| 0590 BMP-2 and NF-ΚB Signaling During Osteoblastic Differentiation in Culture | ||
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L. COOPER, C.-B. CUI, J. GUO, and R. MERCER, University of North Carolina, Chapel Hill, USA Objective: Osseous wound healing in dentistry often occurs following acute or chronic inflammatory tissue responses. The goal of this investigation was to examine the scope of inflammatory signaling through NF-κB on osteoinduction in the MC3T3-E1 osteoblastic cell line. Methods: MC3T3-E1 (passage 4) were cultured in α-MEM media for transient transfection of NF-kB(p65), IκBα, SBE12-Luc, SMAD1, LRP5 and TOPflash encoding plasmids using Effectine. Osteoblastic differentiation was achieved by supplementing media with ascorbic acid and β-glycerophosphate for 7 – 28 days. Osteoinduction was alternatively signaled by adding 0 – 300 ng/ml rhBMP-2. TNF-α (10 nM) was included to induce inflammatory signaling. Results: Inflammatory processes modeled by inclusion of LPS or TNF-α in the cell culture media of MC3T3-E1 cells limited osteogenic supplement or BMP-2 induced differentiation. Transfection of cells with SBE12-luc (a BMP responsive Luciferace construct) and treatment with TNF-α indicated that TNF-α signaling diminished (4 fold, p <0.05) BMP-2 signaling at the SBE12 reporter. Co-transfection of SBE12-luc and NF-κB(p65) encoding plasmids further demonstrated the direct effect of NF-κB signaling on BMP-2 mediated signaling. SBE12-Luc expression induced by co-transfection of a SMAD1-encoding plasmid was blocked by the addition of NF-κB (p65) encoding plasmid. Transfection of IκBα increased SBE12-luc expression following BMP-2 treatment or SMAD1 co-transfection. Additional preliminary studies of LRP5 and RUNX2 contributions to osteoinduction showed inhibition of these additional and key osteoinduction signaling pathaways by NF-κB (p65) action. Conclusion: The effective blocking of BMP-2 mediated signaling by NF-κB is one example of the central role NF-κB plays in this process. Understanding the intricate regulation of osteoblastic differentiation for wound repair requires a more complete description of inflammatory signaling effects on this complex process. | ||
| Seq #87 - Cell differentiation 2:00 PM-3:15 PM, Thursday, March 22, 2007 Ernest N. Morial Convention Center Exhibit Hall I2-J | ||
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