Fumitremorgin C Attenuates Osteoclast Formation and Function via Suppressing RANKL-Induced Signaling Pathways

Abstract

Excessive bone resorption conducted by osteoclasts is considered as the main cause of osteoclast-related bone diseases such as osteoporosis. Therefore, the suppression of excessive osteoclast formation and function is one of the strategies to treat osteoclast-related bone diseases. Fumitremorgin C (Fum) is a mycotoxin extracted from Aspergillus fumigatus. It has been shown to have extensive pharmacological properties, but its role in the treatment of osteoclast-related bone diseases remains unclear. In this study, we aim to find out whether Fum can inhibit the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and function. The results showed that Fum could significantly attenuate osteoclast formation and function at concentrations from 2.5 to 10 µM. The protein expression of bone resorption factors such as NFATc1, cathepsin K, V-ATPase-d2, and c-Fos was suppressed with the treatment of Fum at a concentration of 10 µM. In addition, Fum was also shown to suppress the activity of NF-κB, intracellular reactive oxygen species level, and MAPK pathway. Taken together, the present study showed that Fum could attenuate the formation and function of osteoclast via suppressing RANKL-induced signaling pathways, suggesting that Fum might be a potential novel drug in the treatment of osteoclast-related bone diseases.

Keywords: MAPK; NFATc1; fumitremorgin C; osteoclast; osteoporosis.

Figure 1

Fum attenuates RANKL-induced osteoclastogenesis. (A) Quantification of TRAcP-positive osteoclasts under varying concentrations of Fum (n = 3). **p < 0.01 relative to the Fum-untreated control. (B) Representative images of osteoclasts under the treatment of different concentrations of Fum for 5 days. (C) Proliferation of BMMs treated with Fum at varying concentrations for 48 h was detected using the MTS assay (n = 3). (D) The average area of the actin belt was measured via Image-J (n = 3). **p < 0.01 relative to the Fum-untreated control. (E) Representative images of the actin belt of osteoclasts were captured via confocal microscopy. Scale bar = 100 µm. Fum, fumitremorgin C; TRAcP, tartrate-resistant acid phosphatase; BMMs, bone marrow macrophages; RANKL, receptor activator of nuclear factor-κB ligand.

Figure 2

Fum principally inhibits RANKL-induced osteoclast formation at the middle stage. (A) Representative images of osteoclasts under the treatment of 10 μM Fum in different stages during the process of osteoclastogenesis. (B) The time periods in the presence of Fum. (C) The number of TRAcP-positive osteoclasts under the treatment of Fum was counted (n = 3). **p < 0.01 relative to the Fum-untreated control. Scale bar = 100 μm. Fum, fumitremorgin C; RANKL, receptor activator of nuclear factor-κB ligand; TRAcP, tartrate-resistant acid phosphatase.

Figure 3

Fum suppresses the resorptive function of osteoclast. (A) Representative images of TRAcP-positive multinucleated osteoclasts under the treatment of Fum in the hydroxyapatite-coated plates. (B) The number of TRAcP-positive osteoclasts under the treatment of Fum. n = 3. *p < 0.05, **p < 0.01 relative to the Fum-untreated control. (C) Representative images of osteoclast resorptive function on hydroxyapatite-coated surfaces. (D) The percentage of osteoclast resorptive area on hydroxyapatite-coated surfaces. n = 3. **p < 0.01 relative to the Fum-untreated control. Scale bars = 100 μm. Fum, fumitremorgin C; RANKL, receptor activator of nuclear factor-κB ligand; TRAcP, tartrate-resistant acid phosphatase.

Figure 4

Fum suppresses ROS generation induced by RANKL. (A) Representative images of ROS generation in BMMs in the absence or presence of Fum. Scale bar = 100 μm. (B) Quantification of ROS-positive cells per field (n = 3). (C) Quantitative analyses of fluorescence intensity averaged on osteoclast precursors (n = 3). **p < 0.01 relative to the Fum-untreated control. Fum, fumitremorgin C; ROS, reactive oxygen species; RANKL, receptor activator of nuclear factor-κB ligand; TRAcP, tartrate-resistant acid phosphatase.

Figure 5

Fum attenuates osteoclast formation through inhibiting NF-κB activity and MAPK signaling. (A) The results of NF-κB luciferase reporter assay showed that Fum significantly suppressed NF-κB activity at the concentrations of 5 µM Fum and higher. The luciferase activity of NF-κB was detected by a BMG luminescence reader (n = 3). **p < 0.01 relative to the Fum-untreated control. (B) Representative WB images of the inhibiting effects of Fum on MAPK signaling pathways including P38, ERK, and JNK. BMMs were pretreated with Fum at 10 μM for 60 min and then cultured in the complete medium containing RANKL for 0, 10, 20, 30, and 60 min. The total protein was extracted after intervention. (C) The ratios of IκBα band intensity relative to the β-actin bands. n = 3. (D–F) The ratios of p-P38, p-ERK, and p-JNK band intensity relative to total P38, ERK, and JNK bands (n = 3). The intensity of the bands were analyzed using Image-J. *p < 0.05, **p < 0.01 relative to the Fum-untreated control. Fum, fumitremorgin C; RANKL, receptor activator of nuclear factor-κB ligand; NF-κB, nuclear factor-κB; MAPK, mitogen-activated protein kinases; BMMs, bone marrow macrophages; WB, western blot; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Figure 6

Fum suppresses NFATc1 activity, the upstream and downstream proteins of NFATc1. (A) The results of the NFATc1 luciferase assay showed that Fum obviously inhibited the activity of NFATc1 dose-dependently. The luciferase activity of NFATc1 was detected by a BMG luminescence reader (n = 3). *p < 0.05, **p < 0.01 relative to the Fum-untreated control. (B) Representative WB images of NFATc1 and its upstream and downstream proteins containing cathepsin K, c-Fos, and V-ATPase-d2. (C–F) The ratios of the band intensity of NFATc1, c-Fos, V-ATPase-d2, and cathepsin K relative to β-actin (n = 3). The intensity of the bands was analyzed through Image-J. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the Fum-untreated control. Fum, fumitremorgin C; NFATc1, nuclear factor of activated T cells 1; RANKL, receptor activator of nuclear factor-κB ligand; WB, western blot.

Figure 7

The model for inhibiting the effects of Fum on RANKL-induced NFATc1 activation during osteoclastogenesis. ROS production and the NF-κB and MAPKs pathways are activated after RANKL binding to RANK, leading to the increase of c-Fos and the activation of NFATc1. As a result, the bone resorption molecules, including cathepsin K and V-ATPase-d2, are up-regulated. The present study showed for the first time that Fum attenuates RANKL-induced osteoclastogenesis and bone resorption via inhibiting the activation of NFATc1, indicating a mechanism for inhibiting the effects of Fum on RANKL-induced osteoclasts formation and function. Fum, fumitremorgin C; ROS, reactive oxygen species; RANKL, receptor activator of nuclear factor-κB ligand; NFATc1, nuclear factor of activated T cells 1; NF-κB, nuclear factor-κB; MAPK, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.