ABSTRACT:The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca 2+ , and RANKL-induced activation of NF-B and AP-1 activities.

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.

IntroductionMethotrexate (MTX) induces macrophage apoptosis in vitro, but there is not much evidence for increased synovial macrophage apoptosis in MTX-treated patients. Macrophage apoptosis is reported, however, during clinical response to anti-tumor necrosis factor-alpha (TNF-α) treatments. This implies that TNF-α promotes macrophage survival and suggests that TNF-α may protect against MTX-induced apoptosis. We, therefore, investigated this proposal and the macrophage signaling pathways underlying it.MethodsCaspase-3 activity, annexin-V binding/7-aminoactinomycin D (7-AAD) exclusion and cell-cycle analysis were used to measure steps in apoptosis of primary murine macrophages and cells of the RAW264.7 macrophage cell line that had been exposed to clinically-relevant concentrations of MTX and TNF-α.ResultsMTX induces apoptosis in primary murine macrophages at concentrations as low as 100 nM in vitro. TNF-α, which has a context-dependent ability to increase or to suppress apoptosis, efficiently suppresses MTX-induced macrophage apoptosis. This depends on NF-κB signaling, initiated through TNF Receptor Type 1 ligation. Macrophage colony stimulating factor, the primary macrophage survival and differentiation factor, does not activate NF-κB or protect macrophages from MTX-induced apoptosis. A weak NF-κB activator, Receptor Activator of NF-κB Ligand (RANKL) is likewise ineffective. Blocking NF-κB in TNF-α-exposed macrophages allowed pro-apoptotic actions of TNF-α to dominate, even in the absence of MTX. MTX itself does not promote apoptosis through interference with NF-κB signaling.ConclusionsThese findings provide another mechanism by which TNF-α sustains macrophage numbers in inflamed tissue and identify a further point of clinical complementarity between MTX and anti-TNF-α treatments for rheumatoid arthritis.

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Induction of UCP2 is an early response to hyperoxia in pulmonary macrophages. The mechanism is post-transcriptional. UCP2 induction follows a transient rise in mitochondrial ROS generation. The subsequent falls in Δψm and mitochondrial O₂ⁱ⁻ support the notion that regulable UCP2 expression in macrophages acts to contain mitochondrial ROS generation. That, in turn, may limit inappropriate pro-inflammatory activation in HALI.