Placotylene A, an inhibitor of the receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation, from a Korean sponge Placospongia sp

Abstract

A new inhibitor, placotylene A (1), of the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, and a regioisomer of placotylene A, placotylene B (2), were isolated from a Korean marine sponge Placospongia sp. The chemical structures of placotylenes A and B were elucidated on the basis of 1D and 2D NMR, along with MS spectral analysis and revealed as an iodinated polyacetylene class of natural products. Placotylene A (1) displayed inhibitory activity against RANKL-induced osteoclast differentiation at 10 μM while placotylene B (2) did not show any significant activity up to 100 μM, respectively.

Figure 1

The chemical structures of placotylenes A and B.

Figure 2

COSY (bold) and key HMBC (arrow) correlations of 1.

Figure 3

Effect of placotylene A (1) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. (A) Bone marrow-derived macrophages (BMMs) were cultured for 4 days with RANKL (10 ng/mL) and macrophage-colony stimulating factor (M-CSF) (30 ng/mL) in the presence of different concentrations of 1. The 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 (nuclei ≥3). ** P <0.01, *** P <0.001. (C) Cytotoxicity of 1 on BMMs. The effect of 1 on the viability on BMMs was evaluated using the CCK-8 assay.

Figure 4

Placotylene A (1) abolishes RANKL-stimulated NFATc1 transcriptional and translational expression. (A) BMMs were pretreated with DMSO or 1 (10 μM) for 30 min and then stimulated with RANKL (10 ng/mL) for the indicated number of days. The expressed mRNA levels were analyzed by comparing real-time PCR with the DMSO control. ** P < 0.05, *** P < 0.005. (B) BMMs were pretreated with DMSO or 1 (10 μM) for 1 h and then stimulated with RANKL (10 ng/mL) for the indicated time. The cell lysates were analyzed by western blotting with anti-NFATc1, and anti-actin antibodies as indicated.

Figure 5

Placotylene A (1)-mediated inhibition of osteoclast differentiation was restored by NFATc1 over-expression. (A) BMMs were transduced with the indicated retroviruses harboring vehicle with GFP tag or Ca-NFATc1 expression construct with GFP tag using a retroviral method for 24 h. Infected BMMs were cultured with M-CSF (30 ng/mL) and RANKL (10 ng) for 4 days in the presence or absence of placotylene A (10 μM). The expressed GFP of the infected cells was visualized by fluorescence microscope. (B) TRAP-positive multinucleated osteoclasts were visualized by TRAP staining. (C) TRAP positive cells were counted as osteoclasts (nuclei ≥ 3). ** P < 0.01. (D) TRAP activity was measured at 405 nm. ** P < 0.01.