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ERK Inhibition Increases RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells by Stimulating AMPK Activation and RANK Expression and Inhibiting Anti-Osteoclastogenic Factor Expression

Choi, Eun-Bi, Agidigbi, Taiwo Samuel, Kang, In-Soon, Kim, Chaekyun·2022·International Journal of Molecular Sciences
DOI 10.3390/ijms232113512PMC PMC9656104
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Abstract

Bone absorption is necessary for the maintenance of bone homeostasis. An osteoclast (OC) is a monocyte–macrophage lineage cell that absorbs bone tissue. Extracellular signal-regulated kinases (ERKs) are known to play important roles in regulating OC growth and differentiation. In this study, we examined specific downstream signal pathways affected by ERK inhibition during OC differentiation. Our results showed that the ERK inhibitors PD98059 and U0126 increased receptor activator of NF-κB ligand (RANKL)-induced OC differentiation in RAW 264.7 cells, implying a negative role in OC differentiation. This is supported by the effect of ERK2-specific small interfering RNA on increasing OC differentiation. In contrast to our findings regarding the RAW 264.7 cells, the ERK inhibitors attenuated the differentiation of bone marrow-derived cells into OCs. The ERK inhibitors significantly increased the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) but not the activation of p38 MAPK, Lyn, and mTOR. In addition, while the ERK inhibition increased the expression of the RANKL receptor RANK, it decreased the expression of negative mediators of OC differentiation, such as interferon regulatory factor-8, B-cell lymphoma 6, and interferon-γ. These dichotomous effects of ERK inhibition suggest that while ERKs may play positive roles in bone marrow-derived cells, ERKs may also play negative regulatory roles in RAW 264.7 cells. These data provide important information for drug development utilizing ERK inhibitors in OC-related disease treatment.

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Figure 1 Inhibition of extracellular signal-regulated kinase (ERK) increased osteoclast (OC) differentiation in RAW 264.7 cells. Cells were treated with PD98059 and U0126 in the presence of 50 ng/mL receptor activator of NF-κB ligand (RANKL) for 4 days. (A,B) Effect of ERK inhibitors on the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated OCs (n = 4 independent experiments, each performed in triplicate). (C) Effect of PD98059 on TRAP activity of OCs (n = 3). (D,E) Effect of PD98059 and U0126 on ERK phosphorylation of RAW 264.7-derived OCs (n = 3). (F) Actin ring formation of OCs, which were stained by Alexa 555-phalloidin (F-actin) and DAPI (nuclei), respectively (n = 3). (G) Dose effect of PD98059 on OC differentiation (n = 3 independent experiments, each performed in triplicate). (H) Number of nuclei per OC (n = 3). Each result represents the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (B,C,E,H) or dimethyl sulfoxide (DMSO) (E), and # p < 0.05 vs. 2 μM PD98059 (E).
Figure 1 Inhibition of extracellular signal-regulated kinase (ERK) increased osteoclast (OC) differentiation in RAW 264.7 cells. Cells were treated with PD98059 and U0126 in the presence of 50 ng/mL receptor activator of NF-κB ligand (RANKL) for 4 days. (A,B) Effect of ERK inhibitors on the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated OCs (n = 4 independent experiments, each performed in triplicate). (C) Effect of PD98059 on TRAP activity of OCs (n = 3). (D,E) Effect of PD98059 and U0126 on ERK phosphorylation of RAW 264.7-derived OCs (n = 3). (F) Actin ring formation of OCs, which were stained by Alexa 555-phalloidin (F-actin) and DAPI (nuclei), respectively (n = 3). (G) Dose effect of PD98059 on OC differentiation (n = 3 independent experiments, each performed in triplicate). (H) Number of nuclei per OC (n = 3). Each result represents the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (B,C,E,H) or dimethyl sulfoxide (DMSO) (E), and # p < 0.05 vs. 2 μM PD98059 (E).CC BY · PubMed Central
Figure 2 Inhibition of ERK decreased OC differentiation in bone marrow monocytes (BMMs). Cells were cultured with 30 ng/mL macrophage colony-stimulating factor (M-CSF) for 3 days and then treated with PD98059 and U0126 in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL for 4 days. (A,B) Effect of PD98059 on OC differentiation from BMMs (n = 3 independent experiments, each performed in triplicate). (C,D) Effect of U0126 on OC differentiation from BMMs (n = 3 independent experiments, each performed in triplicate). Each result represents the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. DMSO.
Figure 2 Inhibition of ERK decreased OC differentiation in bone marrow monocytes (BMMs). Cells were cultured with 30 ng/mL macrophage colony-stimulating factor (M-CSF) for 3 days and then treated with PD98059 and U0126 in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL for 4 days. (A,B) Effect of PD98059 on OC differentiation from BMMs (n = 3 independent experiments, each performed in triplicate). (C,D) Effect of U0126 on OC differentiation from BMMs (n = 3 independent experiments, each performed in triplicate). Each result represents the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. DMSO.CC BY · PubMed Central
Figure 3 Downregulation of ERK with ERK2 siRNA increased OC differentiation in RAW 264.7 cells. Cells were transfected with ERK2 siRNA and differentiated into OCs in the presence of 50 ng/mL RANKL for 4 days. (A–C) Effect of ERK2 siRNA on the expression of ERK (n = 5 for mRNA and n = 3 for protein). (D,E) Effect of ERK2 siRNA on OC differentiation (n = 3 independent experiments, each performed in triplicate). (F,G) Relative size of OC and OC area per well of OCs of ERK2 siRNA-transfected cells (n = 3). (H–J) Quantitative analyses of mRNA expression of OC markers, DC-STAMP and cathepsin K, and NFATc1 was measured by qRT-PCR (n = 3). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. scRNA.
Figure 3 Downregulation of ERK with ERK2 siRNA increased OC differentiation in RAW 264.7 cells. Cells were transfected with ERK2 siRNA and differentiated into OCs in the presence of 50 ng/mL RANKL for 4 days. (A–C) Effect of ERK2 siRNA on the expression of ERK (n = 5 for mRNA and n = 3 for protein). (D,E) Effect of ERK2 siRNA on OC differentiation (n = 3 independent experiments, each performed in triplicate). (F,G) Relative size of OC and OC area per well of OCs of ERK2 siRNA-transfected cells (n = 3). (H–J) Quantitative analyses of mRNA expression of OC markers, DC-STAMP and cathepsin K, and NFATc1 was measured by qRT-PCR (n = 3). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. scRNA.CC BY · PubMed Central
Figure 4 Inhibition of ERK increased expression of RANK. RAW 264.7 cells were treated with 5 μM PD98059 and 2 μM U0126, or were transfected with ERK2 siRNA, and differentiated into OCs in the presence of 50 ng/mL RANKL for 4 days. (A,B) Quantitative analyses of mRNA expression by ERK inhibitors and ERK2 siRNA (n = 3). (C–E) Protein expression of RANK in ERK inhibitor-treated and ERK2 siRNA-transfected cells (n = 3). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. control or scRNA.
Figure 4 Inhibition of ERK increased expression of RANK. RAW 264.7 cells were treated with 5 μM PD98059 and 2 μM U0126, or were transfected with ERK2 siRNA, and differentiated into OCs in the presence of 50 ng/mL RANKL for 4 days. (A,B) Quantitative analyses of mRNA expression by ERK inhibitors and ERK2 siRNA (n = 3). (C–E) Protein expression of RANK in ERK inhibitor-treated and ERK2 siRNA-transfected cells (n = 3). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. control or scRNA.CC BY · PubMed Central
Figure 5 Inhibition of ERK increased adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation. Cell lysates were prepared from PD98059-treated OCs and ERK2 siRNA-transfected OCs, and activation of kinases was measured by Western blotting. Phosphorylation of p38 MAPK (n = 4) (A), mTOR (n = 3) (B), Lyn (n = 3) (C), and AMPK (n = 6) (D). (E,F) AMPK phosphorylation was determined in OCs of ERK2 siRNA-transfected cells (n = 5). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. control or scRNA, and # p < 0.05 and ## p < 0.01.
Figure 5 Inhibition of ERK increased adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation. Cell lysates were prepared from PD98059-treated OCs and ERK2 siRNA-transfected OCs, and activation of kinases was measured by Western blotting. Phosphorylation of p38 MAPK (n = 4) (A), mTOR (n = 3) (B), Lyn (n = 3) (C), and AMPK (n = 6) (D). (E,F) AMPK phosphorylation was determined in OCs of ERK2 siRNA-transfected cells (n = 5). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. control or scRNA, and # p < 0.05 and ## p < 0.01.CC BY · PubMed Central
Figure 6 Inhibition of ERK suppressed the expression of inhibitory mediators of OC differentiation. RAW 264.7 cells treated with 5 μM PD98059 and 2 μM U0126 or transfected with ERK2 siRNA, and differentiated into OCs in the presence of 50 ng/mL RANKL for 4 days. Quantitative analyses of mRNA expression of IRF8 (A,B), Bcl6 (C,D), and IFN-γ (E,F) (n = 3). (G–I) Protein expression of Bcl6 in PD98059-treated and ERK2 siRNA-transfected cells (n = 3). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. control or scRNA.
Figure 6 Inhibition of ERK suppressed the expression of inhibitory mediators of OC differentiation. RAW 264.7 cells treated with 5 μM PD98059 and 2 μM U0126 or transfected with ERK2 siRNA, and differentiated into OCs in the presence of 50 ng/mL RANKL for 4 days. Quantitative analyses of mRNA expression of IRF8 (A,B), Bcl6 (C,D), and IFN-γ (E,F) (n = 3). (G–I) Protein expression of Bcl6 in PD98059-treated and ERK2 siRNA-transfected cells (n = 3). Each result represents the mean ± SD. * p < 0.05 and ** p < 0.01 vs. control or scRNA.CC BY · PubMed Central
Figure 7 Proposed molecular mechanism that ERK regulates OC differentiation. Inhibition of ERK increased OC differentiation in RAW 264.7 cells by increasing RANK expression and AMPK phosphorylation and inhibiting the expression of negative mediators of osteoclastogenesis, such as IRF8, Bcl6, and IFN-γ.
Figure 7 Proposed molecular mechanism that ERK regulates OC differentiation. Inhibition of ERK increased OC differentiation in RAW 264.7 cells by increasing RANK expression and AMPK phosphorylation and inhibiting the expression of negative mediators of osteoclastogenesis, such as IRF8, Bcl6, and IFN-γ.CC BY · PubMed Central

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Metadata

Authors
Choi, E.-B., Agidigbi, T. S., Kang, I.-S., & Kim, C.
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2022-11-04
Volume
23
Issue
21
Pages
13512
ISSN
1422-0067
DOI
10.3390/ijms232113512
PMC
PMC9656104
Cited by
22
References
4
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
National Research Foundation of Korea (2020R1F1A1068065, 2022R1I1A1A01055015)

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Choi, E.-B., Agidigbi, T. S., Kang, I.-S., & Kim, C. (2022). ERK Inhibition Increases RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells by Stimulating AMPK Activation and RANK Expression and Inhibiting Anti-Osteoclastogenic Factor Expression. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms232113512

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