Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation.

Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.     

Mechanical stretch inhibits myoblast-to-adipocyte differentiation through Wnt signaling

Myoblasts are able to differentiate into other mesenchymal lineages including adipocytes and osteoblasts. However, it is not known how this differentiation is normally regulated in intact animals and humans. Here, we subjected cultured C2C12 myoblasts to cyclic mechanical stretch (20% elongation) during differentiation into adipocytes. Mechanical stretch inhibited the myoblast-to-adipocyte differentiation significantly, concurrent with an enhanced expression of Wnt10b mRNA. Inhibition of the Wnt signaling by incubation of the myoblasts with a soluble Wnt ligand, sFRP-2, abolished the inhibitory function of mechanical stretch on adipogenesis. These findings provide evidence that mechanical stretch inhibits myoblast-to-adipocyte differentiation possibly through Wnt signaling.     

Nobiletin promotes osteogenic differentiation of human osteoblastic cell line (MG-63) through activating the BMP-2/RUNX-2 signaling pathway

Nobiletin (NOB) is polymethoxy flavonoids, which plentifully there in Citrus depressa and they demonstrate numerous pharmacological effects. NOB has an anti-proliferative effect, attenuates ovalbumin-treated eosinophilic airway inflammation and Type II collagen treated arthritis. NOB noticeably inhibits bone resorption and renovates bone loss in mice model, but role of NOB in bone metabolism is unclear. Human bone is a important organ that sustains its homeostasis among bone resorpting osteoclasts and bone developing osteoblasts. The balances of among these two kind of cell outcomes are implicated in bone remodeling. The current study designed to explore possessions of NOB on differentiation and proliferation of MG-63 cells and contribution of morphogenetic protein signaling. Cell proliferation was analyzed by MTT, mineralization analysis by alizarin red staining and morphogenetic signaling protein by RT-PCR. No stimulus outcome of NOB on cell proliferation was found at days of 1, 3 and 7. Accumulation of calcium was augmented after that treatment of NOB. The mRNA expression of BMP-2, COL-I, ALP, OCN, RUNX2 and COL1A1 augmented markedly with NOB supplement. Hence, NOB can stimulate osteogenic differentiation of MG-63, almost certainly by promoting RUNX2 and BMP-2 signaling and this result might provide to its action on stimulation of osteoblast development, differentiation and augments of bone mass.     

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.     

Tumorhead, a Xenopus gene product that inhibits neural differentiation through regulation of proliferation

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.     

Collagen-derived dipeptide prolyl-hydroxyproline promotes osteogenic differentiation through Foxg1

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.