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EFFECT OF VECTOR-AVERAGED GRAVITY ON SUB- CELLULAR LOCALIZATION OF MITOGEN-ACTIVATED PROTEIN KINASE IN MOUSE OSTEOBLAST-LIKE MC3T3-E1 CELLS. A. Sato¹, M. Fujita¹, M. Kanematsu¹, S. Kamigaichi², M. Takaoki², M. Narato³ , H. Kumagai³, and Y. Taniguchi⁴. ¹Space Utilization Research Programme, ²Space Utilization Research Center, NASDA, ³Advanced Engineering Services Co. Ltd., and ⁴Toray Research Center., Japan.

     We previously reported that microgravity inhibited EGF-induced c-fos gene expression but unaffected the phosphorylation of mitogen-activated protein kinase (MAPK) in MC3T3-E1 cells. To probe the mechanism, we investigated effects of vector-averaged gravity on redistribution of phosphorylated MAPK (phospho-MAPK) from the cytosol to nucleus in cells. The cells were cultured in α-MEM supplemented with 1% FCS and 10 mM HEPES for 24hr, then treated with 100 ng/ml EGF for 10 min under clinostat (rotation speed, 30 rpm) or stationary culture condition. Western blot analysis of MAPK and phospho-MAPK revealed that no significant difference in phosphorylation level of p44/42 MAPK of the cytosolic and nuclear fractions of the EGF-treated cells was observed between cells grown in simulated microgravity and those grown in stationary control. Immunofluoresence data indicated that p44/42 MAPK localized predominantly in the cytoplasm, particularly the perinuclear region in cells, and phospho-p44/42 MAPK detected in nucleus. Simulated microgravity slightly increased in the cytosolic and nuclear p44/42 MAPK and decreased in nuclear phospho-p44/42 MAPK. These results raise the possibility that gravity may affect translocation of cytosolic phospho-p44/42 MAPK into nucleus in MC3T3-E1 cells.