Cell differentiation and p38(MAPK) cascade are inhibited in human osteoblasts cultured in a three-dimensional clinostat.

A three-dimensional (3D) clinostat is a device for multidirectional G force generation. By controlled rotation of two axes, a 3D clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10(-3) G over time. We cultured a human osteoblast cell line in a 3D clinostat and examined the growth properties and differentiation of the cells, including morphology, histological detection of calcification, and mitogen-activated protein kinase (MAPK) cascades. In a normal 1 G condition, alkaline phosphatase (AlPase) activity was detected on day 7 of culture, bone nodules were formed on day 12, and calcium deposits were seen on day 20. In the 3D clinostat, the cells looked larger and bulged. AlPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found in the 3D clinostat up to day 21. The expression levels of core-binding factor A1 (a transcription factor for bone formation) and osteocalcin (a bone matrix protein) increased in the control culture but decreased in culture in 3D clinostat. Phosphorylation of p38(MAPK) (p38) was repressed in culture in 3D clinostat, whereas total p38 as well as total and phosphorylated forms of extracellular signal-regulated kinases and stress-activated protein kinase/jun N-terminal kinase were not changed in the 3D clinostat. When a p38 inhibitor, SB 203580, was added to the culture medium in a normal 1 G environment, AlPase activity and formation of bone nodules and calcium deposits were strongly inhibited. On the other hand, they were inhibited only partially by a MAPK kinase inhibitor, U-0126. On the basis of these results, it is concluded that (1) osteoblast differentiation is inhibited in culture in a 3D clinostat and (2) this inhibition is mainly due to the suppression of p38 phosphorylation.

Crosstalk between glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle.

Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.

Physical stress by magnetic force accelerates differentiation of human osteoblasts.

We examined the effect of magnetic force on differentiation of cultured human osteoblasts. Magnetic microparticles (MPs) were introduced into the cytoplasm of a human osteoblast cell line and the cells were cultured in a magnetic field (MF) in group MP-MF. Three groups of controls were used: cells without MPs were cultured out of MF (group C), cells without MPs were cultured in MF (group MF), and cells with MPs were cultured out of MF (group MP). The cells in group MP-MF became larger and were elongated along the axis of the magnetic poles. Appearance of alkaline phosphatase (AlPase) activity, formation of bone nodules, and calcium deposition were accelerated depending on the intensity of the magnetic field. It takes longer culture in the other three groups to exhibit these changes. Core-binding factor A1 (Cbfa1: transcription factor for osteoblast differentiation) and osteocalcin (a bone-matrix protein involved in controlling osteogenesis) were expressed earlier or stronger in group MP-MF than the other groups. Then we compared phosphorylation of mitogen-activated protein kinase (MAPK) between group MP-MF and group C. Phosphorylation of p38(MAPK) (p38) was increased in group MP-MF, while total p38 as well as total and phosphorylated forms of MAPK/ERK 1/2 and SAPK/JNK were not changed between the two groups. When a p38 inhibitor, SB 203580, was added to the culture medium in group C, AlPase activity, formation of bone nodules, and calcium deposits were completely inhibited. On the other hand, they were inhibited only partially by a MAPK/ERK 1/2 inhibitor, U-0126. Based on these results, it is concluded that (1) osteoblast differentiation is accelerated by a magnetic force, (2) this acceleration is mainly attributed to the activation of p38 phosphorylation, and (3) the stimulus induced by a magnetic field offers a new approach to osteoblast differentiation.