OLA1, an Obg-like ATPase, suppresses antioxidant response via nontranscriptional mechanisms.

Oxidative stress has been implicated in diverse disease states and aging. To date, induction of cellular responses to combat oxidative stress has been characterized largely at the transcriptional level, with emphasis on Nrf2-mediated activation of antioxidant response elements. In this study, we demonstrate that OLA1, a novel Obg-like ATPase, functions as a negative regulator of the cellular antioxidant response independent of transcriptional processes. Knockdown of OLA1 in human cells elicited an increased resistance to oxidizing agents including tert-butyl hydroperoxide (tBH) and diamide without affecting cell proliferation, baseline apoptosis, or sensitivity to other cytotoxic agents that target the mitochondria, cytoskeleton, or DNA. Conversely, overexpression of OLA1 increased cellular sensitivity to tBH and diamide. When challenged with oxidants, OLA1-knockdown cells had decreased production of intracellular reactive oxygen species and exhibited less depletion of reduced glutathione. Surprisingly, knockdown of OLA1 caused only minimal genomic response; no changes were found in the mRNA levels of genes encoding antioxidant enzymes, enzymes that produce antioxidants (including glutathione), or other genes known to respond to Nrf2. Moreover, when de novo protein synthesis was blocked by cycloheximide in OLA1-knockdown cells, they continued to demonstrate increased resistance to both tBH and diamide. These data demonstrate that OLA1 suppresses the antioxidant response through nontranscriptional mechanisms. The beneficial effects observed upon OLA1-knockdown suggest that this regulatory ATPase is a potential novel target for antioxidative therapy.

System x(c)- and thioredoxin reductase 1 cooperatively rescue glutathione deficiency.

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in gamma-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.

Glutathione is essential for early embryogenesis--analysis of a glutathione synthetase knockout mouse.

Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene (Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival.

Glutathione protects cells against arsenite-induced toxicity.

To understand the role of glutathione (GSH) in the protection of cells from arsenite toxicity, we studied the mechanism of apoptotic cell death in cells genetically unable to synthesize GSH (GCS-2 cells). Arsenite stimulated an increase in protein ubiquitination in GCS-2 cells while the wild-type cells were unaffected. Arsenite treatment increased lipid peroxidation and induced ubiquitination of molecular chaperone Hsp90 and impaired its ability to bind cochaperone p50(Cdc-37) and client proteins Plk-1 and Cdk-4 in GCS-2 cells. Treatment with arsenite also partially inhibited proteasome activity in GCS-2 cells. In these cells stably transfected with GFP(u) (a reporter consisting of a short degron fused to the COOH-terminus of GFP), intracellular fluorescence increased, suggesting the accumulation of GFP aggregates. GCS-2 cells underwent apoptosis accompanied by release of cytochrome c into the cytoplasm. Taken together, these data suggest that a possible mechanism of arsenite-induced apoptosis is the accumulation of ubiquitinated proteins and impairment of the protein degradative pathway. Further, protection from arsenite-induced ubiquitination is mediated by GSH and to a lesser extent by available reducing equivalents in the cells.

Survival and cell death in cells constitutively unable to synthesize glutathione.

We examined the role of GSH in survival and cell death using GCS-2 cells that are deficient in glutamate cysteine ligase (gamma-glutamyl cysteine synthetase, gammaGCS), an enzyme essential for GSH synthesis. Cells maintained in 2.5 mM GSH have GSH levels that are approximately 2% of wild type and grow indefinitely; however, they express both pro- and anti-apoptotic Bcl-2 family members and have detectable levels of cytoplasmic cytochrome C. Withdrawal of GSH from the medium results in a fall in intracellular GSH to undetectable levels, decreased mitochondrial dehydrogenase activity, decreased anti-apoptotic factor RNAs, increased pro-apoptotic factor RNAs, additional cytochrome C release, and a fall in ATP levels; however, cells continue to grow for another 24h. At 48 h, these trends continue with the exception that mitochondrial membrane potential and ATP levels rise; DNA fragmentation begins at 48 h. Thus, severe reduction of GSH to 2% of wild type produces a metastable state compatible with survival, but complete absence of GSH triggers apoptosis.

Arsenic inhibition of the JAK-STAT pathway.

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is an essential cascade for mediating normal functions of different cytokines in the development of the hematopoietic and immune systems. Chronic exposure to arsenic has been found to cause immunotoxicity and has been associated with the suppression of hematopoiesis (anemia and leukopenia). Here, we report the novel finding of arsenic-mediated inactivation of the JAK-STAT signaling pathway by its direct interaction with JAK tyrosine kinase. Pretreatment with sodium arsenite strongly inhibited IL-6-inducible STAT3 tyrosine phosphorylation in HepG2 cells and did not affect its serine phosphorylation. As a result, sodium arsenite completely abolished STAT activity-dependent expression of suppressors of cytokine signaling (SOCS). Both cellular and subcelluar experiments showed that the inhibition of JAK-STAT signaling resulted from JAK tyrosine kinase's direct interaction with arsenite, and that arsenic's suppression of JAK tyrosine kinase activity also occurred in the interferon gamma (IFNgamma) pathway. The ligand-independent inhibition by arsenic indicates that JAK was the direct target of arsenic action. Other inflammatory stimulants, stress agents, and metal cadmium failed to induce similar effects on the tyrosine phosphorylation of STAT3 as arsenic does. Our experiments also revealed that arsenic inactivation of the JAK-STAT pathway occurred independent of arsenic activation of MAP kinases. Taken together, our findings indicate that arsenic directly inhibits JAK tyrosine kinase activity and suggest that this direct interference in the JAK-STAT pathway may play a role in arsenic-associated pathogenesis.

Identification of two additional members of the membrane-bound dipeptidase family.

We have cloned two mouse cDNAs encoding previously unidentified membrane-bound dipeptidases [membrane-bound dipeptidase-2 (MBD-2) and membrane-bound dipeptidase-3 (MBD-3)] from membrane-bound dipeptidase-1 (MBD-1) deficient mice (Habib, G.M., Shi, Z-Z., Cuevas, A.A., Guo, Q., Matzuk, M.M., and Lieberman, M.W. (1998) Proc. Natl. Acad. Sci. USA 95, 4859-4863). These enzymes are closely related to MBD-1 (EC 3.4.13.19), which is known to cleave leukotriene D4 (LTD4) and cystinyl-bis-glycine. MBD-2 cDNA is 56% identical to MBD-1 with a predicted amino acid identity of 33%. The MBD-3 and MBD-1 cDNAs share a 55% nucleotide identity and a 39% predicted amino acid sequence identity. All three genes are tightly linked on the same chromosome. Expression of MBD-2 and MBD-3 in Cos cells indicated that both are membrane-bound through a glycosylphosphatidyl-inositol linkage. MBD-2 cleaves leukotriene D4 (LTD4) but not cystinyl-bis-glycine, while MBD-3 cleaves cystinyl-bis-glycine but not LTD4. MBD-1 is expressed at highest levels in kidney, lung, and heart and is absent in spleen, while MBD-2 is expressed at highest levels in lung, heart, and testis and at somewhat lower levels in spleen. Of the tissues examined, MBD-3 expression was detected only in testis. Our identification of a second enzyme capable of cleaving LTD4 raises the possibility that clearance of LTD4 during asthma and in related inflammatory conditions may be mediated by more than one enzyme.

Reversible skeletal abnormalities in gamma-glutamyl transpeptidase-deficient mice.

Gamma-glutamyl transpeptidase (GGT) is a widely distributed ectopeptidase responsible for the degradation of glutathione in the gamma-glutamyl cycle. This cycle is implicated in the metabolism of cysteine, and absence of GGT causes a severe intracellular decrease in this amino acid. GGT-deficient (GGT-/-) mice have multiple metabolic abnormalities and are dwarf. We show here that this latter phenotype is due to a decreased of the growth plate cartilage total height resulting from a proliferative defect of chondrocytes. In addition, analysis of vertebrae and tibiae of GGT-/- mice revealed a severe osteopenia. Histomorphometric studies showed that this low bone mass phenotype results from an increased osteoclast number and activity as well as from a marked decrease in osteoblast activity. Interestingly, neither osteoblasts, osteoclasts, nor chondrocytes express GGT, suggesting that the observed defects are secondary to other abnormalities. N-acetylcysteine supplementation has been shown to reverse the metabolic abnormalities of the GGT-/- mice and in particular to restore the level of IGF-1 and sex steroids in these mice. Consistent with these previous observations, N-acetylcysteine treatment of GGT-/- mice ameliorates their skeletal abnormalities by normalizing chondrocytes proliferation and osteoblastic function. In contrast, resorbtion parameters are only partially normalized in GGT-/- N-acetylcysteine-treated mice, suggesting that GGT regulates osteoclast biology at least partly independently of these hormones. These results establish the importance of cysteine metabolism for the regulation of bone remodeling and longitudinal growth.

Interactions between leukotriene C4 and interleukin 13 signaling pathways in a mouse model of airway disease.

CONTEXT: During an asthmatic episode, leukotriene C4 (LTC4) and interleukin 13 (IL-13) are released into the airways and are thought to be central mediators of the asthmatic response. However, little is known about how these molecules interact or affect each other's signaling pathway. OBJECTIVE: To determine if the LTC4 and IL-13 signaling pathways interact with each other's pathways. DESIGN: We examined airway responsiveness, cysteinyl LTs (Cys-LTs), and Cys-LT and IL-13 receptor transcript levels in wild-type mice and in mice that were deficient in gamma-glutamyl leukotrienase (an enzyme that converts LTC4 to LTD4), STAT6 (signal transducer and activator of transcription 6 [a critical molecule in IL-13 signaling]), and IL-4Ralpha (a subunit of the IL-13 receptor). RESULTS: Wild-type (C57BL/129SvEv) and gamma-glutamyl leukotrienase-deficient mice showed increased airway responsiveness after intranasal instillation of IL-13; similar results were observed after intranasal instillation of IL-13 or LTC4 in a second wild-type strain (BALB/c). Interleukin 13 treatment reduced levels of Cys-LTs in bronchoalveolar lavage fluid. This change was unaccompanied by changes in other arachidonic acid metabolites or in RNA transcript levels of enzymes associated with Cys-LT synthesis. Interleukin 13 treatment also increased transcript levels of the Cys-LT 1 and Cys-LT 2 receptors, while LTC4 increased transcript levels of the alpha1 chain of the IL-13 receptor. Furthermore, IL-4Ralpha-deficient mice had increased airway responsiveness to LTC4 but not to IL-13, whereas STAT6-deficient mice failed to respond to either agonist. CONCLUSIONS: These findings indicate that LTC4 and IL-13 are dependent on or signal through STAT6 to increase airway responsiveness and that both agonists regulate expression of each other's receptors.

Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway.

Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.

Formation and urinary excretion of arsenic triglutathione and methylarsenic diglutathione.

Taking advantage of mice deficient in gamma-glutamyl transpeptidase that are unable to metabolize glutathione (GSH), we have identified two previously unrecognized urinary metabolites of arsenite: arsenic triglutathione and methylarsenic diglutathione. Following administration of sodium arsenite to these mice, approximately 60-70% of urinary arsenic is present as one of these GSH conjugates. We did not detect the dimethyl derivative, dimethyl arsenic GSH; however, dimethyl arsenic (DMAV) represented approximately 30% of urinary arsenic. Administration of buthionine sulfoximine, an inhibitor of GSH synthesis, to wild-type mice reduced urinary arsenic excretion by more than 50%, indicating the GSH dependence of arsenic metabolism, transport, or both. Rodents deficient in three known ABC family transporters (MRP1, MRP2, and MDR1a/1b) exhibited urinary arsenic levels similar or greater than those in wild-type rodents; however, administration of MK571, an MRP inhibitor, reduced urinary arsenic excretion by almost 50%. MK571-treated mice showed approximately 50% reduction of AsIII, MMAV, and AsV as compared to untreated wild-type controls, while DMAV levels were unchanged. These findings suggest that arsenic excretion is in part dependent on GSH and on an MRP transporter other than MRP1 or 2.

Gamma-glutamyl leukotrienase, a novel endothelial membrane protein, is specifically responsible for leukotriene D(4) formation in vivo.

The metabolism of cysteinyl leukotrienes in vivo and the pathophysiological effects of individual cysteinyl leukotrienes are primarily unknown. Recently we identified an additional member of the gamma-glutamyl transpeptidase (GGT) family, gamma-glutamyl leukotrienase (GGL), and developed mice deficient in this enzyme. Here we show that in vivo GGL, and not GGT as previously believed, is primarily responsible for conversion of leukotriene C(4) to leukotriene D(4), the most potent of the cysteinyl leukotrienes and the immediate precursor of leukotriene E(4). GGL is a glycoprotein consisting of two polypeptide chains encoded by one gene and is attached at the amino terminus of the heavy chain to endothelial cell membranes. In mice it localizes to capillaries and sinusoids in most organs and in lung to larger vessels as well. In contrast to wild-type and GGT-deficient mice, GGL-deficient mice do not form leukotriene D(4) in vivo either in blood when exogenous leukotriene C(4) is administered intravenously or in bronchoalveolar lavage fluid of Aspergillus fumigatus extract-induced experimental asthma. Further, GGL-deficient mice show leukotriene C(4) accumulation and significantly more airway hyperreponsiveness than wild-type mice in the experimental asthma, and induction of asthma results in increased GGL protein levels and enzymatic activity. Thus GGL plays an important role in leukotriene D(4) synthesis in vivo and in inflammatory processes.

Bleomycin-induced pulmonary fibrosis is attenuated in gamma-glutamyl transpeptidase-deficient mice.

To investigate repair mechanisms in bleomycin-induced pulmonary fibrosis, we used mice deficient in gamma-glutamyl transpeptidase (GGT-/-), a key enzyme in glutathione (GSH) and cysteine metabolism. Seventy-two hours after bleomycin (0.03 U/g), GGT-/- mice displayed a different inflammatory response to wild-type mice as judged by a near absence of neutrophils in lung tissue and bronchoalveolar lavage and a less pronounced rise in matrix metalloproteinase-9. Inflammation in GGT-/- mice consisted mainly of lymphocytes and macrophages. At 1 month, lungs from bleomycin-treated GGT-/- mice exhibited minimal areas of fibrosis compared with wild-type mice(light microscopy fibrosis index: 510 +/- 756 versus 1975 +/- 817, p < 0.01). Lung collagen content revealed a significant increase in bleomycin-treated wild-type (15.1 +/- 3.8 versus 8.5 +/- 0.7 microg hydroxy(OH)-proline/mg dry weight, p < 0.01) but not in GGT-/- (10.4 +/- 1.7 versus 8.8 +/- 0.8). Control lungs from GGT-/- showed a significant reduction of cysteine (0.03 +/- 0.005 versus 0.055 +/- 0.001, p < 0.02) and GSH levels (1.24 +/- 0.055 versus 1.79 +/- 0.065, p < 0.002). These values decreased after 72 hours of bleomycin in both GGT-/- and wild-type but reached their respective control values after 1 month. Supplementation with N-acetyl cysteine partially ameliorated the effects of GGT deficiency. These findings suggest that increased neutrophils and matrix metalloproteinase-9 during the early inflammatory response and adequate thiol reserves are key elements in the fibrotic response after bleomycin-induced pulmonary injury.