Earlier work in our laboratory demonstrated that naturally occurring reveromycin A (Rev A) causes apoptosis in osteoclasts without accompanying necrosis. Rev A death effects in both normal and diseased joint cells were investigated in this study. A dose of 10 μM Rev A did not cause apoptosis nor necrosis in monolayer chondrocytes, even at pH 6.8, a pH mimicking that of an inflamed joint. In contrast, at the acidic pH Rev A did induce significant apoptosis (fourfold increase at 48 h of treatment, P < 0.005) in normal synoviocytes without accompanying necrosis. Western blot of the normal synoviocyte proteins revealed that cytochrome c levels were not significantly changed over the time course of treatment nor did caspase 8 activity increase; therefore, Rev A appears to exert this apoptotic effect through a mechanism independent of the classical intrinsic and extrinsic pathways. Fibroblast-like synoviocytes isolated from rheumatoid arthritis patients (RAFLS) as well as normal human fibroblast-like synoviocytes (NHFLS), cells known to play key roles in arthritic joint pathology, were also subjected to Rev A treatment at both physiologic and acidic pH's. Neither apoptosis nor necrosis was induced in either RAFLS or NHFLS. Parallel mitomycin C treatment of NHFLS induced both apoptosis and necrosis. Comparative structure-activity analyses of Rev A and mitomycin C revealed that Rev A is less likely to cross the cell membrane at near neutral pH. Collectively the data reveal that a physiological dose of Rev A under acidic conditions induces normal synoviocytes to undergo apoptosis while pathologic fibroblast-like synoviocytes are resistant to apoptosis and necrosis.

Reveromycin A (Rev A) is a natural compound that specifically causes apoptosis in osteoclasts and hence may have potential as an osteoporosis therapeutic. Earlier work in our laboratory demonstrated that Rev A did not cause significant cell death in chondrocytes. To date no studies have been conducted on Rev A activity in synoviocytes or the disease‐causing cells of rheumatoid arthritis, human fibroblast‐like synoviocytes (HFLS‐RA). LDH release assays were used to measure necrosis levels and caspase 3 was quantitated from cell extracts. Rev A‐induced cell death in both cell lines was characterized under the more acidic pH of 6.8, which mimics the pH of an inflamed joint and provides a pH believed to be important for Rev A action.10 microM Rev A‐treated normal synoviocytes showed distinct apoptotic changes with a 2‐fold increase in caspase 3 activity compared to untreated. At 0.1 and 1.0 microM Rev A, caspase 3 activity did not increase. Significant necrosis was not observed at any of the concentrations. These preliminary results suggest that 10 microM Rev A may have undesirable apoptotic effects in normal synoviocytes of the joint under acidic conditions. However at lower, potentially relevant therapeutic doses, apoptotic events do not appear to occur. Untreated and 10 microM Rev A‐treated HFLS‐RA at both 7.4 and 6.8 pH's demonstrated normal morphology under the phase contrast microscope and did not demonstrate significantly different levels of apoptosis or necrosis compared to untreated, suggesting that the typically apoptosis‐resistant HFLS‐RA behave in a similar fashion when exposed to Rev A. Funding provided by an Indiana Academy of Science senior research grant, the Hodson Summer Research Institute, and the Indiana Wesleyan University Division of Natural Sciences.

Bisphosphonate treatments can produce an unwanted side effect of bisphosphonate‐related osteonecrosis of the jaw (BRONJ). One of the most potent bisphosphonates, zolendronate (ZOL), is used to treat postmenopausal osteoporosis, Paget’s disease, and cancer metastasis to bone. Individuals affected with BRONJ are unable to heal from dental procedures, leading to painful oral lesions. The goal of the work presented here was to investigate the ability of GGOH to rescue both murine MC3T3E1 osteoblasts and human periodontal ligament fibroblasts from ZOL‐induced apoptosis. Apoptosis was measured using the APOPercentage® assay. In osteoblasts, 72 hours of 50 micromolar ZOL treatment induced apoptosis by 12.5‐fold but simultaneous exposure to 10 micromolar GGOH decreased apoptosis by 64% (p<0.05). In a separate set of experiments, a booster of GGOH was administered 24 hours after the initiation of treatment, dramatically reducing ZOL‐induced apoptosis by 98% (p<0.0001). In periodontal ligament fibroblasts, ZOL treatment induced apoptosis 10‐fold and GGOH reduced apoptosis by 82% (p<0.05). The booster of GGOH reduced ZOL‐induced apoptosis by 99%. Collectively the data demonstrates the ability of GGOH to potentially rescue both hard and soft tissues of the oral cavity from ZOL‐induced apoptosis with a supplemental dosage of GGOH 24 hours following initiation of treatment essentially eliminating apoptosis. Grant Funding Source: Supported by grants from the Indiana Academy of Science and the Hodson Summer Research Institute

Osteoporosis is a prevalent disease with several current treatments. However, these treatments can have harmful long‐term side effects on individuals utilizing them. Reveromycin A (Rev A) is a natural compound that specifically causes apoptosis in osteoclasts but not osteoblasts. To further test whether Rev A can be a safe osteoporosis treatment without detrimental effects in the surrounding joint, the compound’s ability to induce apoptosis and necrosis in ATDC5 murine chondrocytes and HIG‐82 rabbit synoviocytes was measured. Inflammatory joint disease is known to accompany osteoporosis; consequently, Rev A‐induced cell death was characterized in each of these two cell lines under both physiological pH and a pH of 6.8 to mimic the pH of an inflamed joint. After treatment, morphology was assessed through phase contrast microscopy, apoptosis was measured through colorimetric caspase 3 activity assay, and necrosis was quantitated by use of a lactate dehydrogenase (LDH) release assay. Six hours of ten micromolar Rev A treatment did not significantly induce chondrocyte apoptosis, chondrocyte necrosis, or synoviocyte necrosis at either pH. Synoviocyte apoptosis was not induced by Rev A at physiological pH, but at a pH of 6.8, Rev A induced apoptosis compared to untreated by 4.9–fold (p<0.005). The results suggest that Rev A may have undesirable apoptotic effects in synoviocytes of the joint under acidic conditions. Grant Funding Source: Supported by grants from the Indiana Academy of Science and the Hodson Summer Research Institute