavastatin induced the expression of TGF-beta, and cycloheximide, a protein synthesis inhibitor, antagonized the increased levels of pitavastatin on Smad3. On the other hand, pitavastatin antagonized dexamethasone- or etoposide-induced apoptosis in a dose-dependent manner, and Smad3 inactivation by dominant negative Smad3 or an inhibition of endogenous TGF-beta action by SB431542 antagonized anti-apoptotic effects of pitavastatin, indicating that pitavastatin suppressed osteoblast apoptosis partly through TGF-beta-Smad3 pathway. In conclusion, the present study has demonstrated for the first time that statin suppressed cell apoptosis partly through TGF-beta-Smad3 pathway in osteoblastic cells.

Hypertrophic cardiomyopathy and ventricular hypertrophy due to hypertension can be differentiated on the basis of quantitative analysis of the transmural gradient in integrated backscatter.