Interaction of Protein Phosphatase 1δ with Nucleophosmin in Human Osteoblastic Cells

International Journal of Oncology

Hirashima

Teramachi

et al. 2013

Self Cite

Double-stranded RNA-dependent protein kinase (PKR) is one of the players in the cellular antiviral responses and is involved in transcriptional stimulation through activation of NF-κB. Treatment of the human osteosarcoma cell line MG63 with the protein phosphatase inhibitor okadaic acid stimulated the expression and phosphorylation of IκBα, as judged from the results of real-time PCR and western blot analysis. We investigated the functional relationship between PKR and signal transduction of NF-κB by establishing PKR-K/R cells that produced a catalytically inactive mutant of PKR. Phosphorylation of eIF-2α, a substrate of PKR, was not stimulated by okadaic acid in the PKR-K/R cells, whereas okadaic acid induced phosphorylation of eIF-2α in MG63 cells. Phosphorylation of NF-κB in MG63 cells was stimulated by okadaic acid; however, okadaic acid did not induce phosphorylation of NF-κB in the PKR-K/R cells. Finally, okadaic acid-induced apoptosis was inhibited in the PKR-K/R cells. Our results suggest that okadaic acid-induced phosphorylation of IκBα was mediated by PKR kinase activity, thus, indicating the involvement of this kinase in the control mechanism governing the activation of NF-κB and induction of apoptosis.

Section: Introductionmentioning

confidence: 99%

Okadaic acid activates the PKR pathway and induces apoptosis through PKR stimulation in MG63 osteoblast-like cells

International Journal of Oncology

Hirashima

Teramachi

et al. 2013

Self Cite

“…Immunoprecipitation was performed as described previously [33], using specific antibodies for IRF4 and IRF8.…”

Section: Methodsmentioning

confidence: 99%

Stimulation of Osteoclast Formation by RANKL Requires Interferon Regulatory Factor-4 and Is Inhibited by Simvastatin in a Mouse Model of Bone Loss

Nakashima

2013

PLoS ONE

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Diseases of bone loss are a major public health problem. Here, we report the novel therapeutic action of simvastatin in osteoclastogenesis and osteoprotection, demonstrated by the ability of simvastatin to suppress osteoclast formation in vitro and in vivo. We found that in vitro, IRF4 expression is upregulated during osteoclast differentiation induced by RANKL (receptor activator of nuclear factor-κB ligand), while simvastatin blocks RANKL-induced osteoclastogenesis and decreases expression of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1), IRF4 and osteoclast markers. We also show that IRF4 acts in cooperation with NFATc2 and NF-κB on the promoter region of NFATc1 to accelerate its initial transcription during the early stage of osteoclastogenesis. Moreover, our study using IRF4 siRNA knockdown directly demonstrates the requirement for IRF4 in NFATc1 mRNA transcription and its necessity in RANKL-induced osteoclast differentiation. Our results suggest that the reduction in osteoclastogenesis is partly due to the inhibition of IRF4 production in RANKL-induced osteoclast differentiation. To investigate the in vivo effects of simvastatin in RANKL-treated mice, we examined the bone mineral density (BMD) of a mouse model of bone loss, and found that simvastatin significantly reduced bone loss by suppressing osteoclast numbers in vivo, even in the presence of high concentrations of RANKL. These results suggest that the depletion of osteoclasts is not due to the reduction in RANKL produced by osteoblasts in vivo. The results are consistent with the hypothesis that simvastatin blocks RANKL-induced IRF4 expression in osteoclastogenesis. We propose that the expression of IRF4 by osteoclasts could be a promising new therapeutic target in bone-loss diseases.

Roles of PKR in differentiation and apoptosis of bone-related cells

2016

Anat Sci Int

Double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine protein kinase which is activated by double-stranded RNAs and related to several signal transduction pathways. To examine the effects of PKR on bone metabolism, we established PKR-K/R mutant cells in which amino acid lysine at 296 is substituted with arginine. PKR regulated apoptosis in osteoblastic cells via nuclear factor kappa-B (NF-κB) cascade. MC3T3-E1 cells cultured with osteoblast differentiation medium differentiated into osteoblasts, while the mutant cells did not differentiate into osteoblasts. RAW246.7 cells triggered with receptor activator of NF-κB ligand (RANKL) formed tartrate-resistant acid phosphatase-positive multinucleated giant cells, whereas PKR-K/R mutant RAW cells did not. Differentiation of osteoblasts and osteoclasts was caused by NF-κB activation and signal transducer and activator of transcription 1 (STAT1) ubiquitination and degradation. We also demonstrated involvement of PKR in chondrocyte differentiation. PKR prevented tumor necrosis factor-α- and interleukin 1α-induced bone resorption in calvaria and artificially induced periodontal disease in rat. Our findings indicate that PKR regulates bone metabolism in vitro and in vivo.