Fluid shear-induced NFkappaB 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 (NFkappaB) 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 NFkappaB to the nucleus within 1 h of the onset of shear, with NFkappaB returning to the cytoplasm after 2 h of continuous flow. NFkappaB translocation in response to shear was inhibited by the protease inhibitor, Nalpha-p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFkappaB. Block of NFkappaB 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 NFkappaB translocation in response to shear. However, addition of the intracellular Ca2+ chelator BAPTA completely blocked NFkappaB translocation. While block of Ca2+ entry with channel blockers failed to inhibit NFkappaB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca2+ release with the PLC inhibitor U73122 completely abrogated the NFkappaB response to shear. These data indicate that NFkappaB 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 NFkappaB translocation.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D051546:Cyclooxygenase 2; D003599:Cytoskeleton / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010477:Perfusion; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013314:Stress, Mechanical; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D016328:NF-kappa B; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D010738:Type C Phospholipases; D002118:Calcium