Microgravity Science and Technology

, Volume 23, Issue 1, pp 19–27

Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

  • Zhongquan Dai
  • Yingjun Tan
  • Fen Yang
  • Lina Qu
  • Hongyu Zhang
  • Yumin Wan
  • Yinghui Li
Original Article

Abstract

Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure alteration inhibits the function performing.

Keywords

Parabolic flight Microgravity Osteoblast Microfilament ERK1/2 

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References

  1. Bucaro, M.A., Fertala, J., Adams, C.S., Steinbeck, M., Ayyaswamy, P., Mukundakrishnan, K., Shapiro, I.M., Risbud, M.V.: Bone cell survival in microgravity: evidence that modeled microgravity increases osteoblast sensitivity to apoptogens. Ann. N. Y. Acad. Sci. 1027, 64–73 (2004)CrossRefGoogle Scholar
  2. Carmeliet, G., Bouillon, R.: The effect of microgravity on morphology and gene expression of osteoblasts in vitro. FASEB J. 13, S129–S134 (1999)Google Scholar
  3. Fitzgerald, J., Hughes-Fulford, M.: Gravitational loading of a simulated launch alters mRNA expression in osteoblasts. Exp. Cell Res. 228(1), 168–171 (1996)CrossRefGoogle Scholar
  4. Fitzgerald, J., Hughes-Fulford, M.: Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts. FASEB J. 13(3), 553–557 (1999)Google Scholar
  5. Furutsu, M., Kawashima, K., Negishi, Y., Endo, H.: Bidirectional effects of hypergravity on the cell growth and differentiated functions of osteoblast-like ROS17/2.8 cells. Biol. Pharm. Bull. 23(10), 1258–1261 (2000)Google Scholar
  6. Gebken, J., Luders, B., Notbohm, H., Klein, H.H., Brinckmann, J., Muller, P.K., Batge, B.: Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2). J. Biochem. (Tokyo) 126(4), 676–82 (1999)Google Scholar
  7. Guignandon, A., Vico, L., Alexandre, C., Lafage-Proust, M.H.: Shape changes of osteoblastic cells under gravitational variations during parabolic flight, relationship with PGE2 synthesis. Cell Struct. Funct. 20, 369–375 (1995)CrossRefGoogle Scholar
  8. Hashemi, B.B., McClure, J.E., Pierson, D.L.: Gravity sensitivity of T-cell activation: the actin cytoskeleton, ASGSB Annual Meeting Abstracts (2002)Google Scholar
  9. Hatton, J.P., Pooran, M., Li, C.F., Luzzio, C., Hughes-Fulford, M.: A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK1/2 pathway. J. Bone Miner Res. 18(1), 58–66 (2003)CrossRefGoogle Scholar
  10. Hughes-Fulford, M.: Altered cell function in microgravity. Exp. Gerontol. 26(2–3), 247–256 (1991)CrossRefGoogle Scholar
  11. Hughes-Fulford, M.: The role of signaling pathways in osteoblast gravity perception. J. Gravit. Physiol. 9(1), 257–260 (2002)Google Scholar
  12. Hughes-Fulford, M.: Function of the cytoskeleton in gravisensing during spaceflight. Adv. Space Res. 32(8), 1585–1593 (2003)CrossRefGoogle Scholar
  13. Hughes-Fulford, M., Lewis, M.L.: Effects of microgravity on osteoblast growth activation. Exp. Cell Res. 224(1), 103–109 (1996)CrossRefGoogle Scholar
  14. Ishikawa, Y., Mogami, Y., Miyamoto, Y.: Analysis of the gravity response mechanism of actin filament in osteoblast. Biol. Sci. Space 19(2), 78–79 (2005)Google Scholar
  15. Kacena, M.A., Todd, P., Landis, W.J.: Osteoblasts subjected to spaceflight and simulated space shuttle launch conditions. In Vitro Cell. Dev. Biol. Anim. 39(10), 454–459 (2003)CrossRefGoogle Scholar
  16. Kacena, M.A., Todd, P., Gerstenfeld, L.C., Landis, W.J.: Experiments with osteoblasts cultured under hypergravity conditions. Microgravity Sci. Technol. 15(1), 28–34 (2004)CrossRefGoogle Scholar
  17. Kumei, Y., Shimokawa, H., Ohya, K., Katano, H., Akiyama, H., Hirano, M., Morita, S.: Small GTPase Ras and Rho expression in rat osteoblasts during spaceflight. Ann. N. Y. Acad. Sci. 1095, 292–9 (2007)CrossRefGoogle Scholar
  18. Loesberg, W.A., Walboomers, X.F., Van Loon, J.J., Jansen, J.A.: The effect of combined hypergravity and microgrooved surface topography on the behaviour of fibroblasts. Cell Motil. Cytoskelet. 63(7), 384–94 (2006)CrossRefGoogle Scholar
  19. Loesberg, W.A., Walboomers, X.F., van Loon, J.J., Jansen, J.A.: Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography. Cell Motil. Cytoskelet. 65(2), 116–29 (2008)CrossRefGoogle Scholar
  20. Moes, M., Maarten, J.A., Bijvelt, J.J., Boonstra, J.: Actin dynamics in mouse fibroblasts in microgravity. Microgravity Sci. Technol. 19(5–6), 180–183 (2007)CrossRefGoogle Scholar
  21. Morita, S., Nakamura, H., Kumei, Y., Shimokawa, H., Ohya, K., Shinomiya, K.: Hypergravity stimulates osteoblast phenotype expression: a therapeutic hint for disuse bone atrophy. Ann. N. Y. Acad. Sci. 1030, 158–161 (2004)CrossRefGoogle Scholar
  22. Reunanen, N., Foschi, M., Han, J., Kahari, V.M.: Activation of extracellular signal-regulated kinase 1/2 inhibits type I collagen expression by human skin fibroblasts. J. Biol. Chem. 275, 34634–34639 (2000)CrossRefGoogle Scholar
  23. Saito, M., Soshi, S., Fujii, K.: Effect of hyper- and microgravity on collagen post-translational controls of MC3T3-E1 osteoblasts. J. Bone Miner. Res. 18(9), 1695–705 (2003)CrossRefGoogle Scholar
  24. Samaj, J., Baluska, F., Hirt, H.: From signal to cell polarity: mitogenactivated protein kinases as sensors and effectors of cytoskeleton dynamicity. J. Exp. Bot. 55, 189–198 (2004)CrossRefGoogle Scholar
  25. Sato, A., Fujita, M., Kanematsu, M., Kamigaichi, S., Takaoki, M., Narato, M., Kumagai, H., Taniguchi, Y.: Effect of vector-averaged gravity on sub-cellular localization of mitogen-activated protein kinase in mouse osteoblast-like MC3T3-E1 cells. ASGSB Annual Meeting Abstracts (2000)Google Scholar
  26. Tairbekov, M.G.: Mechanisms of the gravitational sensitivity of cells. J. Gravit. Physiol. 11(2), P181–183 (2004)Google Scholar
  27. Tjandrawinata, R.R., Vincent, V.L., Hughes-Fulford, M.: Vibrational force alters mRNA expression in osteoblasts. FASEB J. 11(6), 493–497 (1997)Google Scholar
  28. Uva, B., Masini, M.A., Sturla, M., Prato, P., Tagliafierro: Effect of microgravity on the cytoskeleton of cultured nervous cells. Gravit. Space Biol. Bull. 14(1), 53 (2000)Google Scholar
  29. Vercoutere, W., Parra, M., Roden, C., Wing, A., Damsky, C., Holton, N., Globus, R., Almeida, E.: Hypergravity stimulates osteoblast proliferation Via matrix-intergrin-signaling pathway. ASGSB Meeting Abstracts (2003)Google Scholar
  30. Waki, H., Shimizu, T., Katahira, K., Nagayama, T., Yamasaki, M., Katsuda, S.: Effects of microgravity elicited by parabolic flight on abdominal aortic pressure and heart rate in rats. J. Appl. Physiol. 93, 1893–1899 (2002)Google Scholar
  31. Zhongquan, D., Yinghui, L., Bai, D., Weiquan, L., Yuguo, Z., Pengpeng, L.: Effects of clinorotation on COL1A1-EGFP gene expression. Sci. China, Ser. C Chem. Life Sci. 47(3), 203–210 (2004)Google Scholar
  32. Zhongquan, D., Yinghui, L., Bai, D., Zhang, X., Tan, Y., Wan, Y.: Actin microfilaments participate in the regulation of the COL1A1 promoter activity in ROS17/2.8 cells under simulated microgravity. Adv. Space Res. 38(6), 1159–1167 (2006)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Zhongquan Dai
    • 1
    • 2
  • Yingjun Tan
    • 2
  • Fen Yang
    • 2
  • Lina Qu
    • 2
  • Hongyu Zhang
    • 2
  • Yumin Wan
    • 2
  • Yinghui Li
    • 2
  1. 1.Faculty of Aerospace MedicineFourth Military Medical UniversityXi’anChina
  2. 2.State Key Laboratory of Space Medicine Fundamentals and ApplicationChina Astronaut Research and Training CenterBeijingChina

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