title: Fluid shear-induced NFkappaB translocation in osteoblasts is mediated by intracellular calcium release.
abstract: 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.
mesh_terms: 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
chemical_list: D007527:Isoenzymes; D016328:NF-kappa B; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D010738:Type C Phospholipases; D002118:Calcium