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Objective In this study, we aimed to clarify the precise mechanism underlying lipopolysaccharide (LPS)-induced osteoclastogenesis in periodontal disease with a special reference to double-stranded RNA-dependent protein kinase (PKR). Material and methods We dissected the role of PKR in LPS-induced osteoclast differentiation and function using primary mouse bone marrow cells and RAW264.7 pre-osteoclastic cell line. We used a rat experimental periodontitis (PD) model induced by ligature placement with a Porphyromonas gingivalis LPS injection (PD rat) and analyzed the therapeutic effects of C16, a PKR inhibitor, on bone loss in PD rats. Results Protein kinase is strongly upregulated and phosphorylated by LPS in the osteoclasts. The inhibition of PKR suppressed LPS-stimulated osteoclast formation and activation. PKR inhibition also suppressed the LPS-mediated activation of NF-κB and MAPK, which are critical pathways for osteoclastogenesis. High expressions of PKR were detected in osteoclasts of PD rats, and the treatment with C16 effectively prevented alveolar bone destruction in PD rats. Conclusions PKR plays a pivotal role in LPS-induced bone loss in PD and, thus, has potential as a therapeutic target for PD.

Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF-α plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF-α-induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF-α-induced osteoclastogenesis. The TNF-α-induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pre-treatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF-α-induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF-α-induced activation of NF-κB and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-κB and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF-α-induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF-α-induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF-α-induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF-α-induced osteoclast differentiation in vitro and in vivo.