Jeffrey M. Reece

Reactive Oxygen Species as a Signal in Glucose-Stimulated Insulin Secretion

One of the unique features of β-cells is their relatively low expression of many antioxidant enzymes. This could render β-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H2O2. In both models, insulin secretion could be stimulated by provision of either exogenous H2O2 or diethyl maleate, which raises intracellular H2O2 levels. Provision of exogenous H2O2 scavengers, including cell permeable catalase and N-acetyl-l-cysteine, inhibited glucose-stimulated H2O2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H2O2, had no effect on GSIS. Because oxidative stress is an important risk factor for β-cell dysfunction in diabetes, the relationship between glucose-induced H2O2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H2O2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function.

Transcription factor Nrf2 activation by inorganic arsenic in cultured keratinocytes: involvement of hydrogen peroxide

Inorganic arsenic is a well-documented human carcinogen that targets the skin. The induction of oxidative stress, as shown with arsenic, may have a bearing on the carcinogenic mechanism of this metalloid. The transcription factor Nrf2 is a key player in the regulation of genes encoding for many antioxidative response enzymes. Thus, the effect of inorganic arsenic (as sodium arsenite) on Nrf2 expression and localization was studied in HaCaT cells, an immortalized human keratinocyte cell line. We found, for the first time, that arsenic enhanced cellular expression of Nrf2 at the transcriptional and protein levels and activated expression of Nrf2-related genes in these cells. In addition, arsenic exposure caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2-mediated oxidative response genes. Arsenic simultaneously increased the expression of Keap1, a regulator of Nrf2 activity. The coordinated induction of Keap1 expression and nuclear Nrf2 accumulation induced by arsenic suggests that Keap1 is important to arsenic-induced Nrf2 activation. Furthermore, when cells were pretreated with scavengers of hydrogen peroxide (H(2)O(2)) such as catalase-polyethylene glycol (PEG-CAT) or Tiron, arsenic-induced nuclear Nrf2 accumulation was suppressed, whereas CuDIPSH, a cell-permeable superoxide dismutase (SOD) mimic compound that produces H(2)O(2) from superoxide (*O(2)(-)), enhanced Nrf2 nuclear accumulation. These results indicate that H(2)O(2), rather than *O(2)(-), is the mediator of nuclear Nrf2 accumulation. Additional study showed that arsenic causes increased cellular H(2)O(2) production and that H(2)O(2) itself has the ability to increase Nrf2 expression at both the transcription and protein levels in HaCaT cells. Taken together, these data clearly show that arsenic increases Nrf2 expression and activity at multiple levels and that H(2)O(2) is one of the mediators of this process.

Role of the Cytoskeleton in Calcium Signaling in NIH 3T3 Cells AN INTACT CYTOSKELETON IS REQUIRED FOR AGONIST-INDUCED [Ca2+] i SIGNALING, BUT NOT FOR CAPACITATIVE CALCIUM ENTRY

Treatment of NIH 3T3 cells with cytochalasin D (10 μm, 1 h at 37 °C) disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. Calcium mobilization by ATP or by platelet-derived growth factor was abolished, while the ability of thapsigargin (2 μm) to empty calcium stores and activate calcium influx was unaffected. Similar experiments with nocodazole to depolymerize the tubulin network yielded identical results. Platelet-derived growth factor induced an increase in inositol phosphates, and this increase was undiminished in the presence of cytochalasin D. Therefore, the blockade of agonist responses by this drug does not result from decreased phospholipase C. Injection of inositol 1,4,5-trisphosphate (IP3) released calcium to the same extent in control and cytochalasin D-treated cells. Confocal microscopic studies revealed a significant rearrangement of the endoplasmic reticulum after cytochalasin D treatment. Thus, disruption of the cytoskeleton blocks agonist-elicited [Ca2+] i mobilization, but this effect does not result from a lower calcium storage capacity, impaired function of the IP3 receptor, or diminished phospholipase C activity. We suggest that cytoskeletal disruption alters the spatial relationship between phospholipase C and IP3 receptors, impairing phospholipase C-dependent calcium signaling. Capacitative calcium entry was not altered under these conditions, indicating that the coupling between depletion of intracellular calcium stores and calcium entry does not depend on a precise structural relationship between intracellular stores and plasma membrane calcium channels.

Interleukin-10 Protects Lipopolysaccharide-Induced Neurotoxicity in Primary Midbrain Cultures by Inhibiting the Function of NADPH Oxidase

The role of anti-inflammatory cytokines in Parkinsons disease is not completely understood. In this study, using mesencephalic neuron-glia cultures, we report that both pretreatment and post-treatment of rat mesencephalic neuron-glia cultures with interleukin (IL)-10, a natural immune modulator, reduced lipopolysaccharide (LPS)-induced DA neurotoxicity. The main purpose of this study was to elucidate the molecular mechanism underlying IL-10-elicited neuroprotection. IL-10 significantly inhibited LPS-induced production of tumor necrosis factor-α, nitric oxide, and extracellular superoxide in microglia cells. In addition, using reconstituted neuron and glia cell cultures, IL-10 was shown to be neuroprotective only in the presence of microglia. More importantly, IL-10 failed to protect DA neurons in cultures from mice lacking NADPH oxidase (PHOX), a key enzyme for extracellular superoxide production in immune cells, suggesting the critical role of PHOX in IL-10 neuroprotection. This conclusion was further supported by the finding that IL-10 inhibited LPS-induced translocation of the cytosolic subunit of NADPH oxidase p47phox to the membrane. When the Janus tyrosine kinase (JAK) 1 signaling pathway was blocked, IL-10 failed to attenuate LPS-induced superoxide production, indicating that the JAK1 signaling cascade mediates the inhibitory effect of IL-10. Together, our results suggest that IL-10 inhibits LPS-induced DA neurotoxicity through the inhibition of PHOX activity in a JAK1-dependent mechanism.

MPP+-induced COX-2 activation and subsequent dopaminergic neurodegeneration

The importance of cyclooxygenase‐2 (COX‐2) in mediating Parkinsons disease (PD) was suggested in reports, indicating that COX‐2 selective inhibitors or genetic knockout reduce 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced dopaminergic (DA) neurotoxicity in a mouse model of PD. However, cell types and mechanisms underlying the activation of COX‐2 have not been clearly elucidated in these animal studies. Using primary neuron‐glia cultures, we aimed to determine 1) whether microglia participate in 1‐methyl‐4‐phenylpryridinium (MPP+)‐induced COX‐2 activation and 2) whether the activation of COX‐2 contributes to subsequent neurotoxicity. MPP+, in a concentration‐dependent manner, increased prostaglandin E2 (PGE2) production in mixed neuron‐microglia cultures but not in enriched neuron, microglia, or astroglia cultures nor in mixed neuron‐astroglia cultures. MPP+‐induced PGE2 increase was completely abolished by treatment with DuP697, a COX‐2 selective inhibitor. DuP697 also significantly reduced MPP+‐induced DA neurotoxicity as determined by DA uptake assay. Immunocytochemistry and confocal microscopy studies showed enhanced COX‐2 expression in both microglia and neurons after MPP+ treatment. However, neuronal increase in COX‐2 expression was not totally dependent on the production of PGE2 from microglia, since microglia deficient in COX‐2 only attenuated, but did not completely block, MPP+‐increased PGE2 production in mixed neuron‐microglia cultures, suggesting that part of PGE2 production was originated from neurons. Together, these results indicate that MPP+‐induced COX‐2 expression and subsequent PGE2 production depend on interactions between neurons and microglia. Microgliosis may also be responsible for the COX‐2 activation in neurons, leading to the enhanced DA neurotoxicity, which, in turn, reinforces microgliosis. Thus inhibition of microgliosis and COX‐2 activity may stop this vicious circle and be valuable strategies in PD therapy.

MAC1 mediates LPS-induced production of superoxide by microglia: The role of pattern recognition receptors in dopaminergic neurotoxicity

Microglia‐derived superoxide is critical for the inflammation‐induced selective loss of dopaminergic (DA) neurons, but the underlying mechanisms of microglial activation remain poorly defined. Using neuron‐glia and microglia‐enriched cultures from mice deficient in the MAC1 receptor (MAC1−/−), we demonstrate that lipopolysaccharide (LPS) treatment results in lower TNFα response, attenuated loss of DA neurons, and absence of extracellular superoxide production in MAC1−/− cultures. Microglia accumulated fluorescently labeled LPS in punctate compartments associated with the plasma membrane, intracellular vesicles, and the Golgi apparatus. Cytochalasin D (CD), an inhibitor of phagocytosis, blocked LPS internalization. However, microglia derived from Toll‐like receptor 4 deficient mice and MAC1−/− mice failed to show a significant decrease in intracellular accumulation of labeled LPS, when compared with controls. Pretreatment with the scavenger receptor inhibitor, fucoidan, inhibited 79% of LPS accumulation in microglia without affecting superoxide, indicating that LPS internalization and superoxide production are mediated by separate phagocytosis receptors. Together, these data demonstrate that MAC1 is essential for LPS‐induced superoxide from microglia, implicating MAC1 as a critical trigger of microglial‐derived oxidative stress during inflammation‐mediated neurodegeneration.

Cadmium-induced malignant transformation in rat liver cells: role of aberrant oncogene expression and minimal role of oxidative stress

Our study examined the role of oxidative stress and aberrant gene expression in malignant transformation induced by chronic, low‐level cadmium exposure in non‐tumorigenic rat liver epithelial cell line, TRL 1215. Cells were cultured in 1.0 μM cadmium (as CdCl2) for up to 28 weeks and compared to passage‐matched control cells. The level of cadmium used for transformation produced no evidence of increased superoxide (O2− ·) or hydrogen peroxide (H2O2) levels in the early stages of exposure (≤24 hr). The chronic cadmium exposed liver epithelial cells (CCE‐LE) were hyperproliferative with a growth rate about 3‐fold higher than control cells. CCE‐LE cells produced highly aggressive tumors upon inoculation into mice confirming malignant transformation. Analysis of cellular reactive oxygen species (ROS) showed that CCE‐LE cells possessed markedly lower basal levels of intracellular O2− ·and H2O2 and were very tolerant to high‐dose (50 μM) cadmium‐induced ROS. Time course studies showed the production of ROS by high‐dose cadmium was abolished well in advance of malignant transformation. In contrast, marked overexpression of the oncogenes c‐myc and c‐jun occurred in transformed CCE‐LE cells as evidenced by up to 10‐fold increases in both transcript and protein. A significant increase in DNA‐binding activity of the transcription factors AP‐1 and NF‐κB occurred in CCE‐LE cells. Increases in oncogene expression and transcription factor activity occurred concurrently with malignant transformation. Thus, cadmium‐induced ROS occurs as an early, high‐dose event but is abolished well in advance of malignant transformation. Low‐level chronic cadmium triggers oncogene overexpression possibly by altering critical transcription factor activity. Such changes in cellular gene expression likely culminate in the loss of growth control and cadmium‐induced neoplastic transformation in CCE‐LE cells, whereas generation of ROS by cadmium seemed to play a minimal role in this transformation.

Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor

It has been reported that extracellular matrix (ECM) molecules regulate monocyte activation by binding with a 67 kDa nonintegrin laminin receptor (LR). As microgliosis is a pivotal factor in propelling the progress of chronic neurodegeneration in the brain, we hypothesized that LR may regulate the microgliosis and subsequent neurotoxicity. Using 1‐methyl‐4‐phe‐nylpyridinium (MPP+) ‐treated C57 mice primary mesencephalic neuron‐glia cultures as an in vitro Parkinsons disease (PD) model, we observed that MPP+ treatment increased LR expression only in the mixed neuron‐glia but not in microglia‐enriched or microglia‐depleted cultures, indicating that MPP+‐induced increase of LR expression is associated with neuron‐microglia interaction. Using confocal microscopic examination, we found that LR was localized in the microglia, which were F4/80 positive. Treatment with the antibody (Ab) against LR (LR‐Ab) or YIGSR, a synthetic pentapeptide inhibitor for LR, significantly attenuated the MPP+‐increased F4/80 immunoreactivity (24 h) and dopaminergic (DA) neurotoxicity. LR‐Ab also attenuated MPP+‐increased microglial phagocytotic activity (48 h) and the superoxide production (4 days). Further study demonstrated that exogenous laminin (1–10 µg/ml) treatment induced microglial activation and DA neurotoxicity, in a dose‐dependent manner, which was partially attenuated by the LR‐Ab. We concluded that by regulating cell‐ECM interaction, LR plays important roles in mediating microgliosis and subsequent DA neurotoxicity. Laminin is a potential ligand for activating this LR receptor. This study also suggests that laminin/LR is a potential target for developing new therapeutic drugs against neurodegenerative disorders such as PD.—Wang, T., Zhang, W., Pei, Z., Block, M., Wilson, B., Reece, J. M., Miller, D. S., and Hong, J.‐S. Reactive microgliosis participates in MPP+‐induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor. FASEB J. 20, 906–915 (2006)

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.