J Bone Metab.  2020 Feb;27(1):53-63. 10.11005/jbm.2020.27.1.53.

Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca2⁺-Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression

• Park KH
• Gu DR
• Kim MS
• Lee SH

• KMID: 2471314
• DOI: http://doi.org/10.11005/jbm.2020.27.1.53

Abstract

BACKGROUND
Rosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated.
METHODS
The effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RANKL-induced Ca2⁺-oscillation was also investigated.
RESULTS
RMF-E remarkably inhibited TRAP+-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca2⁺-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2.
CONCLUSIONS
RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.

Keyword

Osteoclasts; Bone diseases; NFATc1; Calcium signaling; Osteogenesis

MeSH Terms

Acid Phosphatase
Antigens, Differentiation
Arthritis
Arthritis, Rheumatoid
Blotting, Western
Bone Diseases
Calcium Signaling
Cytoplasm*
Far East
In Vitro Techniques
Osteoclasts
Osteogenesis
Osteoporosis
Periodontitis
Phospholipases
Protein Kinases
Protein-Tyrosine Kinases
Real-Time Polymerase Chain Reaction
Rosa*
T-Lymphocytes*
Transcription Factors
Acid Phosphatase
Antigens, Differentiation
Phospholipases
Protein Kinases
Protein-Tyrosine Kinases
Transcription Factors

Figure

• 

  Fig. 1 Effects of Rosae Multiflorae fructus extract (RMF-E) on cell viability. (A) Bone marrow-derived macrophages (BMMs) were cultured with the indicated concentrations of RMF-E, in the presence of macrophage colony-stimulating factor (M-CSF) (30 ng/mL) for 1 day. (B) BMMs were cultured with/without 30 µg/mL RMF-E, in the presence of M-CSF, for 4 days. Cell viability was measured as described in the materials and methods. Data are presented as the mean±standard deviation of 3 independent experiments. NS, not significant.

• 

  Fig. 2 Effect of Rosae Multiflorae fructus extract (RMF-E) on osteoclast differentiation and resorption activity. Bone marrow-derived macrophages were cultured with the indicated concentrations of RMF-E, under receptor activator of nuclear factor-κB ligand (100 ng/mL) and macrophage-colony stimulating factor (30 ng/mL) treatment, for 4 days. (A) Osteoclasts were fixed and stained for tartrate-resistant acid phosphatase (TRAP). (B) TRAP+-multinuclear cells with ≥3 nuclei were counted as mature osteoclasts. (C) Total TRAP activity from TRAP+-mono-, di-, and multi-nuclear cells were measured at an absorbance of 405 nm (A405 nm). (D) F-actin rings were stained with rhodamine-phalloidin in osteoclasts treated with/without RMF-E (20 µg/mL). (E) The resorption activity of osteoclasts treated with/without RMF-E (20 µg/mL) was measured by pit formation on hydroxyapatite-coated plates, after 7 days. Data are expressed as the mean±standard deviation and are representative of 3 independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs. the non-treated control (NC, 0 µg/mL RMF-E) (Scale bar=200 µm). NS, not significant.

• 

  Fig. 3 Effects of Rosae Multiflorae fructus extract (RMF-E) on the expression of osteoclast differentiation marker genes and transcription factors. Bone marrow-derived macrophages were cultured with receptor activator of nuclear factor-κB ligand (RANKL) (100 ng/mL) and macrophage colony-stimulating (M-CSF) treatment in the presence or absence of RMF-E (20 µg/mL) for 4 days or the indicated times (for c-Fos). (A) Osteoclast differentiation marker gene expression was examined by real-time polymerase chain reaction. Messenger RNA (mRNA) levels were normalized with Gapdh and expressed as fold change of mRNA level. (B, C) Whole lysate (30 µg) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting. c-Fos (B) and nuclear factor-activated T cells c1 (NFATc1) (C) expression were detected using the anti-c-Fos and NFATc1 antibody, respectively. Fold change normalized by actin is presented in the lower panel. Data are expressed as the mean±standard deviation and are representative of 3 independent experiments. *P<0.05 and **P<0.01 vs. the control group (0 µg/mL RMF-E).

• 

  Fig. 4 Effects of Rosae Multiflorae fructus extract (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-induced intracellular signaling. Bone marrow-derived macrophages (BMMs) were pretreated with/without RMF-E (20 µg/mL) for 2 hr in the presence of macrophage colony-stimulating factor (M-CSF) (30 ng/mL), and then RANKL (100 ng/mL)-treated, to stimulate intracellular signaling, at indicated times. Lysate (30 µg) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting. (A) The activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK], c-JUN N-terminal kinase [JNK], and p38) and IκBα was examined using their respective antibodies. (B) Twenty four-hr RANKL-stimulated BMMs were acutely treated with RMF-E (20 µg/mL). RANKL-stimulated Ca2+-oscillation by was measured using the fluorescence Ca2+ indicator (Fura-2, AM). (C) Bruton's tyrosine kinase (BTK) and phospholipase C-γ2 (PLCγ2) activation were detected using the anti p-BTK/BTK and p-PLCγ2/PLCγ2 antibody, respectively. Fold change normalized by their non-phosphorylated proteins is presented in the right panel. Data are expressed as mean±standard deviation and are representative of 3 independent experiments. *P<0.05 and **P<0.01 vs. the control group (0 µg/mL RMF-E).

• 

  Fig. 5 Schematic diagram of the effect of Rosae Multiflorae fructus extract (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. The RANKL/RANK interaction may lead to mitogen-activated protein kinases (MAPKs) activation, followed by induction of c-Fos expression, and it also leads to Bruton's tyrosine kinase (BTK) and phospholipase C-γ2 (PLCγ2) activation inducing calcium signaling, followed by induction of nuclear factor-activated T cells c1 (NFATc1) expression and activation. RMF-E may inhibit NFATc1 induction via suppression of c-Fos and modulation of the BTK/PLCγ signaling pathways regulating Ca2+ signaling, resulting in the inhibition of RANKL-induced osteoclastogenesis. The red line indicates the inhibition pathway of RMF-E. TRAF6, tumor necrosis factor receptor associated factor 6; ITAM, immunoreceptor tyrosine-based activation motif.

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