Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration(Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.