CELL DIFFERENTIATION AND p38MAPK CASCADE ARE INHIBITED IN HUMAN OSTEOBLASTS CULTURED IN A THREE-DIMENSIONAL CLINOSTAT

Abstract

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.

The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells

“…In this study, we further demonstrated that SMG could enhance the multipotential differentiation capacity of BMSCs which enriches the previous studies. We used a clinostat for simulating microgravity, which could generate a multidirectional G force that placed the cells in a constant direction changing the gravity environment (Yuge et al 2003). As a result, the cells had no time to respond to normal gravity.…”

Effects of compressive force on the differentiation of pluripotent mesenchymal cells

“…It is known that p38 MAPK plays an important role in the differentiation of various cells, such as the adipocytic differentiation of 3T3L1 cells (Engelman et al, 1998), myogenic differentiation of C2C12 cells (Cuenda and Cohen, 1999), and neuronal differentiation (Morooka and Nishida, 1998;Iwasaki et al, 1999). Recently, the role of p38 MAPK in osteoblast differentiation has been reported (Suzuki et al, 2002;Yuge et al, 2003). Moreover, p38 MAPK and the Smad pathway play crucial roles in Runx2 expression following induction by transforming growth factor-beta and BMP (Lee et al, 2002).…”

Effects of simulated weightlessness on cellular morphology and biological characteristics of cell lines SGC-7901 and HFE-145

“…However, many studies have examined other human or animal cells under these conditions. Cell morphology changes were observed under weightlessness or simulated weightlessness in human osteoblast cells (Yuge et al, 2003) and erythrocytes from humans and rabbits (Shen et al, 1997). However, no significant changes have been observed for monkey kidney cells (Sato et al, 2001).…”