Fluid shear-induced NFκB translocation in osteoblasts is mediated by intracellular calcium release

Bone formation in response to exogenous mechanical loading is dependent on prostaglandin synthesis by the inducible isoform of cyclooxygenase, COX-2. While several transcription factors target the COX-2 gene, we examined the role of nuclear factor kappa B (NFκB) on COX-2 upregulation in osteoblasts in response to fluid shear due to its involvement in immune and inflammatory responses in other cell types. Application of 12 dyn/cm² laminar flow to MC3T3-E1 osteoblast-like cells resulted in translocation of NFκB to the nucleus within 1 h of the onset of shear, with NFκB returning to the cytoplasm after 2 h of continuous flow. NFκB translocation in response to shear was inhibited by the protease inhibitor, Nα -p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFκB. Block of NFκB translocation with these inhibitors blocked the shear-induced upregulation of COX-2. We found that disruption of the actin cytoskeleton with cytochalasin D or microtubules with nocodozol did not alter NFκB translocation in response to shear. However, addition of the intracellular Ca²⁺ chelator BAPTA completely blocked NFκB translocation. While block of Ca²⁺ entry with channel blockers failed to inhibit NFκB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca²⁺ release with the PLC inhibitor U73122 completely abrogated the NFκB response to shear. These data indicate that NFκB translocation to the nucleus is essential for the fluid shear-induced increase in COX-2. Further, these studies suggest that intracellular Ca²⁺ release, but not the cytoskeletal architecture, is important to NFκB translocation.