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Possible Involvement of Fluid Flow Detection by Osteoblasts in Their Activation Process.  M. Takaoki1, H. Park2, N. Murakami1, D.Shiba2 and J-I. Gyotoku2. 1Space Utilization Center, 2ISS Science Project Office, JAXA, Tsukuba, Japan.

Decreased interstitial flow under microgravity has been thought to be a cause of bone mineral loss during space flight.  Although mechanical stimulations such as flow significantly affect cellular activities, the molecular nature of putative mechanoreceptor has been unclear. While studying effects of mechanical stimuli on the expression of an immediate early gene c-fos by an osteoblast-like line MC3T3-E1, we noticed that mere manual handling of culture flasks affected the response far greater than intended mechanical stimulation such as centrifugation. Carrying culture flasks in conventional laboratory practices may induce culture fluid flow even with extremely gentle handling. Moreover, such procedures bring with cooling down of culture fluid to the room temperature, which may also affect the response. We then developed a simple technique to minimize the effects of such problems.  Temperature change and fluid flow were independently applied to the cells in culture flasks.  It was shown that culture fluid flow, generated by manual rocking of the flask at stable temperature, induced transient c-fos expression. Temperature change at standing still also induced similar response.

   It has been reported that kidney cells could sense fluid flow by molecules named polycystins -1 and -2 located on primary cilia. MC3T2-E1 cells also had primary cilia and expressed polycystin-2, suggesting analogous events in bone cells. Polycystins are TRP channel super family proteins. Some TRP channel proteins are known to be either hot or cold temperature receptors.  The finding that fluid flow and temperature shift separately induced similar response, suggested possible involvement of receptors molecules related both mechanical and thermal perceptions in osteoblast activation.