A marine fungus-derived nitrobenzoyl sesquiterpenoid suppresses receptor activator of NF-κB ligand-induced osteoclastogenesis and inflammatory bone destruction

Abstract

Background and purpose: Osteoclasts are unique cells to absorb bone. Targeting osteoclast differentiation is a therapeutic strategy for osteolytic diseases. Natural marine products have already become important sources of new drugs. The naturally occurring nitrobenzoyl sesquiterpenoids first identified from marine fungi in 1998 are bioactive compounds with a special structure, but their pharmacological functions are largely unknown. Here, we investigated six marine fungus-derived nitrobenzoyl sesquiterpenoids on osteoclastogenesis and elucidated the mechanisms.

Experimental approach: Compounds were first tested by RANKL-induced NF-κB luciferase activity and osteoclastic TRAP assay, followed by molecular docking to characterize the structure-activity relationship. The effects and mechanisms of the most potent nitrobenzoyl sesquiterpenoid on RANKL-induced osteoclastogenesis and bone resorption were further evaluated in vitro. Micro-CT and histology analysis were used to assess the prevention of bone destruction by nitrobenzoyl sesquiterpenoids in vivo.

Key results: Nitrobenzoyl sesquiterpenoid 4, with a nitrobenzoyl moiety at C-14 and a hydroxyl group at C-9, was the most active compound on NF-κB activity and osteoclastogenesis. Consequently, nitrobenzoyl sesquiterpenoid 4 exhibited suppression of RANKL-induced osteoclastogenesis and bone resorption from 0.5 μM. It blocked RANKL-induced IκBa phosphorylation, NF-κB p65 and RelB nuclear translocation, NFATc1 activation, reduced DC-STAMP but not c-Fos expression during osteoclastogenesis in vitro. Nitrobenzoyl sesquiterpenoid 4 also ameliorated LPS-induced osteolysis in vivo.

Conclusion and implications: These results highlighted nitrobenzoyl sesquiterpenoid 4 as a novel inhibitor of osteoclast differentiation. This marine-derived sesquiterpenoid is a promising lead compound for the treatment of osteolytic diseases.

Keywords: DC-STAMP; NF-κB; NFATc1; nitrobenzoyl sesquiterpenoids; osteoclast; osteolysis.

FIGURE 1

Inhibition of RANKL‐induced NF‐κB luciferase activity and osteoclastogenesis by nitrobenzoyl sesquiterpenoids (NS1–6 and 7), respectively. Structures of NS1–6 and 7 (a). RAW264.7 cells which had been transfected with an NF‐κB luciferase reporter construct were cultured with NS1–6, 7 (4 μM) or NF‐κB inhibitor BAY11‐7082 (5 μM) for 4 h, and then stimulated by RANKL (100 ng·ml⁻¹). Six hours later the luciferase activity (b) was measured. Cell viability of NS1–6 or 7 (4 μM) for 12 h in RAW264.7 cells (c) were measured by MTT assay. RAW264.7 cells treated with NS1–6 or 7 (1 μM) and RANKL for 5 days, TRAP‐positive multinucleated cells (nuclei >3, purple) were regarded as osteoclasts, some osteoclasts were indicated by red arrows (d). Quantification of osteoclasts treated with NS1–6 and 7 were shown (e). Cell viability of NS1–6 or 7 (1 μM) for 5 days (f) on RAW264.7 cells were measured by MTT assay. Values are expressed as the means ± SD (n = 5 independent experiments). ^# P < 0.05 relative to untreated controls, *P < 0.05 compared to RANKL‐treated controls. Scale bars, 500 μm

FIGURE 2

Molecular docking of nitrobenzoyl sesquiterpenoid 4 with NF‐κB p65. (a) Binding sites of the molecule nitrobenzoyl sesquiterpenoid 4 (NS4) with the NF‐κB p65 protein. Hydrophobic, polar and the exposed regions of the receptor are depicted in green, purple, and red colours, respectively. (b) The interaction details of the predicted binding mode of NS4 with p65. The contact residues are displayed and labelled by type and number, along with detailed interaction types, distance and energy

FIGURE 3

Nitrobenzoyl sesquiterpenoid 4 (NS4) suppresses RANKL‐induced osteoclast differentiation and functions. Representative images of osteoclasts from RAW264.7 cells treated with NS4 (0.5–2 μM) for 5 days, TRAP‐positive multinucleated cells (nuclei >3, purple) were regarded as osteoclasts and indicated by red arrows (a, left) and quantified (a, right). BMMs were cultured with M‐CSF (50 ng·ml⁻¹) and NS4 (0.5, 1 or 2 μM), then stimulated by 100 ng·ml⁻¹ of RANKL for 3 days. TRAP‐positive multinucleated cells (nuclei >5) were regarded as osteoclasts (b, left) and quantified (b, right). Representative images of bone resorption area on the hydroxyapatite‐coated surfaces by osteoclasts from pre‐osteoclastic RAW264.7 cells (c) or BMMs (d) are shown. The release of Ca⁺⁺ from the hydroxyapatite‐coated plates to the culture medium after 2 μM NS4 administration were also measured (e). The resorption by osteoclasts on bone slice was also inhibited by 2‐μM NS4 (f). The resorption area by osteoclasts from RAW264.7 cells (c, right) or BMMs (d, f, right) was quantified as a percentage to the total area of hydroxyapatite‐coated surface or bone slice surface, and some resorption areas are indicated by red arrows. Cell viability of NS4 (g) at 0.5–2 μM in RAW264.7 cells for 5 days were measured by MTT assay. Cell viability of NS4 at different concentrations on BMMs for 24 h (h) and 5 days (i) were measured by cellcounting kit 8 assay. Values are expressed as the means ± SD (n = 5 independent experiments). ^# P < 0.05 relative to untreated controls, *P < 0.05 relative to RANKL‐treated controls. Scale bars, 500 μm

FIGURE 4

Nitrobenzoyl sesquiterpenoid 4 (NS4) suppresses NF‐κB signalling pathway induced by RANKL. RAW264.7 cells stably transfected with an NF‐κB luciferase reporter construct were cultured with NS4 (0.5, 1, and 2 μM) for 4 h and then stimulated by RANKL, 6 h later the luciferase activity (a) was measured. RAW264.7 cells were cultured with NS4 (0.5, 1, or 2 μM) for 4 h, and then stimulated by RANKL (100 ng·ml⁻¹) for 30 min. Total proteins, cytosolic, and nuclear proteins were extracted and analysed by western blotting using antibodies against p‐IκBa, β‐actin, p65, RelB, and lamin A/C. The relative protein or nuclear protein expression levels of p‐IκBa (b, c) and p65 (d, e) to β‐actin, or p65 (f, g) and RelB (i, j) to lamin A/C were determined using ImageJ software. RAW264.7 cells were cultured with NS4 (2 μM) for 4 h and then stimulated by RANKL (100 ng·ml⁻¹) for 30 min. The nuclear translocation of NF‐κB p65 (h) was imaged by immunofluorescence analysis. Values are expressed as the means ± SD (n = 5 independent experiments). ^# P < 0.05, relative to untreated controls, *P < 0.05 relative to RANKL‐treated controls. Scale bars, 10 μm

FIGURE 5

Nitrobenzoyl sesquiterpenoid 4 (NS4) prevents RANKL‐induced NFATc1 but not c‐Fos activation. RAW264.7 cells were treated with NS4 (0.5, 1 or 2 μM) for 4 h, followed by the stimulation with 100 ng·ml⁻¹ of RANKL for 24 h. Nuclear proteins were then extracted and analysed by western blotting using antibodies against c‐Fos, lamin A/C, β‐actin and NFATc1. The relative protein expression of c‐Fos (a, b) and NFATc1 (e, f) to lamin A/C were determined using ImageJ software. RAW264.7 cells, which had been transfected with NFATc1 luciferase reporter construct, were treated with NS4 (0.5–4 μM) and cyclosporin A (CsA; 1 μM) for 4 h, and then stimulated by 100 ng·ml⁻¹ of RANKL. After 24 h, the luciferase activity (c) was detected. Expression of NFATc1 mRNA (d) followed by RANKL stimulation for 24 h was examined. Values are expressed as the means ± SD (n = 5 independent experiments). ^# P < 0.05, relative to untreated controls, *P < 0.05 relative to RANKL‐treated controls

FIGURE 6

Nitrobenzoyl sesquiterpenoid 4 (NS4) suppresses RANKL‐induced osteoclast‐related genes and DC‐STAMP. RAW264.7 cells were treated with NS4 (0.5, 1, and 2 μM) for 4 h, and then stimulated by 100 ng·ml⁻¹ of RANKL for 24 h. The expression of osteoclast‐related genes TRAP, β3‐integrin (a) and DC‐STAMP (b) were analysed by using real time‐PCR. Total proteins were extracted and analysed by western blotting after RANKL stimulation for 48 h using antibodies against DC‐STAMP and β‐actin (c, d). The protein expression level of DC‐STAMP relative to β‐actin was established using ImageJ software. Values are expressed as the means ± SD (n = 5 independent experiments). ^# P < 0.05 relative to untreated controls, *P < 0.05 relative to RANKL‐treated controls

FIGURE 7

NF‐κB p65 R246A mutation reduces the nuclear translocation of p65 and the inhibition of nitrobenzoyl sesquiterpenoid 4 (NS4) on NFATc1‐Luc activity during osteoclastogenesis. RAW264.7 cells, transfected with NF‐κB p65 R246A (mut) or control WT plasmids (wt) or empty vector, were stimulated with or without RANKL (100 ng·ml⁻¹) for 30 min. Proteins were extracted and analysed by western blotting using antibodies against β‐actin, p65 and lamin A/C. The expression levels of total p65 protein (a, b), cytoplasmic p65 protein to β‐actin (c, d), or nuclear p65 protein level (e, f) to lamin A/C were determined using ImageJ software. RAW264.7 cells, which stably transfected with a NFATc1 luciferase reporter construct, were transiently transfected with NF‐κB p65 R246A or control WT plasmids and stimulated by 100 ng·ml⁻¹ of RANKL. After 24 h, the NFATc1 luciferase activity (g) was detected. Values are expressed as the means ± SD (n = 5 independent experiments). ^# P < 0.05,relative to unstimulated controls, *P < 0.05, relative to vector or RANKL‐treated controls

FIGURE 8

Nitrobenzoyl sesquiterpenoid 4 (NS4) prevents inflammatory bone loss induced by LPS in vivo. Groups of mice injected with PBS (n = 6) or LPS (n = 6) but without treatment with NS4 or injected with LPS and treated with 1 mg·kg⁻¹ (n = 6) or 5 mg·kg⁻¹ NS4 (n = 6) were used. Representative 3D reconstructions of transverse (a, above) and longitudinal (a, below) sections of femur from each group by micro‐CT were shown. The parameters of trabecular bone including BMD, BV/TV, Tb. Sp, Tb. N, Cor. Th, Tb. Th and ConnD (b) were analysed. Sections of femur from each group were stained with H&E (c, above) and TRAP (c, middle and below). BV/TV (d), number of osteoclasts (e), and osteoclast surface/bone surface (f) were analysed. In addition, three groups (n = 5) of mice were treated with PBS, LPS and NS4 (5 mg·kg⁻¹) as described above and i.p. injected calcein 7 and 3 days prior to killing. Bone formation indicated by calcein staining was examined by fluorescence microscopy (g) and MAR was calculated (h). ^# P < 0.05 relative to PBS treated controls, *P < 0.05 relative to LPS treated controls