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ERK5 activation is essential for osteoclast differentiation.

Amano S, Chang YT, Fukui Y - PLoS ONE (2015)

Bottom Line: Therefore, activation of ERK5 is required for the induction of c-Fos.These events were confirmed in experiments using M-CSF-dependent bone marrow macrophages.Taken together, the present results show that activation of the MEK5/ERK5 pathway with M-CSF is required for osteoclast differentiation, which may induce differentiation through the induction of c-Fos.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, Keyakidai, Sakado City, Japan.

ABSTRACT
The MEK/ERK pathways are critical for controlling cell proliferation and differentiation. In this study, we show that the MEK5/ERK5 pathway participates in osteoclast differentiation. ERK5 was activated by M-CSF, which is one of the essential factors in osteoclast differentiation. Inhibition of MEK5 by BIX02189 or inhibition of ERK5 by XMD 8-92 blocked osteoclast differentiation. MEK5 knockdown inhibited osteoclast differentiation. RAW264.7D clone cells, which are monocytic cells, differentiate into osteoclasts after stimulation with sRANKL. ERK5 was activated without any stimulation in these cells. Inhibition of the MEK5/ERK5 pathway by the inhibitors also blocked the differentiation of RAW264.7D cells into osteoclasts. Moreover, expression of the transcription factor c-Fos, which is indispensable for osteoclast differentiation, was inhibited by treatment with MEK5 or ERK5 inhibitors. Therefore, activation of ERK5 is required for the induction of c-Fos. These events were confirmed in experiments using M-CSF-dependent bone marrow macrophages. Taken together, the present results show that activation of the MEK5/ERK5 pathway with M-CSF is required for osteoclast differentiation, which may induce differentiation through the induction of c-Fos.

No MeSH data available.


Related in: MedlinePlus

MEK5 siRNA inhibited the formation of TRAP (+) MNCs in 4B12 cells.(A) 4B12 cells were transfected with GFP siRNA or MEK5 siRNA. The 4B12 cells (2.5 × 104) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 3 days, MEK5 gene expression was measured by qRT-PCR. Similar results were obtained in two independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA. (B) 4B12 cells (5 × 105) were transfected with siRNAs for GFP or MEK5. The levels of MEK1, 2, and 5 were analyzed by Western blot analysis. Similar results were obtained in two independent experiments. (C) 4B12 cells were transfected with siRNAs for GFP or MEK5. The cells were stimulated with M-CSF (10 ng/ml). After 30 min, the phosphorylation of ERK5 was monitored. Similar results were obtained in two independent experiments. (D) 4B12 cells (5 × 105) were transfected with GFP or MEK5 siRNAs. The transfected cells (5 × 103) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 6 days, the number of TRAP-positive MNCs was counted. Similar results were obtained in three independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA.
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pone.0125054.g004: MEK5 siRNA inhibited the formation of TRAP (+) MNCs in 4B12 cells.(A) 4B12 cells were transfected with GFP siRNA or MEK5 siRNA. The 4B12 cells (2.5 × 104) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 3 days, MEK5 gene expression was measured by qRT-PCR. Similar results were obtained in two independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA. (B) 4B12 cells (5 × 105) were transfected with siRNAs for GFP or MEK5. The levels of MEK1, 2, and 5 were analyzed by Western blot analysis. Similar results were obtained in two independent experiments. (C) 4B12 cells were transfected with siRNAs for GFP or MEK5. The cells were stimulated with M-CSF (10 ng/ml). After 30 min, the phosphorylation of ERK5 was monitored. Similar results were obtained in two independent experiments. (D) 4B12 cells (5 × 105) were transfected with GFP or MEK5 siRNAs. The transfected cells (5 × 103) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 6 days, the number of TRAP-positive MNCs was counted. Similar results were obtained in three independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA.

Mentions: We also tested the effects of MEK5 siRNA on the differentiation of 4B12 cells into TRAP (+) MNCs. Knockdown of MEK5 using a MEK5-specific siRNA selectively inhibited the expression of MEK5 at the mRNA and protein levels, but it did not affect the levels of MEK1 and MEK2 (Fig 4A and 4B). After M-CSF treatment, the phosphorylation of ERK5 was inhibited by MEK5 knockdown (Fig 4C). As expected, MEK5 knockdown suppressed the formation of TRAP (+) MNCs (Fig 4D). These results suggest that the MEK5/ERK5 pathway is required for the differentiation of 4B12 cells into osteoclasts.


ERK5 activation is essential for osteoclast differentiation.

Amano S, Chang YT, Fukui Y - PLoS ONE (2015)

MEK5 siRNA inhibited the formation of TRAP (+) MNCs in 4B12 cells.(A) 4B12 cells were transfected with GFP siRNA or MEK5 siRNA. The 4B12 cells (2.5 × 104) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 3 days, MEK5 gene expression was measured by qRT-PCR. Similar results were obtained in two independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA. (B) 4B12 cells (5 × 105) were transfected with siRNAs for GFP or MEK5. The levels of MEK1, 2, and 5 were analyzed by Western blot analysis. Similar results were obtained in two independent experiments. (C) 4B12 cells were transfected with siRNAs for GFP or MEK5. The cells were stimulated with M-CSF (10 ng/ml). After 30 min, the phosphorylation of ERK5 was monitored. Similar results were obtained in two independent experiments. (D) 4B12 cells (5 × 105) were transfected with GFP or MEK5 siRNAs. The transfected cells (5 × 103) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 6 days, the number of TRAP-positive MNCs was counted. Similar results were obtained in three independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA.
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pone.0125054.g004: MEK5 siRNA inhibited the formation of TRAP (+) MNCs in 4B12 cells.(A) 4B12 cells were transfected with GFP siRNA or MEK5 siRNA. The 4B12 cells (2.5 × 104) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 3 days, MEK5 gene expression was measured by qRT-PCR. Similar results were obtained in two independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA. (B) 4B12 cells (5 × 105) were transfected with siRNAs for GFP or MEK5. The levels of MEK1, 2, and 5 were analyzed by Western blot analysis. Similar results were obtained in two independent experiments. (C) 4B12 cells were transfected with siRNAs for GFP or MEK5. The cells were stimulated with M-CSF (10 ng/ml). After 30 min, the phosphorylation of ERK5 was monitored. Similar results were obtained in two independent experiments. (D) 4B12 cells (5 × 105) were transfected with GFP or MEK5 siRNAs. The transfected cells (5 × 103) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 6 days, the number of TRAP-positive MNCs was counted. Similar results were obtained in three independent experiments. *P<0.05 when compared with 4B12 cells transfected with GFP siRNA.
Mentions: We also tested the effects of MEK5 siRNA on the differentiation of 4B12 cells into TRAP (+) MNCs. Knockdown of MEK5 using a MEK5-specific siRNA selectively inhibited the expression of MEK5 at the mRNA and protein levels, but it did not affect the levels of MEK1 and MEK2 (Fig 4A and 4B). After M-CSF treatment, the phosphorylation of ERK5 was inhibited by MEK5 knockdown (Fig 4C). As expected, MEK5 knockdown suppressed the formation of TRAP (+) MNCs (Fig 4D). These results suggest that the MEK5/ERK5 pathway is required for the differentiation of 4B12 cells into osteoclasts.

Bottom Line: Therefore, activation of ERK5 is required for the induction of c-Fos.These events were confirmed in experiments using M-CSF-dependent bone marrow macrophages.Taken together, the present results show that activation of the MEK5/ERK5 pathway with M-CSF is required for osteoclast differentiation, which may induce differentiation through the induction of c-Fos.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, Keyakidai, Sakado City, Japan.

ABSTRACT
The MEK/ERK pathways are critical for controlling cell proliferation and differentiation. In this study, we show that the MEK5/ERK5 pathway participates in osteoclast differentiation. ERK5 was activated by M-CSF, which is one of the essential factors in osteoclast differentiation. Inhibition of MEK5 by BIX02189 or inhibition of ERK5 by XMD 8-92 blocked osteoclast differentiation. MEK5 knockdown inhibited osteoclast differentiation. RAW264.7D clone cells, which are monocytic cells, differentiate into osteoclasts after stimulation with sRANKL. ERK5 was activated without any stimulation in these cells. Inhibition of the MEK5/ERK5 pathway by the inhibitors also blocked the differentiation of RAW264.7D cells into osteoclasts. Moreover, expression of the transcription factor c-Fos, which is indispensable for osteoclast differentiation, was inhibited by treatment with MEK5 or ERK5 inhibitors. Therefore, activation of ERK5 is required for the induction of c-Fos. These events were confirmed in experiments using M-CSF-dependent bone marrow macrophages. Taken together, the present results show that activation of the MEK5/ERK5 pathway with M-CSF is required for osteoclast differentiation, which may induce differentiation through the induction of c-Fos.

No MeSH data available.


Related in: MedlinePlus