J Bone Metab.  2012 Nov;19(2):121-127. 10.11005/jbm.2012.19.2.121.

Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation

• Kim JY
• Kim YK
• Choi MK
• Oh J
• Kwak HB
• Kim JJ

• KMID: 1975985
• DOI: http://doi.org/10.11005/jbm.2012.19.2.121

Abstract

OBJECTIVES
Osteoporosis is a disease of bones that is thought to result from an imbalance between bone resorption and bone formation. Although osteoporosis itself has no symptoms, osteoporosis caused by osteoclasts leads to an increased risk of fracture. Here we examined the effects of cornus officinalis on receptor activator of nuclear factor-kappaB ligand (RANKL)-mediated osteoclast differentiation.
METHODS
We evaluated the effects of cornus officinalis on RANKL-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs) and performed a cytotoxicity assay, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot analysis.
RESULTS
Cornus officinalis significantly inhibits RANKL-mediated osteoclast differentiation in a dose-dependent manner, but without cytotoxicity against BMMs. The mRNA expression of tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in BMMs treated with RANKL was considerably inhibited by cornus officinalis treatment. Also, cornus officinalis inhibits the protein expression of c-Fos and NFATc1. Cornus officinalis greatly inhibits RANKL-induced phosphorylation of p38 and c-JUN N-terminal kinase (JNK). Also, cornus officinalis significantly suppresses RANKL-induced degradation of I-kappaB.
CONCLUSIONS
Taken together, our results suggest that cornus officinalis may be a useful the treatment of osteoporosis.

Keyword

Cornus officinalis; Osteoclast; Osteoporosis; RANKL

MeSH Terms

Acid Phosphatase
Blotting, Western
Bone Resorption
Cornus
Cytoplasm
Isoenzymes
JNK Mitogen-Activated Protein Kinases
Macrophages
Osteoclasts
Osteogenesis
Osteoporosis
Phosphorylation
RANK Ligand
Receptor Activator of Nuclear Factor-kappa B
RNA, Messenger
T-Lymphocytes
Acid Phosphatase
Isoenzymes
JNK Mitogen-Activated Protein Kinases
RANK Ligand
RNA, Messenger
Receptor Activator of Nuclear Factor-kappa B

Figure

• 

  Fig. 1 Cornus officinalis inhibits osteoclast differentiation. (A) Bone marrow macrophages (BMMs) were cultured for 4 d with macrophage colony-stimulating factor (M-CSF; 30 ng/mL) and receptor activator of nuclear factor-kappaB ligand (RANKL; 100 ng/mL) in the presence or absence of Cornus officinalis. After 4 d, cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton x-100, and stained for tartrate-resistant acid phosphatase (TRAP). (B) TRAP-positive cells were counted as osteoclasts. (C) BMMs were cultured 3 d with M-CSF (30 ng/mL) in the presence or absence of Cornus officinalis. After 3 d, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2 tetrazolium hydroxide (XTT) solution was added to each well and then incubated for 4-6 h. The plate was measured at 450 nm using a microplate reader. Experiments were performed in triplicate and similar results were obtained in two independent experiments.

• 

  Fig. 2 Cornus officinalis inhibits osteoclast-specific gene expression. bone marrow-derived macrophages (BMMs) were treated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. cDNA was synthesized by using total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using cDNA. PCR products were separated by electrophoresis in an agarose gel and visualized with ethidium bromide staining. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide; TRAP, tartrate-resistant acid phosphatase; OSCAR, osteoclast-associated receptor; NFATc1, nuclear factor of activated T cells cytoplasmic 1, GAPDH, glyceraldehyde-3-phosphate dehydrogenase

• 

  Fig. 3 Cornus officinalis inhibits the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Bone marrow-derived macrophages (BMMs) were pretreated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. The cells were lysed and proteins were revolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide

• 

  Fig. 4 Cornus officinalis inhibits receptor activator of nuclear factor-kappaB ligand (RANKL) signaling pathways. Bone marrow-derived macrophages (BMMs) were pretreated with or without Cornus officinalis (100 µg/mL) and further stimulated with RANKL (100 n/mL) for the indicated time. The cells were lysed and proteins were revolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide; JNK, c-JUN N-terminal kinase

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