Reveromycin A‐Induced Apoptosis in Osteoclasts Is Not Accompanied by Necrosis

Abstract

Reveromycin A (RM-A), a small natural product isolated from Streptomyces bacteria, is a potential osteoporosis therapeutic in that it specifically induces apoptosis in osteoclasts but not osteoblasts. The purpose of the study presented here was to further elucidate the intracellular mechanisms of RM-A death effects in mature osteoclasts. A specific clone of RAW264.7 murine macrophages that was previously characterized for its ability to acquire an osteoclast nature on differentiation was differentiated in the presence of receptor activator of nuclear factor kappa B ligand (RANKL). Subsequent staining was performed for tartrate-resistant acid phosphatase to confirm their osteoclast character. These osteoclasts were treated with ten micromolar RM-A for 2, 4, 6, 24, and 48 h at a pH of 5.5. Peak apoptosis induction occurred at 4-6 h as measured by caspase 3 activity. Lactate dehydrogenase release assay revealed no significant RM-A-induced necrosis. Western blot analysis of cytoplasmic extracts demonstrated activation of caspase 9 (2.3-fold at 2 h and 2.6-fold at 4 h, each P < 0.05) and no significant changes in Bcl-XL . In nuclear extracts, NFκB levels significantly increased on differentiation with RANKL but then remained constant through RM-A treatment. Over the extended time course studied, RM-A-induced apoptosis in osteoclasts was not accompanied by necrosis, suggesting that RM-A would likely have limited effects on immediate, neighboring bone cell types. This specific cell death profile is promising for potential clinical investigations of RM-A as a bone antiresorptive.

Death effects of reveromycin A in normal and disease‐associated cells of the joint

“…The negative values suggest greater solubility in the aqueous phase and hence lower lipophilicity at these pH's . The logD value of +2.39 for Rev A at a pH of 5.5 is consistent with Rev A‐induced apoptosis in osteoclasts as osteoclasts were tested at this pH, a pH reflecting that observed in vivo at the bone‐osteoclast interface . Osteoclast apoptosis was not observed at neutral pH .…”

“…Synovial fluid pH in an inflamed, arthritic joint can reach a moderately acidified state of pH 6.8 . In an effort to reflect the acidic environment of the inflamed joint as well as an acidic environment thought to be important for REV A entry into and action within osteoclasts, normal chondrocytes were treated with REV A at a pH of 6.8. As predicted ethanol induced a rounded, apoptotic cell morphology (Figure A) that quantitatively represented an eightfold increase in caspase 3 activity ( P < 0.05, Figure B).…”

“…25 The logD value of +2.39 for Rev A at a pH of 5.5 is consistent with Rev A-induced apoptosis in osteoclasts as osteoclasts were tested at this pH, a pH reflecting that observed in vivo at the bone-osteoclast interface. 1,3 Osteoclast apoptosis was not observed at neutral pH. 3 These structure-activity prediction analyses indicated that Rev A is less likely to cross a cell membrane than mitomycin C, especially at near neutral pH, thereby at least in part explaining the inability of Rev A to cause death effects in NHFLS.…”

“…At pH 7.4 Rev A likely exists as a dicarboxylate with less relative membrane permeability. At pH 5.5, as measured at the osteoclast ruffled border, Rev A is most likely a monocarboxylate, allowing greater relative membrane permeability …”

“…In an earlier study, we reported that reveromycin A (Rev A), an antibiotic isolated from a soil bacteria, Streptomyces reveromyceticus , induced apoptosis in osteoclasts via caspase 9 activation but did not trigger necrosis over an extended period of treatment . This phenomenon of osteoclast reduction in the absence of potential inflammation‐inducing necrosis is favorable for development of Rev A as a bone‐resorptive disease therapy or adjunct.…”