Role of intracellular labile iron, ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide.

To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H2O2-resistant Jurkat T cell line "HJ16" was developed by gradual adaptation of parental "J16" cells to high concentrations of H2O2. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H2O2-induced oxidative damage and necrotic cell death (up to 3mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H2O2-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H2O2 concentrations higher than 0.1mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H2O2 concentrations higher than 0.1mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H2O2 treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H2O2 treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H2O2 treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H2O2 has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H2O2 treatment.

Myeloid cell expression of the RNA-binding protein HuR protects mice from pathologic inflammation and colorectal carcinogenesis.

The innate immune response involves a variety of inflammatory reactions that can result in inflammatory disease and cancer if they are not resolved and instead are allowed to persist. The effective activation and resolution of innate immune responses relies on the production and posttranscriptional regulation of mRNAs encoding inflammatory effector proteins. The RNA-binding protein HuR binds to and regulates such mRNAs, but its exact role in inflammation remains unclear. Here we show that HuR maintains inflammatory homeostasis by controlling macrophage plasticity and migration. Mice lacking HuR in myeloid-lineage cells, which include many of the cells of the innate immune system, displayed enhanced sensitivity to endotoxemia, rapid progression of chemical-induced colitis, and severe susceptibility to colitis-associated cancer. The myeloid cell-specific HuR-deficient mice had an exacerbated inflammatory cytokine profile and showed enhanced CCR2-mediated macrophage chemotaxis. At the molecular level, activated macrophages from these mice showed enhancements in the use of inflammatory mRNAs (including Tnf, Tgfb, Il10, Ccr2, and Ccl2) due to a lack of inhibitory effects on their inducible translation and/or stability. Conversely, myeloid overexpression of HuR induced posttranscriptional silencing, reduced inflammatory profiles, and protected mice from colitis and cancer. Our results highlight the role of HuR as a homeostatic coordinator of mRNAs that encode molecules that guide innate inflammatory effects and demonstrate the potential of harnessing the effects of HuR for clinical benefit against pathologic inflammation and cancer.

HuR controls lung branching morphogenesis and mesenchymal FGF networks.

Lung development is controlled by regulatory networks governing mesenchymal-epithelial interactions. Transcription factors and signaling molecules are known to participate in this process, yet little is known about the post-transcriptional regulation of these networks. Here we demonstrate that the RNA-binding protein (RBP) HuR is an essential regulator of mesenchymal responses during lung branching. Its epiblast-induced deletion blocked the morphogenesis of distal bronchial branches at the initiation of the pseudoglandular stage. The phenotype originated from defective mesenchymal responses since the conditional restriction of HuR deletion in epithelial progenitors did not affect distal branching or the completion of lung maturation. The loss of HuR resulted in the reduction of the key inducer of bud outgrowth and endodermal branching, FGF10 and one of its putative transcriptional regulators, Tbx4. Furthermore, exogenous FGF10 could rescue the branching defect of affected lung buds. HuR was found to bind and control the Fgf10 and Tbx4 mRNAs; as a result its deletion abolished their inducible post-transcriptional regulation by the mesenchymal regulator FGF9. Our data reveals HuR as the first RBP identified to play a dominant role in lung development and as a key post-transcriptional regulator of networks guiding tissue remodeling during branching morphogenesis.

The RNA-binding protein Elavl1/HuR is essential for placental branching morphogenesis and embryonic development.

HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuR's requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuR's mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuR's absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.

Suppression of UVA-mediated release of labile iron by epicatechin--a link to lysosomal protection.

UVA (320-380 nm) radiation generates an oxidative stress in cells and leads to an immediate release of potentially damaging labile iron pools in human skin cells. Treatment of cultured skin fibroblasts for several hours with physiologically relevant concentrations of either epicatechin (EC), a flavonoid plant constituent present in foods, or methylated epicatechin (3'-O-methyl epicatechin, MeOEC), its major human metabolite, prevents this iron release. The similarity of the effectiveness of EC and MeOEC argues against chelation as the mechanism of iron removal. Evidence based on measurements of lysosomal integrity strongly supports the hypothesis that the catechins protect against lysosomal destruction by UVA. Such damage would normally lead to protease release, which has been previously shown to cause ferritin degradation and release of labile iron.

Caged-iron chelators a novel approach towards protecting skin cells against UVA-induced necrotic cell death.

Exposure of human skin cells to solar UVA radiation leads to an immediate dose-dependent increase of labile iron that subsequently promotes oxidative damage and necrotic cell death. Strong iron chelators have been shown to suppress cell damage and necrotic cell death by moderating the amount of labile iron pool (LIP), but chronic use would cause severe side effects owing to systemic iron depletion. Prodrugs that become activated in skin cells at physiologically relevant doses of UVA, such as "caged-iron chelators", may provide dose- and context-dependent release. Herein, we describe prototypical iron chelator compounds derived from salicylaldehyde isonicotinoyl hydrazone and pyridoxal isonicotinoyl hydrazone and demonstrate that the intracellular LIP and subsequent necrotic cell death of human skin fibroblasts is significantly decreased upon exposure to a combination of the prototypical compounds and physiologically relevant UVA doses. Iron regulatory protein bandshift and calcein fluorescence assays reveal decreased intracellular LIP following irradiation of caged-chelator-treated cells, but not in control samples where either UVA light, or caged-chelator is absent. Furthermore, flow cytometry shows that these compounds have no significant toxicity in the skin fibroblasts. This novel light-activated prodrug strategy may therefore be used to protect skin cells against the deleterious effects of sunlight.

Towards multifunctional antioxidants: synthesis, electrochemistry, in vitro and cell culture evaluation of compounds with ligand/catalytic properties.

Numerous human diseases are linked to a biochemical condition known as oxidative stress (OS). Antioxidants are therefore becoming increasingly important as potential disease prevention and therapeutic agents. Since OS is a multi-stressor event, agents combining a range of different antioxidant properties, such as redox catalysis and metal binding, might be more effective and selective than mono-functional agents. Selenium derivatives of aniline and pyridine combine redox activity with metal binding properties. These multifunctional agents have a distinct electrochemical profile, and exhibit good catalytic activity in the glutathione peroxidase mimic and metallothionein assays. They also show antioxidant activity in a skin cell model of UVA-induced stress. These compounds might therefore provide the basis for novel agents combining two or more distinct antioxidant properties.

Susceptibility of skin cells to UVA-induced necrotic cell death reflects the intracellular level of labile iron.

The mechanism of resistance of keratinocytes to ultraviolet A (UVA) (320-400 nm)-induced oxidative damage has not yet been elucidated. Here, we examined the possible link between the intracellular level of the labile iron pool (LIP) and the susceptibility to UVA-induced cell death using a series of human skin fibroblast and keratinocyte cell lines as a model. Resistance of keratinocytes to UVA-induced cell death was confirmed by flow cytometry and in fibroblasts necrosis was found to be the primary mode of cell death induced by UVA. The percentage of necrosis in fibroblasts also correlated with the extent of intracellular ATP depletion, a hallmark of necrotic cell death. The evaluation of the intracellular level of LIP by calcein assay revealed that both "basal" and "UVA-induced" levels of LIP in keratinocytes were several fold lower than in fibroblasts. Accordingly the dose to give an equivalent level of necrosis was several fold lower in fibroblasts than in keratinocytes. Furthermore, the modulation of "basal" or "UVA-induced" level of LIP by either Desferal and/or hemin treatment significantly affected the extent of UVA-induced necrotic cell death and ATP depletion in all the cell lines. Cellular susceptibility to UVA-induced necrotic cell death appears to reflect the intracellular level of LIP.

Novel poly(ADP-ribose) polymerase-1 inhibitor, AG14361, restores sensitivity to temozolomide in mismatch repair-deficient cells.

PURPOSE: Mismatch repair (MMR) deficiency confers resistance to temozolomide, a clinically active DNA-methylating agent. The purpose of the current study was to investigate the reversal mechanism of temozolomide resistance by the potent novel poly(ADP-ribose) polymerase (PARP)-1 inhibitor, AG14361, in MMR-proficient and -deficient cells. EXPERIMENTAL DESIGN: The effects of AG14361, in comparison with the methylguanine DNA methyltransferase inhibitor, benzylguanine, on temozolomide-induced growth inhibition were investigated in matched pairs of MMR-proficient (HCT-Ch3, A2780, and CP70-ch3) and -deficient (HCT116, CP70, and CP70-ch2) cells. RESULTS: AG14361 enhanced temozolomide activity in all MMR-proficient cells (1.5-3.3-fold) but was more effective in MMR-deficient cells (3.7-5.2-fold potentiation), overcoming temozolomide resistance. In contrast, benzylguanine only increased the efficacy of temozolomide in MMR-proficient cells but was ineffective in MMR-deficient cells. The differential effect of AG14361 in MMR-deficient cells was not attributable to differences in PARP-1 activity or differences in its inhibition by AG14361, nor was it attributable to differences in DNA strand breaks induced by temozolomide plus AG14361. MMR-deficient cells are resistant to cisplatin, but AG14361 did not sensitize any cells to cisplatin. PARP-1 inhibitors potentiate topotecan-induced growth inhibition, but AG14361 did not potentiate topotecan in MMR-deficient cells more than in MMR-proficient cells. CONCLUSIONS: MMR defects are relatively common in sporadic tumors and cancer syndromes. PARP-1 inhibition represents a novel way of selectively targeting such tumors. The underlying mechanism is probably a shift of the cytotoxic locus of temozolomide to N(7)-methylguanine and N(3)-methyladenine, which are repaired by the base excision repair pathway in which PARP-1 actively participates.