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

Departments of 1 Anatomy, 2 Physiology and Biophysics, and 3 Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana 46202 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 Ca 2+ concentration ([Ca 2+ ] 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/cm 2 ). Before application of shear, cells were treated with two Ca 2+ channel inhibitors or various blockers of intracellular Ca 2+ release for 0.5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca 2+ ] i response, neither gadolinium nor nifedipine, an L-type channel Ca 2+ 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 Ca 2+ chelator, or thapsigargin, which empties intracellular Ca 2+ 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 (IP 3 )-induced intracellular Ca 2+ 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 IP 3 -mediated intracellular Ca 2+ release is required for modulating flow-induced responses in MC3T3-E1 cells. actin cytoskeleton; intracellular calcium, c-fos; cyclooxygenase-2; phospholipase C