Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shear increase the expression of the early r esponse gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, tw o proteins linked to the anabolic response of bone to mechanical stimulatio n, in vivo. These increases in gene expression are dependent on shear-induc ed actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteobl ast-like cells respond to shear with a rapid increase in intracellular Ca2 concentration ([Ca2+](i)) that we postulate is important to subsequent cel lular responses to shear. To test this hypothesis, MC3T3-E1 cells were grow n on glass slides coated with fibronectin and subjected to laminar fluid fl ow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca2+ channel inhibitors or various blockers of intracellular Ca2+ release for 0.5-1 h. Although gadolinium, a mechanosensitive channel blocker, signi ficantly reduced the [Ca2+](i) response, neither gadolinium nor nifedipine, an L-type channel Ca2+ channel blocker, were able to block shear-induced s tress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. Ho wever, 1,2-bis(2-minophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intra cellular Ca2+ chelator, or thapsigargin, which empties intracellular Ca2+ s tores, completely inhibited stress fiber formation and c-fos/CQX-2 producti on in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP3)-induced intracel lular Ca2+ release, also completely suppressed actin reorganization and c-f os/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inact ive isoform of U-73122, did not inhibit these shear-induced responses. Thes e results suggest that IP3-mediated intracellular Ca2+ release is required for modulating flow-induced responses in MC3T3-E1 cells.