title: Response to mechanical strain in an immortalized pre-osteoblast cell is dependent on ERK1/2.
abstract: Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.
authors.affiliation: Department of Medicine, Emory University School of Medicine and the Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA. xfan@emory.edu
authors.forename: Xian
authors.initials: X
authors.lastname: Fan
authors.forename: Jill A
authors.initials: JA
authors.lastname: Rahnert
authors.forename: Tamara C
authors.initials: TC
authors.lastname: Murphy
authors.forename: Mark S
authors.initials: MS
authors.lastname: Nanes
authors.forename: Edward M
authors.initials: EM
authors.lastname: Greenfield
authors.forename: Janet
authors.initials: J
authors.lastname: Rubin
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics; D002461:Cell Line, Transformed; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D053244:Osteoprotegerin; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D000076182:Sp7 Transcription Factor; D013314:Stress, Mechanical; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D006023:Glycoproteins; D008562:Membrane Glycoproteins; C102260:NOR 4; D053244:Osteoprotegerin; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C493716:Runx2 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D014157:Transcription Factors; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000469:Alkaline Phosphatase; D002117:Calcitriol