title: Fluid flow induces Rankl expression in primary murine calvarial osteoblasts.
abstract: Mechanical loading of bone generates fluid flow within the mineralized matrix that exerts fluid shear stress (FSS) on cells. We examined effects of FSS on receptor activator of nuclear factor kappa B ligand (RANKL), a critical factor for osteoclast formation. Primary murine osteoblasts were subjected to pulsatile FSS (5 Hz, 10 dynes/cm(2)) for 1 h and then returned to static culture for varying times (post-FSS). Protein levels were measured by Western analysis and mRNA by Northern analysis, RT-PCR and quantitative PCR. There were 20- to 40-fold increases in RANKL mRNA at 2-4 h post-FSS. RANKL protein was induced by 2 h post-FSS and remained elevated for at least 8 h. Effects were independent of cyclooxygenase-2 activity. Small increases (up to three-fold) in mRNA of the decoy receptor for RANKL, osteoprotegerin, were seen. Five min of FSS, followed by static culture, was as effective in stimulating RANKL mRNA as 4 h of continuous FSS. FSS induced cAMP activity, and H-89, a protein kinase A (PKA) inhibitor, blocked the FSS induction of RANKL. H-89 also inhibited the PKC pathway, but specific PKC inhibitors, GF109203X and Go6983, did not inhibit FSS-induced RANKL. FSS induced phosphorylation of ERK1/2, and PD98059, an inhibitor of the ERK pathway, inhibited the FSS induction of RANKL mRNA 60%-90%. Thus, brief exposure to FSS resulted in sustained induction of RANKL expression after stopping FSS, and this induction was dependent on PKA and ERK signaling pathways. Increased RANKL after mechanical loading may play a role in initiating bone remodeling.
authors.affiliation: Department of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
authors.forename: Meenal
authors.initials: M
authors.lastname: Mehrotra
authors.forename: Masatomo
authors.initials: M
authors.lastname: Saegusa
authors.forename: Sunil
authors.initials: S
authors.lastname: Wadhwa
authors.forename: Olga
authors.initials: O
authors.lastname: Voznesensky
authors.forename: Donald
authors.initials: D
authors.lastname: Peterson
authors.forename: Carol
authors.initials: C
authors.lastname: Pilbeam
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D053244:Osteoprotegerin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D053245:RANK Ligand; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D012212:Rheology; D015398:Signal Transduction; D012886:Skull / Q000166:cytology*
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D052246:Cyclooxygenase 2 Inhibitors; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D051546:Cyclooxygenase 2; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases