Double Stranded RNA‐Dependent Protein Kinase is Necessary for TNF‐α‐Induced Osteoclast Formation In Vitro and In Vivo

Abstract

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 pretreatment 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.

TAK1 inhibition subverts the osteoclastogenic action of TRAIL while potentiating its antimyeloma effects

“…The generation of OCs from RAW264.7 cells 16 or mouse bone marrow cells 17 was performed as previously described. The cells were cultured in Eagle's minimal essential medium a modification (SigmaAldrich) supplemented with 10% FBS, L-glutamine, and 50 mg/mL penicillin/streptomycin.…”

Inhibition of PKR protects against H2O2-induced injury on neonatal cardiac myocytes by attenuating apoptosis and inflammation

“…However, most of the previous studies revealed the roles of PKR in confronting the chronic cellular stresses, including regulating metabolic homeostasis23, mediating TNF-α induced osteoclast formation26 and systolic overload-induced congestive heart failure21, et al . In this study, we investigated in the face of H 2 O 2 treatment on NCM, an acute and drastic cellular stress, whether PKR is involved.…”

Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli

“…Moreover, p38 MAPK signalling pathways is also implicated in mediating RANKL-induced osteoclastogenesis, suggested the increased TNF-α mRNA abundance induced by RANKL is mediated by activating this signalling cascade leading to TNF-α transcriptional stabilities. Besides that, most recently, Shinohara et al 141 have reported that double-stranded RNA-dependent protein kinase (PKR) association is necessary for TNF-a induced RAW-OCs formation by p38MAPK, and ERK signalling ( Figure 2).…”

“…Specifically, Zou et al 136 explained this autocrine manner as RANKL induction of osteoclastogenesis is accompanied by a rapid and transient increase in TNF-α mRNA abundance in the precursor cell; specific anti-TNF-α antibodies or antibodies directed against TNF receptor type I inhibit the osteoclastogenic activity of RANKL in RAW264.7cells. Besides that, most recently, Shinohara et al141 have reported that double-stranded RNA-dependent protein kinase (PKR) In fact, AU-rich elements (AREs) of 3′ untranslated region (3′-UTR), as cis-element, involved in the regulation of TNF-α mRNA transcription 137,138.…”