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Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro
Junpei Teramachi1, Hiroyuki Morimoto, Ryoko Baba
1Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto, Tokushima 770-8504, Japan.
Abstract:
Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and α-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic-polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-κB protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important roles in the differentiation of osteoclasts.
Insights
Double-stranded RNA-dependent protein kinase (PKR) is essential for osteoclast differentiation. Inhibiting PKR or using a PKR mutant blocked the formation of these bone-resorbing cells, revealing a key role in bone biology.
Area of Science:
- Cell Biology
- Molecular Biology
- Bone Biology
Background:
- Double-stranded RNA-dependent protein kinase (PKR) is a crucial mediator of cellular stress responses and has known roles in host defense and cell proliferation.
- Previous research established PKR's involvement in osteoblast differentiation and calcification.
- The specific role of PKR in osteoclast differentiation remained largely uncharacterized.
Purpose of the Study:
- To investigate the function of PKR in the differentiation process of osteoclasts.
- To determine if PKR signaling is necessary for the formation of functional osteoclasts.
Main Methods:
- Established a stable RAW264.7 cell line expressing a dominant-negative PKR mutant (PKR-K/R).
- Utilized RANKL stimulation to induce osteoclast differentiation in wild-type and PKR-K/R cells.
- Employed RT-PCR and Western blotting to analyze gene and protein expression of osteoclast markers and signaling pathways.
- Administered a specific PKR inhibitor (2-aminopurine) to assess its impact on osteoclastogenesis.
- Performed immunohistochemical analysis on mouse bone tissue.
Main Results:
- RAW264.7 cells expressing the PKR mutant (PKR-K/R) failed to differentiate into TRAP-positive multinuclear osteoclasts upon RANKL stimulation.
- A specific PKR inhibitor, 2-aminopurine, significantly suppressed RANKL-induced osteoclast differentiation.
- Expression of key osteoclast markers (cathepsin K, calcitonin receptor) and fusion-related genes was downregulated in PKR-deficient cells.
- NF-κB signaling was suppressed, while STAT1 expression was elevated in PKR-K/R cells.
- PKR was confirmed to be localized in mature osteoclasts in mouse bone tissue.
Conclusions:
- PKR plays a critical and indispensable role in osteoclast differentiation and function.
- PKR signaling is essential for the expression of osteoclast-specific genes and the activation of key regulatory pathways like NF-κB.
- Targeting PKR may offer a novel therapeutic strategy for modulating bone resorption in diseases characterized by excessive osteoclast activity.
