Microarray Profile of Gene Expression During Osteoclast Differentiation in Modelled Microgravity

Abstract

Microgravity (µXg) leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast (OCL) is the multinucleated bone-resorbing cell. In this study, we used the NASA developed ground-based rotating wall vessel bioreactor (RWV), rotary cell culture system (RCCS) to simulate µXg conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to normal gravity control (Xg). Gene expression profiling of RAW 264.7 OCL progenitor cells in modelled µXg by Agilent microarray analysis revealed significantly increased expression of critical molecules such as cytokines/growth factors, proteases and signalling proteins, which play an important role in enhanced OCL differentiation/function. Transcription factors such as c-Jun, MITF and CREB implicated in OCL differentiation are upregulated; however no significant change in the levels of NFATc1 expression in preosteoclast cells subjected to modelled µXg. We also identified high-level expression of calcium-binding protein, S100A8 (calcium-binding protein molecule A8/calgranulin A) in preosteoclast cells under µXg. Furthermore, modelled µXg stimulated RAW 264.7 cells showed elevated cytosolic calcium (Ca(2+)) levels/oscillations compared to Xg cells. siRNA knock-down of S100A8 expression in RAW 264.7 cells resulted in a significant decrease in modelled µXg stimulated OCL differentiation. We also identified elevated levels of phospho-CREB in preosteoclast cells subjected to modelled µXg compared to Xg. Thus, modelled µXg regulated gene expression profiling in preosteoclast cells provide new insights into molecular mechanisms and therapeutic targets of enhanced OCL differentiation/activation to prevent bone loss and fracture risk in astronauts during space flight missions.

Mechanotransduction as an Adaptation to Gravity

“…Osteoclast differentiation is enhanced in microgravity (116). Microarray analysis of osteoclasts in the modified rotating wall vessel RCCS showed an upregulation of genes involved in osteoclast differentiation (117). Several studies indicate that bone loss under microgravity is caused by a decrease in osteoblast function and an increase in osteoclastic activity (118, 119).…”

RhoA GTPase interacts with beta-catenin signaling in clinorotated osteoblasts

“…1A). The unloading condition (0.005 g) was simulated by rotating the cell culture chamber (Ibidi, Verona, WI, USA) longitudinally at 15 rpm [33]. Two ports of the cell culture chamber were connected with syringe filters (Whatman, Piscataway, NJ, USA) to allow gas exchange.…”

“…Clinorotation has been widely used as a simulated model of unloading or microgravity for in vitro studies [22–26]. The method does not generate any weightlessness condition but it moves a fixed direction of gravitational forces by rotating a culture chamber, which results in a vector-averaged reduction in the apparent gravity on the cell culture.…”

Physiological Effects of Microgravity on Bone Cells

“…Irradiation results in an early increase in osteoclast number and activity, which may most likely contribute to radiationinduced osteoporosis [90]. Microarray analysis of osteoclasts in the modified rotating wall vessel RCCS shows a significant upregulation in genes involved in osteoclast differentiation [91].…”

“…Several elevated growth factors in preosteoclast cells under microgravity may alter RANKL and OPG levels in bone environments and facilitate enhanced osteoclast activity [91]. The inhibitory effect of OPG on osteoclast differentiation comes from the fact that it prevents the binding of RANKL to its receptor RANK [97].…”