Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation.
OBJECTIVE
While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-kappaB (NF-kappaB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins.


METHODS
Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-kappaB were measured using enzyme-linked immunosorbent assay.


RESULTS
The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-kappaB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-kappaB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts.


CONCLUSIONS
These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.
Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai, Japan. e-nemoto@umin.ac.jp
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Nemoto
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Honda
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Kanaya
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Takada
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Shimauchi
D016475:3T3 Cells; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D003739:Dental Cementum / Q000166:cytology* / Q000378:metabolism*; D015870:Gene Expression; D015850:Interleukin-6 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D051379:Mice; D016328:NF-kappa B / Q000096:biosynthesis; D053472:Nod Signaling Adaptor Proteins / Q000819:agonists / Q000096:biosynthesis*; D010006:Osteoblasts / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D051193:Toll-Like Receptors / Q000819:agonists / Q000096:biosynthesis*; D015854:Up-Regulation
D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D016328:NF-kappa B; D053472:Nod Signaling Adaptor Proteins; D051193:Toll-Like Receptors; D001205:Ascorbic Acid