Inhibition of Osteoclast Differentiation and Promotion of Osteogenic Formation by Wolfiporia extensa Mycelium

Abstract

Osteoporosis, Greek for "porous bone," is a bone disease characterized by a decrease in bone strength, microarchitectural changes in the bone tissues, and an increased risk of fracture. An imbalance of bone resorption and bone formation may lead to chronic metabolic diseases such as osteoporosis. Wolfiporia extensa, known as "Bokryung" in Korea, is a fungus belonging to the family Polyporaceae and has been used as a therapeutic food against various diseases. Medicinal mushrooms, mycelium and fungi, possess approximately 130 medicinal functions, including antitumor, immunomodulating, antibacterial, hepatoprotective, and antidiabetic effects, and are therefore used to improve human health. In this study, we used osteoclast and osteoblast cell cultures treated with Wolfiporia extensa mycelium water extract (WEMWE) and investigated the effect of the fungus on bone homeostasis. Subsequently, we assessed its capacity to modulate both osteoblast and osteoclast differentiation by performing osteogenic and anti-osteoclastogenic activity assays. We observed that WEMWE increased BMP-2-stimulated osteogenesis by inducing Smad-Runx2 signal pathway axis. In addition, we found that WEMWE decreased RANKL-induced osteoclastogenesis by blocking c-Fos/NFATc1 via the inhibition of ERK and JNK phosphorylation. Our results show that WEMWE can prevent and treat bone metabolic diseases, including osteoporosis, by a biphasic activity that sustains bone homeostasis. Therefore, we suggest that WEMWE can be used as a preventive and therapeutic drug.

Keywords: Osteoporosis; Wolfiporia extensa; mycelium; osteoblasts; osteoclasts.

Fig. 1. Wolfiporia extensa mycelium water extract (WEMWE) impairs RANKL-mediated osteoclast differentiation.

(A) After overnight cell seeding, BMM cells were treated for 4 days with M-CSF (50 ng/ml) and RANKL (30 ng/ml) in the presence of WEMWE (0.01, 0.03, 0.1, and 0.3 μg/ml) or vehicle (dimethyl sulfoxide; DMSO). Multinucleated osteoclasts were visualized using TRAP staining. (B) TRAP-positive multinuclear cells were counted using an inverted microscope (left panel) and TRAP activity was measured using a spectrophotometer (right panel). *p < 0.05; **p < 0.01; ***p < 0.001 (versus vehicle control). (C) Effect of WEMWE on the viability of BMMs was evaluated using the CCK-8 assay.

Fig. 2. WEMWE inhibits RANKL-induced expression of c-Fos/NFATc1 by modulating ERK and JNK phosphorylation.

(A) Total RNA of BMM cells that had finished differentiation was isolated using TRIzol reagent and mRNA expression was measured using real-time PCR. Gapdh was used as an internal control. (B) The effect of WEMWE on the protein expression level of RANKL-induced transcription factors was evaluated using Western blot analysis. Actin was used as an internal control. (C) The indicated signaling molecules expression levels were quantified using Western blot analysis. Following serum starvation for 1 d, BMM cells were pre-treated with vehicle or WEMWE (0.3 μg/ml) for 1 h prior to RANKL stimulation (30 ng/ml) for the indicated times. Actin was used as an internal control. One representative result from three independent experiments yielding similar results is shown. The experiment was performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001 (versus vehicle control).

Fig. 3. WEMWE promotes BMP-2-induced osteoblast differentiation.

(A) After overnight cell seeding, C2C12 cells were treated for 4 days with vehicle (DMSO) or WEMWE (1, 3, 10, and 30 μg/ml) in the presence of BMP-2 (50 ng/ml). Osteoblast differentiation was visualized via alkaline phosphatase staining. (B) ALP activity was monitored by measuring absorbance at 405 nm. (C) Effect of WEMWE on the viability of C2C12 cells was evaluated using the CCK-8 assay.

Fig. 4. WEMWE stimulates BMP-2–induced expression of Runx2.

(A) C2C12 cells were stimulated in the presence of BMP-2 (50 ng/ml) with either vehicle (water) as a control or WEMWE (30 μg/ml) for the indicated times. The mRNA expression levels were assessed using real-time PCR. GAPDH was used as an internal control. The sample was performed triplicate. *p < 0.05; **p < 0.01; ***p < 0.001 (versus vehicle control). (B) Effects of WEMWE on the expression levels of Runx2 and ALP were evaluated by immunoblot analysis. GAPDH was used as an internal control. (C) WEMWE induces BMP-2– mediated phosphorylation of Smad signaling molecules. Following 24 h serum starvation, C2C12 cells were pre-treated with vehicle as a control or WEMWE (30 μg/ml) for 1 h prior to BMP-2 stimulation (50 ng/ml) for the indicated times. The expression levels of the signaling molecules were evaluated by Western blotting. Actin was used as an internal control. One representative result from three independent experiments yielding similar results is shown.