Toll-like receptor 3 signaling in osteoblasts

Objective: Many microbial constituents are recognized by toll-like receptors (TLRs) expressed on various cells. The osteoblast is a cell type of mesenchymal origin that is responsible for bone matrix deposition, or bone formation. In response to TLR ligands, macrophages produce several inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interferon-beta (IFN-beta) to activate immune responses. Double-stranded RNA (poly(I):poly(C)) act as ligands of TLR3. To explore a part of potential roles of various TLRs in osteoblast, we have analyzed TLR3 signaling and its potential function.

Methods: Osteoblastic MC3T3-E1 (E1) cell were treated with poly(I):poly(C) for appropriate time, and then total RNA and protein were extracted. These were subjected to RT-PCR and western blot analysis respectively. After 30 days poly(I):poly(C) treatment, mineralization of E1 cells was visualized by von Kossa staining. Moreover, alkaline phosphatase (ALP) transcription activity in osteosarcoma cell line MG-63 was assessed by luciferase reporter assay.

Results: We showed here that osteoblastic E1 cells expressed TLR1, 3, 4, 5, 6 and 7. Moreover, poly(I):poly(C) dsRNA up-regulated IFN-beta production in E1 cells. Moreover, poly (I):poly(C) induced tyrosine phosphorylation of the transcription factor STAT1 in E1 cells. An anti-IFN-beta neutralizing antibody partially inhibited poly(I):poly(C)-induced IFN-beta mRNA, and STAT1 phosphorylation. Unexpectedly, TLR3 stimulation of E1 cells by poly (I:C) strongly induced their mineralization. Furthermore, poly (I:C) increased fibroblast growth factor 2 (FGF2) mRNA expression in E1 cells and stimulated ALP promoter activity in osteosarcoma MG63 cells.

Conclusion: These results suggest that TLR3 stimulation of osteoblasts induces their mineralization and IFN-beta production during viral infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone.