Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca2++ concentration ([Ca2++]i) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm²). 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, significantly reduced the [Ca2++]i response, neither gadolinium nor nifedipine, an L-type channel Ca2++ channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM, an intracellular Ca2++ chelator, or thapsigargin, which empties intracellular Ca2++ stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates d-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca2++ release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP3-mediated intracellular Ca2++release is required for modulating flow-induced responses in MC3T3-E1 cells.
actin cytoskeleton; intracellular calcium, c-fos; cyclooxygenase-2; phospholipase C