J Bone Metab.  2016 Nov;23(4):207-214. 10.11005/jbm.2016.23.4.207.

Attenuation of RANKL-induced Osteoclast Formation via p38-mediated NFATc1 Signaling Pathways by Extract of Euphorbia Lathyris L

• Kang JH
• Lim H
• Jeong JE
• Yim M

• KMID: 2361582
• DOI: http://doi.org/10.11005/jbm.2016.23.4.207

Abstract

BACKGROUND
Osteoclasts are the only cell type capable of breaking down bone matrix, and its excessive activation is responsible for the development of bone-destructive diseases. Euphorbia lathyris L. (ELL) is an herbal plant that belongs to the Euphorbiaceae family. This study investigated the effects of the methanol extract of the aerial part of ELL on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and signaling pathways.
METHODS
Osteoclasts were formed by co-culturing mouse bone marrow with osteoblasts or by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The expression level of mRNA was analyzed by real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR. Western blotting assays were performed to detect the expression or activation level of proteins.
RESULTS
ELL inhibited RANKL-induced osteoclast formation without cytotoxicity. Furthermore, the RANKL-stimulated bone resorption was diminished by ELL. Mechanistically, ELL blocked the RANKL-triggered p38 mitogen-activated protein kinase (MAPK) phosphorylation, which resulted in the suppression of the expression of c-Fos and nuclear factor of activated T cells (NFATc1). In osteoblasts, ELL had little effect on the mRNA expression of RANKL and osteoprotegerin (OPG).
CONCLUSIONS
The present data suggest that ELL has an inhibitory effect on osteoclast differentiation and function via downregulation of the p38/c-Fos/NFATc1 signaling pathways. Thus, ELL could be useful for the treatment of bone diseases associated with excessive bone resorption.

Keyword

Euphorbia; Genes fos; Nfatc1 protein mouse; Osteoclasts; p38 mitogen-activated protein kinases

MeSH Terms

Animals
Blotting, Western
Bone Diseases
Bone Marrow
Bone Matrix
Bone Resorption
Dentin
Down-Regulation
Euphorbia*
Euphorbiaceae
Humans
Macrophage Colony-Stimulating Factor
Macrophages
Methanol
Mice
Osteoblasts
Osteoclasts*
Osteoprotegerin
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Plants
Protein Kinases
RANK Ligand
Real-Time Polymerase Chain Reaction
Reverse Transcription
RNA, Messenger
T-Lymphocytes
Macrophage Colony-Stimulating Factor
Methanol
Osteoprotegerin
Protein Kinases
RANK Ligand
RNA, Messenger
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Euphorbia lathyris L. (ELL) inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and bone resorption. (A) Bone marrow-derived macrophages (BMMs) were cultured with RANKL (100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence of the indicated concentration of methanol extract of ELL for four days and tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated osteoclasts (MNCs) were counted. (B) BMMs were cultured with M-CSF (30 ng/mL) in the presence or absence of methanol extract of ELL (50 µg/mL) for 48 hr and a microtitration (MTT) assay was performed. (C, D) In BMM cultures, mRNA expression level was determined by reverse transcription (RT)-polymerase chain reaction (PCR) (C) or real-time PCR (D). (E) BMMs were differentiated on dentine slices with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for four days and methanol extract of ELL (50 µg/mL) was treated for an additional two days. The number of resorption pits were counted. Scale bar=200 µm. Data are expressed as mean±standard deviation from at least three independent experiments. Veh, vehicle; R, receptor activator of nuclear factor-kappa B ligand (RANKL); CTR, calcitonin receptor; CTK, cathepsin K; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05.

• 

  Fig. 2 Euphorbia lathyris L. (ELL) inhibits osteoclast formation in the co-culture system. (A) Mouse bone marrow cells and calvarial osteoblasts were co-cultured with vehicle or methanol extract of ELL (50 µg/mL) in the presence of 1α,25-dihydroxy-vitamin D3 (1α,25-[OH]2D3 10 nM) for seven days, and the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). TRAP-positive (TRAP+) multinucleated osteoclasts (MNCs) containing three or more nuclei were counted as osteoclasts. (B, C) Mouse calvarial osteoblasts were pretreated with methanol extract of ELL (50 µg/mL) for 30 min and then cultured with 1α,25-(OH)2D3 for 24 hr. The mRNA expression level was determined by real-time polymerase chain reaction (PCR) using specific primers for (B) receptor activator of nuclear factor-kappa B ligand (RANKL) and (C) osteoprotegerin (OPG). Data are expressed as mean±standard deviation from at least three independent experiments. Veh, vehicle; VitD3, 1α,25-dihydroxy-vitamin D3 (1α,25-[OH]2D3); GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05.

• 

  Fig. 3 Euphorbia lathyris L. (ELL) inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced expression of c-Fos and nuclear factor of activated T cells (NFATc1) via p38 mitogen-activated protein kinase (MAPK) activation. (A-E) Bone marrow-derived macrophages (BMMs) were preincubated in the absence or presence of methanol extract of ELL (50 µg/mL) for 30 min, and then treated with or without 100 ng/mL of RANKL for 24 hr (for NFATc1 and c-Fos) or 15 min (for p-p38, p-extracellular signal-regulated kinase [ERK], and inhibitor of kappa B [IκB]). Cell lysates were then subjected to western blot analysis using (A) NFATc1, (B) c-Fos, (C) p-p38, (D) p-ERK, or (E) IκB antibodies. Data are expressed as mean±standard deviation from at least three independent experiments. Veh, vehicle; NFATc1, nuclear factor of activated T cells; RANKL, receptor activator of nuclear factor-kappa B ligand. *P<0.05.

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