Michael W. Lieberman

S-Nitrosothiols signal the ventilatory response to hypoxia

Increased ventilation in response to hypoxia has been appreciated for over a century, but the biochemistry underlying this response remains poorly understood. Here we define a pathway in which increased minute ventilation ([Vdot]E ) is signalled by deoxyhaemoglobin-derived S-nitrosothiols (SNOs). Specifically, we demonstrate that S-nitrosocysteinyl glycine (CGSNO) and S-nitroso-l-cysteine (l-CSNO)—but not S-nitroso-d-cysteine (d-CSNO)—reproduce the ventilatory effects of hypoxia at the level of the nucleus tractus solitarius (NTS). We show that plasma from deoxygenated, but not from oxygenated, blood produces the ventilatory effect of both SNOs and hypoxia. Further, this activity is mediated by S-nitrosoglutathione (GSNO), and GSNO activation by γ-glutamyl transpeptidase (γ-GT) is required. The normal response to hypoxia is impaired in a knockout mouse lacking γ-GT. These observations suggest that S-nitrosothiol biochemistry is of central importance to the regulation of breathing.

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.

Ultraviolet radiation-induced metallothionein-I gene activation is associated with extensive DNA demethylation

Ultraviolet irradiation (UV) of cadmium-sensitive S49 mouse cells induces a large increase in cadmium-resistant variants. About 30%-40% of these variants make metallothionein (MT)-I mRNA while S49 cells do not. S49 cells contain two copies of the MT-I gene; both alleles are heavily methylated but can be conveniently distinguished by the methylation status of a single Hpa II site. In lines expressing MT-I, one allele becomes completely demethylated at all methylation-sensitive restriction sites examined over at least a 2.5 kb region spanning the MT-I gene. Activation of a quiescent gene by UV has implications for understanding the initiation of carcinogenesis.

Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA

Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites.

System xc− 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 γ-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.

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 kinases direct interaction with arsenite, and that arsenics suppression of JAK tyrosine kinase activity also occurred in the interferon γ (IFNγ) 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.

Disruption of γ-Glutamyl Leukotrienase Results in Disruption of Leukotriene D4 Synthesis In Vivo and Attenuation of the Acute Inflammatory Response

ABSTRACT To study the function of γ-glutamyl leukotrienase (GGL), a newly identified member of the γ-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGLtm1) and in both GGL and GGT (GGLtm1-GGTtm1) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGLtm1 show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but ∼70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C4 (LTC4) to LTD4, indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD4. Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC4 and LTD4 have distinctly different functions in the inflammatory process.

Suramin inhibits wound healing following filtering procedures for glaucoma.

BACKGROUND Trabeculectomies are the most frequently performed procedures in surgically treating eyes with glaucoma. Failures are caused by fibrosis in the external ostium of the filtering procedure. In order to inhibit the fibrotic wound healing reaction, a new pharmacological approach using suramin, which inhibits a variety of important growth factors was used. METHODS Pigmented rabbits were used and filtering procedures performed. Suramin was applied with concentrations ranging from 10 mg/ml to 333 mg/ml once during surgery and four times following surgery. The success of the filtering procedure was assessed by intraocular pressure measurements. To evaluate possible intraocular toxic effects, treated eyes were histopathologically evaluated after 4 weeks, and the ciliary body adjacent to the site of application was examined using electron microscopy. RESULTS With concentrations of suramin of 200 mg/ml and 333 mg/ml, the trabeculectomies were patent longer than in the controls and in eyes operated with mitomycin C, which currently is the most frequently used antiproliferative drug to enhance the outcome of surgery in humans. No severe toxic effects to the ciliary epithelium were seen in suramin treated eyes. CONCLUSIONS This study demonstrates for the first time the efficiency of a substance that broadly inhibits the action of growth factors on target cells in the setting of ocular wound healing. In this in vivo model, suramin has been shown to be highly effective in preventing scarring and in having fewer toxic side effects than usually used antimetabolites. These results therefore may suggest a new approach to the surgical treatment of glaucoma.

Pancreatic Necrosectomy Using Covered Esophageal Stents A Novel Approach

Background: Endoscopic necrosectomy for necrotizing pancreatitis has been increasingly used as an alternative to surgical or percutaneous interventions. The use of fully covered esophageal self-expandable metallic stents may provide a safer and more efficient route for internal drainage. The aim of this study was to evaluate the safety and efficacy of endoscopic treatment of pancreatic necrosis with these stents. Methods: A retrospective study at 2 US academic hospitals included patients with infected pancreatic necrosis from July 2009 to November 2012. These patients underwent transgastric placement of fully covered esophageal metallic stents draining the necrosis. After necrosectomy, patients underwent regular sessions of endoscopic irrigation and debridement of cystic contents. The efficacy endpoint was successful resolution of infected pancreatic necrosis without the need for surgical or percutaneous interventions. Results: Seventeen patients were included with the mean age of 41±12 years. A mean of 5.3±3.4 sessions were required for complete drainage and the follow-up period was 237.6±165 days. Etiology included gallstone pancreatitis (6), alcohol abuse (6), s/p distal pancreatectomy (2), postendoscopic retrograde cholangiopancreatography pancreatitis (1), medication-induced pancreatitis (1), and hyperlipidemia (1). Mean size of the necrosis was 14.8 cm (SD 5.6 cm), ranging from 8 to 19 cm. Two patients failed endoscopic intervention and required surgery. The only complication was a perforation during tract dilation, which was managed conservatively. Fifteen patients (88%) achieved complete resolution. Conclusions: Endoscopic necrosectomy with covered esophageal metal stents is a safe and successful treatment option for infected pancreatic necrosis.

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.