Early induction of uncoupling protein-2 in pulmonary macrophages in hyperoxia-associated lung injury.

CONTEXT: High concentrations of inspired oxygen contribute to the pathogenesis of neonatal bronchopulmonary dysplasia and adult acute respiratory distress syndrome. Animal models of hyperoxia-associated lung injury (HALI) are characterized by enhanced generation of reactive oxygen species (ROS) and an adaptive antioxidant response. ROS contribute to pathogenesis, partly through enhancing pro-inflammatory activity in macrophages. Uncoupling protein-2 (UCP2) is an inner mitochondrial membrane protein whose expression lowers mitochondrial superoxide (O2ⁱ⁻) production. UCP2, therefore, has potential to contribute to antioxidant response. It is inducible in macrophages. OBJECTIVES AND METHODS: We hypothesized that induction of UCP2 occurred in response to pulmonary hyperoxia in vivo and that expression localized to pulmonary macrophages. We then investigated mechanisms of UCP2 regulation in hyperoxia-exposed macrophages in vitro and correlated changing UCP2 expression with mitochondrial membrane potential (Δψm) and O2ⁱ⁻ production. RESULTS: UCP2 is induced in lungs of mice within 1 h of hyperoxia exposure. Induction occurs in pulmonary alveolar macrophages in vivo, and can be replicated in vitro in isolated macrophages. UCP2 mRNA does not change. UCP2 increases quickly after the first hyperoxia-induced burst of mitochondrial O2ⁱ⁻ generation. Suppression of Δψm and mitochondrial O2ⁱ⁻ production follow and persist while UCP2 is elevated. DISCUSSION AND CONCLUSIONS: 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.

TNF-α renders macrophages resistant to a range of cancer chemotherapeutic agents through NF-κB-mediated antagonism of apoptosis signalling.

The abundance of macrophages is an independent negative prognostic factor in a range of cancer types, linked to the actions of macrophage products on vasculogenesis and cancer cell survival, motility and metastasis. TNF-α is a macrophage product and a product of some cancer cell types that is also associated with adverse prognosis in clinical and experimental cancers, through enhanced tumour cell growth, survival and metastasis. Macrophages are important targets of TNF-α. We observed that TNF-α partly substituted for the macrophage growth factor, M-CSF, in maintaining macrophage survival by protecting cells from apoptosis. We found that TNF-α afforded similar protection to chemotherapeutic agents and related cytotoxic drugs that acted through a range of apoptosis-initiating pathways, but not where protein synthesis was inhibited. Protection was dependent on intact NF-κB signalling. In addition to NF-κB-dependent factors previously identified as anti-apoptotic, we found an absolute requirement for very early antagonism of mitochondrial cytochrome C release, which sufficed to prevent apoptosis in the face of activation of a range of upstream apoptosis pathways, including p53, DISC-linked, mitochondrial depolarisation and calcium-sensitive pathways. The capacity of TNF-α to preserve macrophage numbers in the face of chemotherapy drugs is a potential contributor to prognosis in TNF-α-expressing cancers, encouraging further testing of anti-TNF-α treatments in these patients.

Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-kappaB-dependent pathway.

INTRODUCTION: Methotrexate (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. METHODS: Caspase-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. RESULTS: MTX 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. CONCLUSIONS: These 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.

Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages.

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.

Effects of various pesticides on human 5alpha-reductase activity in prostate and LNCaP cells.

Certain pesticides are able to disturb the sex steroid hormone system and to act as antiandrogens. While the different underlying mechanisms remain unclear, inhibition of 5alpha-reductase, the enzyme which is indispensable for the synthesis of DHT and thus normal masculinization, appears to be one of the sensitive targets for endocrine disruption. We therefore tested several endocrine disrupters with antiandrogenic effects in vivo for their influence on 5alpha-reductase activity in two different test systems: (a) an enzyme assay with human Lymph Node Carcinoma of Prostate (LNCaP) cells and (b) an enzyme assay with human prostate tissue homogenate. The selected pesticides and industrial compounds were monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), triphenyltin (TPT), diuron, fenarimol, linuron, p,p'DDE, prochloraz and vinclozolin. The synthetic androgen methyltestosterone and the synthetic antiandrogen flutamide, as well as the 5alpha-reductase inhibitor finasteride served as control compounds. The effect of the organotin compounds DBT, TBT and TPT on enzyme activity was approximately the same in both test systems, with IC(50) values ranging between 2.7 and 11.2 microM, while in prostate tissue, methyltestosterone and prochloraz proved to be stronger inhibitors (IC(50) values of 1.9 and 12.4 microM) than in LNCaP cells (IC(50) values of 13.2 and 53.2 microM). The inhibitory impact of finasteride was approximately 130 times stronger in prostate tissue than in LNCaP cells. Fenarimol, flutamide, linuron and p,p'DDE inhibited 5alpha-reductase activity only at very high concentrations (IC(50)> or =24 microM) in prostate homogenates, and not at all in LNCaP cells. On average, the IC(20) values were 3.5 times lower than the IC(50) values. Diuron, MBT and vinclozolin exerted no effect in either of the test systems. The finding of pesticides acting as 5alpha-reductase inhibitors might be of clinical relevance. As a screening tool for putative ED, the tissue assay is the more practical and sensitive method. However, the cell assay can, to some extent, reflect particular cell processes since the living cell is able to compensate moderate toxicological effects of the ED on cell viability, and possibly also their impact on 5alpha-reductase activity.

COMPRENDO: Focus and approach.

Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body's signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that "the dose makes the poison" may not always be the case because some EDCs do not induce the classical dose-response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters--Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates) .

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production.

UNLABELLED: 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 Ca2+, and RANKL-induced activation of NF-kappaB and AP-1 activities. INTRODUCTION: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. MATERIALS AND METHODS: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappaB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. RESULTS: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. CONCLUSION: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Dithioerythritol (DTE) prevents inhibitory effects of triphenyltin (TPT) on the key enzymes of the human sex steroid hormone metabolism.

Organotins are known to induce imposex (pseudohermaphroditism) in marine neogastropods and are suggested to act as specific endocrine disruptors, inhibiting the enzyme-mediated conversion of steroid hormones. Therefore, we investigated the in vitro effects of triphenyltin (TPT) on human 5alpha-reductase type 2 (5alpha-Re 2), cytochrome P450 aromatase (P450arom), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD 3), 3beta-HSD type 2 and 17beta-HSD type 1 activity. First, the present study demonstrates that significant amounts of TPT occurred in the blood of eight human volunteers (0.17-0.67 microg organotin cation/l, i.e. 0.49-1.92 nmolcation/l). Second, TPT showed variable inhibitory effects on all the enzymes investigated. The mean IC(50) values were 0.95 microM for 5alpha-Re 2 (mean of n=4 experiments), 1.5 microM for P450arom (n=5), 4.0 microM for 3beta-HSD 2 (n=1), 4.2 microM for 17beta-HSD 3 (n=3) and 10.5 microM for 17beta-HSD 1 (n=3). To exclude the possibility that the impacts of TPT are mediated by oxidizing essential thiol residues of the enzymes, the putative compensatory effects of the reducing agent dithioerythritol (DTE) were investigated. Co-incubation with DTE (n=3) resulted in dose-response prevention of the inhibitory effects of 100 microM deleterious TPT concentrations on 17beta-HSD 3 (EC(50) value of 12.9 mM; mean of n=3 experiments), 3beta-HSD 2 (0.90 mM; n=3), P450 arom (0.91 mM; n=3) and 17beta-HSD 1 (0.21 mM; n=3) activity. With these enzymes, the use of 10mM DTE resulted in an at least 80% antagonistic effect, whereas, the effect of TPT on 5alpha-Re 2 was not compensated. In conclusion, the present study shows that TPT acts as an unspecific, but significant inhibitor of human sex steroid hormone metabolism and suggests that the inhibitory effects are mediated by the interaction of TPT with critical cysteine residues of the enzymes.