title: Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts.
abstract: 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.
authors.affiliation: Department of Anatomy, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: N X
authors.initials: NX
authors.lastname: Chen
authors.forename: K D
authors.initials: KD
authors.lastname: Ryder
authors.forename: F M
authors.initials: FM
authors.lastname: Pavalko
authors.forename: C H
authors.initials: CH
authors.lastname: Turner
authors.forename: D B
authors.initials: DB
authors.lastname: Burr
authors.forename: J
authors.initials: J
authors.lastname: Qiu
authors.forename: R L
authors.initials: RL
authors.lastname: Duncan
mesh_terms: D016475:3T3 Cells; D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D051546:Cyclooxygenase 2; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation; D016762:Genes, fos; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D000199:Actins; D007527:Isoenzymes; D012333:RNA, Messenger; D004533:Egtazic Acid; D019284:Thapsigargin; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D010738:Type C Phospholipases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium