Methyl Ganoderic Acid DM: A Selective Potent Osteoclastogenesis Inhibitor

Increased osteoclastic bone resorption plays a central role in the pathogenesis of many bone diseases, and osteoclast inhibitors are the most widely used treatments for these diseases. Ganoderic acid DM, the main component of Ganoderma lucidum, has been known for its medicinal effects such as anti-androgen and anti-proliferative activities. In this study, we investigated the inhibitory effects of ganoderic acid DM and its analog (methyl ganoderic acid DM and 7-oxo-methyl ganoderic acid Z) on osteoclastogenesis using RAW264 cell in vitro. Methyl ganoderic acid DM blocked osteoclastogenesis completely at 12.5 μM with low cytotoxicity less than 30%. On the other hands, ganoderic acid DM blocked osteoclastogenesis completely at the higher concentration of 50 μM, but 7-oxo-methyl ganoderic acid Z did not up to 100 μM. These results implicated the carbonyl group at C-3 is essentially for selective osteoclastogenesis inhibitory activity, and methyl esters at C-26 should play an important role in enhancing its osteoclastogenesis inhibitory activity

Direct stimulation of osteoclastogenesis by MIP-1alpha: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.