Machine intelligence identifies soluble TNFa as a therapeutic target for spinal cord injury.

Traumatic spinal cord injury (SCI) produces a complex syndrome that is expressed across multiple endpoints ranging from molecular and cellular changes to functional behavioral deficits. Effective therapeutic strategies for CNS injury are therefore likely to manifest multi-factorial effects across a broad range of biological and functional outcome measures. Thus, multivariate analytic approaches are needed to capture the linkage between biological and neurobehavioral outcomes. Injury-induced neuroinflammation (NI) presents a particularly challenging therapeutic target, since NI is involved in both degeneration and repair. Here, we used big-data integration and large-scale analytics to examine a large dataset of preclinical efficacy tests combining five different blinded, fully counter-balanced treatment trials for different acute anti-inflammatory treatments for cervical spinal cord injury in rats. Multi-dimensional discovery, using topological data analysis (TDA) and principal components analysis (PCA) revealed that only one showed consistent multidimensional syndromic benefit: intrathecal application of recombinant soluble TNFα receptor 1 (sTNFR1), which showed an inverse-U dose response efficacy. Using the optimal acute dose, we showed that clinically-relevant 90 min delayed treatment profoundly affected multiple biological indices of NI in the first 48 h after injury, including reduction in pro-inflammatory cytokines and gene expression of a coherent complex of acute inflammatory mediators and receptors. Further, a 90 min delayed bolus dose of sTNFR1 reduced the expression of NI markers in the chronic perilesional spinal cord, and consistently improved neurological function over 6 weeks post SCI. These results provide validation of a novel strategy for precision preclinical drug discovery that is likely to improve translation in the difficult landscape of CNS trauma, and confirm the importance of TNFα signaling as a therapeutic target.

Inhibition of HIF-prolyl-4-hydroxylases prevents mitochondrial impairment and cell death in a model of neuronal oxytosis.

Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases.

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely ß2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.

Activating transcription factor 4 mediates hyperglycaemia-induced endothelial inflammation and retinal vascular leakage through activation of STAT3 in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: There is convincing evidence that endoplasmic reticulum (ER) stress is implicated in the pathogenesis of diabetes and its complications; however, the mechanisms are not fully understood. This study aimed to dissect the role and signalling pathways of activating transcription factor 4 (ATF4) in ER-stress-associated endothelial inflammation and diabetic retinopathy. METHODS: ER stress and ATF4 activity were manipulated by complementary pharmacological and genetic approaches in cultured retinal endothelial (TR-iBRB) cells. Diabetes was induced by streptozotocin in heterozygous Atf4 knockout and wild-type mice. ER stress markers, inflammatory cytokines and adhesion molecules, activation of the signal transducer and activator of transcription 3 (STAT3) pathway, and retinal vascular permeability were measured. RESULTS: High-glucose treatment resulted in rapid induction of ER stress, activation of ATF4, and increased production of inflammatory factors in TR-iBRB cells. Suppressing ER stress or inhibiting ATF4 activity markedly attenuated high-glucose-induced production of intercellular adhesion molecule 1, TNF-α and vascular endothelial growth factor. Conversely, enhancing ER stress or overexpressing Atf4 was sufficient to induce endothelial inflammation, which was, at least in part, through activation of the STAT3 pathway. Furthermore, knockdown of the Stat3 gene or inhibiting STAT3 activity restored ER homeostasis in cells exposed to high glucose and prevented ATF4 activation, suggesting that STAT3 is required for high-glucose-induced ER stress. Finally, we showed that downregulation of Atf4 significantly ameliorated retinal inflammation, STAT3 activation and vascular leakage in a mouse model of type 1 diabetes. CONCLUSIONS/INTERPRETATION: Taken together, our data reveal a pivotal role of ER stress and the ATF4/STAT3 pathway in retinal endothelial inflammation in diabetic retinopathy.

Drosophila importin alpha1 performs paralog-specific functions essential for gametogenesis.

Importin alpha's mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin alpha's, alpha1's, alpha2's, and alpha3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster we describe the isolation and phenotypic characterization of the first animal importin alpha1 mutant. Animal alpha1's are more similar to ancestral plant and fungal alpha1-like genes than to animal alpha2 and alpha3 genes. Male and female importin alpha1 (Dalpha1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dalpha1 mutant phenotypes were rescued by Dalpha1 transgenes, but not by Dalpha2 or Dalpha3 transgenes. Genetic interactions between the ectopic expression of Dalpha1 and the karyopherins CAS and importin beta1 suggest that high nuclear levels of Dalpha1 are deleterious. We conclude that Dalpha1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dalpha1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin alpha-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin alpha-gene family.

Nationwide data confirms absence of 'July phenomenon' in obstetrics: it's safe to deliver in July.

OBJECTIVE(S): To determine whether operator-dependent obstetric complications occur at higher rates in July at teaching hospitals using a large, nationwide sample of deliveries. STUDY DESIGN: Data for this study were obtained from an administrative dataset, the Nationwide Inpatient Sample, for the years 1998 to 2002. Singleton deliveries and singleton livebirth admissions among Medicaid patients at teaching hospitals with OB/GYN residents working on the Labor and Delivery ward were identified. Outcomes for various complications for these patients in the month of July were compared to those occurring in the months from August to June. RESULTS: The 26,546 women in our cohort who delivered in July were compared to the 272,584 women delivering during August to June. There were no statistically significant differences in the rates of cesarean delivery, urethral/bladder injury, third or fourth degree lacerations, wound complications, postpartum hemorrhage, transfusion, shoulder dystocia, chorioamnionitis or anesthesia-related complications. The 26,175 singleton livebirth admissions in July were compared to 266,158 such admissions in August to June. There were no statistically significant differences in the rates of brachial plexus injury (0.2 vs 0.2%, P=0.824) or birth asphyxia (0.1 vs 0.1%, P=0.643). CONCLUSION(S): This study shows no increased rate of operator-dependent complications of delivery at teaching hospitals nationwide in the month of July.

Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

Resistance of human renal cell carcinoma (RCC) and melanoma to the apoptosis-inducing effects of IFNs was postulated to result from epigenetic silencing of genes by DNA methylation, a common feature of human cancers. To reverse silencing, 5-AZA-deoxycytidine (5-AZA-dC) or selective depletion of DNA methyltransferase 1 (DNMT1) by phosphorothioate oligonucleotide antisense (DNMT1 AS) were employed in cells resistant (<5% terminal deoxynucleotidyl transferase-mediated nick-end labeling positive) to apoptosis induction by IFN-alpha2 and IFN-beta (ACHN, SK-RC-45, and A375). 5-AZA-dC and DNMT1 AS similarly depleted available DNMT1 protein and, at doses that did not cause apoptosis alone, resulted in apoptotic response to IFNs. The proapoptotic tumor suppressor RASSF1A was reactivated by DNMT1 inhibitors in all three cell lines. This was associated with demethylation of its promoter region. IFNs augmented RASSF1A protein expression after reactivation by DNMT1 inhibition. In IFN-sensitive WM9 melanoma cells, expression of RASSF1A was constitutive but also augmented by IFNs. RASSF1A small interfering RNA reduced IFN-induced apoptosis in WM9 cells and in DNMT1-depleted ACHN cells. Conversely, lentiviral expression of RASSF1A but not transduction with empty virus enabled IFN-induced apoptosis. IFN induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL-neutralizing antibody inhibited apoptotic response to IFN in RASSF1A-expressing ACHN cells. Accordingly, RASSF1A markedly sensitized to recombinant TRAIL. Normal kidney epithelial cells, although expressing RASSF1A, did not undergo apoptosis in response to IFN or TRAIL but had >400-fold higher TRAIL decoy receptor 1 expression than transduced ACHN cells (real-time reverse transcription-PCR). Results identified RASSF1A as regulated by IFNs and participating in IFN-induced apoptosis at least in part by sensitization to TRAIL.

Sequence-selective DNA binding drugs mithramycin A and chromomycin A3 are potent inhibitors of neuronal apoptosis induced by oxidative stress and DNA damage in cortical neurons.

Global inhibitors of RNA or protein synthesis such as actinomycin D or cycloheximide abrogate neuronal apoptosis induced by numerous pathological stimuli in vitro and in vivo. The clinical application of actinomycin D or cycloheximide to human neurological disease has been limited by the toxicities of these agents. To overcome these toxicities, strategies must be developed to inhibit selectively the expression of deleterious proapoptotic proteins, while leaving the expression of antiapoptotic, proregeneration, and other critical homeostatic proteins unperturbed. Mithramycin A (trade name Plicamycin) is an aureolic acid antibiotic that has been used in humans to treat hypercalcemia and several types of cancers. This class of agents is believed to act, in part, by selectively inhibiting gene expression by displacing transcriptional activators that bind to G-C-rich regions of promoters. Here we demonstrate that mithramycin A and its structural analog chromomycin A3 are potent inhibitors of neuronal apoptosis induced by glutathione depletion-induced oxidative stress or the DNA-damaging agent camptothecin. We correlate the protective effects of mithramycin A with its ability to inhibit enhanced DNA binding of the transcription factors Sp1 and Sp3 to their cognate "G-C" box induced by oxidative stress or DNA damage. The protective effects of mithramycin A cannot be attributed to global inhibition of protein synthesis. Together, these results suggest that mithramycin A and its structural analogs may be effective agents for the treatment of neurological diseases associated with aberrant activation of apoptosis and highlight the potential use of sequence-selective DNA-binding drugs as neurological therapeutics.

Epidermal growth factor receptor induced apoptosis: potentiation by inhibition of Ras signaling.

Previous studies have shown that certain tumor cell lines which naturally express high levels of the epidermal growth factor receptor (EGFR) undergo apoptosis when exposed to epidermal growth factor. Whether this phenomenon is a direct result of receptor overexpression or some other genetic alteration renders these cells sensitive to apoptosis is yet to be established. We show that experimentally increasing the level of EGFR expression predictably leads to apoptosis in a variety of cell types which requires an active tyrosine kinase but not EGFR autophosphorylation sites. Expression of a dominant negative Ras mutant in EGFR overexpressing cells results in a significant potentiation of EGFR induced apoptosis suggesting that Ras activation is a key survival signal generated by the EGFR. We propose that potentiation of EGFR induced apoptosis by dominant negative Ras results, at least in part, by a block of Akt activation.

L-buthionine sulfoximine potentiates the antitumor effect of 4-hydroperoxycyclophosphamide when administered locally in a rat glioma model.

OBJECTIVE: L-buthionine sulfoximine (BSO) inhibits glutathione synthesis and may modulate tumor resistance to some alkylating agents, but it has not been proven effective in the treatment of intracranial neoplasms. To evaluate this drug for the treatment of brain tumors, we studied the use of BSO for potentiating the antineoplastic effect of 4-hydroxyperoxycyclophosphamide (4-HC) in the rat 9L glioma model. METHODS: The survival of male Fischer 344 rats with intracranial 9L gliomas was measured after implantation of controlled-release polymers containing one of the following: no drug, BSO, 4-HC, or both BSO and 4-HC. The efficacy of intracranial 4-HC treatment was assessed with and without serial systemic intraperitoneal BSO injections. Tissue glutathione levels were measured in the brains, tumors, and livers of animals treated with intraperitoneal injections or local delivery of BSO. RESULTS: The median survival of animals treated with intracranial polymers containing 4-HC was 2.3 times greater than that of controls. This survival benefit was doubled by local delivery of BSO. In contrast, systemic BSO therapy did not improve survival time. In animals that were treated systemically, both liver and tumor glutathione levels were significantly lower than they were in control animals. In the locally treated animals, glutathione levels were reduced in the brain tumor but not in the liver. CONCLUSION: These results demonstrate that local but not systemic delivery of BSO enhances the antineoplastic effect of 4-HC in this rat 9L glioma model. In addition, because local delivery of BSO within the brain did not deplete glutathione levels systemically, this method of treatment may be safer than systemic administration of BSO.

The epidermal growth factor receptor engages receptor interacting protein and nuclear factor-kappa B (NF-kappa B)-inducing kinase to activate NF-kappa B. Identification of a novel receptor-tyrosine kinase signalosome.

The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by a diverse number of stimuli including tumor necrosis factor-alpha, interleukin-1, UV irradiation, viruses, as well as receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). NF-kappaB activation by the tumor necrosis factor receptor (TNFR) involves the formation of a multiprotein complex termed a signalosome. Although previous studies have shown that the activated EGFR can induce NF-kappaB, the mechanism of this activation remains unknown. In this study, we identify components of the signalosome formed by the activated EGFR required to activate NF-kappaB and show that, although the activated EGFR uses mechanisms similar to the TNFR, it recruits a distinct signalosome. We show the EGFR forms a complex with a TNFR-interacting protein (RIP), which plays a key role in TNFR-induced NF-kappaB activation, but not with TRADD, an adaptor protein which serves to recruit RIP to the TNFR. Furthermore, we show that the EGFR associates with NF-kappaB-inducing kinase (NIK) and provide evidence suggesting multiprotein complex formation between the EGFR, RIP, and NIK. Using a dominant negative NIK mutant, we show that NIK activation is required for EGFR-mediated NF-kappaB induction. We also show that a S32/36 IkappaBalpha mutant blocks EGFR-induced NF-kappaB activation. Our studies also suggest that a high level of EGFR expression, a frequent occurrence in human tumors, is optimal for epidermal growth factor-induced NF-kappaB activation. Finally, although protein kinase B/Akt has been implicated in tumor necrosis factor and PDGF-induced NF-kappaB activation, our studies do not support a role for this protein in EGFR-induced NF-kappaB activation.

Ironing-out mechanisms of neuronal injury under hypoxic-ischemic conditions and potential role of iron chelators as neuroprotective agents.

Iron is the most abundant transition metal in the brain, where it functions as an important cofactor in a host of vital metabolic processes and plays an absolutely essential role in cell viability. Free iron is also very toxic when present in high concentrations, thus placing this essential metal at the core of neurotoxic injury in a number of neurological disorders. The pivotal role of iron in cellular homeostasis, including its latent toxicity, necessitates a tight regulation of iron metabolism. Oxygen and iron appear to play an important role in iron homeostasis. They appear to exert their homeostatic role by modulating the proteins involved in a complex interplay between iron sensing, transport, and storage. These key regulatory proteins include ferritin (intracellular storage), transferrin (extracellular transport), transferrin receptor, and iron regulatory protein (sensor of intracellular iron concentration). The interplay of iron and oxygen is most intriguing in the setting of stroke, where hypoxia and free iron appear to interact in causing the subsequent neuronal death.

Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/CREB and increased expression of glycolytic enzymes, p21(waf1/cip1), and erythropoietin.

Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway in non-neuronal cells that culminates in the stabilization of the transcriptional activator hypoxia-inducible factor-1 (HIF-1) and increased expression of gene products that mediate hypoxic adaptation. We examined the hypothesis that iron chelators prevent oxidative stress-induced death in cortical neuronal cultures by inducing expression of HIF-1 and its target genes. We report that the structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic cortical neuronal cultures. The protective effects of iron chelators are correlated with their ability to enhance DNA binding of HIF-1 and activating transcription factor 1(ATF-1)/cAMP response element-binding protein (CREB) to the hypoxia response element in cortical cultures and the H19-7 hippocampal neuronal cell line. We show that mRNA, protein, and/or activity levels for genes whose expression is known to be regulated by HIF-1, including glycolytic enzymes, p21(waf1/cip1), and erythropoietin, are increased in cortical neuronal cultures in response to iron chelator treatment. Finally, we demonstrate that cobalt chloride, which also activates HIF-1 and ATF-1/CREB in cortical cultures, also prevents oxidative stress-induced death in these cells. Altogether, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress.

Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis.

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O-2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O-2 levels appears to activate or modulate cell death, as a recombinant SV expressing the O-2 scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O-2 rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O-2 by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.

p53-independent inhibition of proliferation and p21(WAF1/Cip1)-modulated induction of cell death by the antioxidants N-acetylcysteine and vitamin E.

Epidemiological evidence has suggested an association between diets rich in antioxidants and diminished risks of various types of cancer. Proposed mechanisms for protective effects of antioxidants have involved inhibition of free radical-mediated DNA damage. Recent data suggest that antioxidants may prevent or eliminate cancerous cells through their ability to inhibit proliferation or to induce programmed cell death (PCD). To begin to identify cell cycle and cell death regulatory factors involved in antioxidant-induced growth arrest and PCD, we have studied colorectal carcinoma cells (CRCs) that differ in expression of the tumor suppressor protein p53, and of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). The antioxidants, N-acetylcysteine (NAC) and vitamin E either inhibited proliferation in a p53-independent manner without affecting cell viability or induced cell death. Growth arrest was not associated with upregulation of the CDK inhibitors p21(Waf1/Cip1), p18(ink4c) or p16(ink4a), but was associated with a decrease in reactive oxygen species (ROS). In contrast to previous observations, the absence of p21(Waf1/Cip1) increased susceptibility of CRCs to antioxidant-induced PCD. NAC decreased levels of retinoblastoma protein (Rb) phosphorylation in all cells tested, but Rb was cleaved only in cells which underwent NAC-induced death. Although NAC decreased ROS in all cells studied, cell lines in which PCD occurred had higher baseline levels of ROS than cell lines in which proliferation was blocked. These observations suggest that expression of p21(Waf1/Cip1) and basal levels of ROS are important determinants of outcome after antioxidant treatment.

Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor.

The calcium-sensing receptor (CaR) is a membrane-bound, G-protein-coupled receptor present on parathyroid cells which monitors the level of extracellular calcium (Ca2+o) and transduces signals involved in serum calcium regulation. Expression of CaR protein in tissues with functions unrelated to systemic calcium homeostasis, including the brain, suggests that extracellular calcium (Ca2+o) may act as a first messenger to regulate diverse cellular functions. To test this hypothesis, we examined the effect of increasing Ca2+o on apoptosis induced by Sindbis Virus in AT-3 prostate carcinoma cells. We found a steep increase in cell survival with between 5 and 7 mM added Ca2+o (EC50 = 6.1 mM). Magnesium, a less potent agonist of the calcium sensing receptor, was also protective (EC50 = 23.4 mM). Northern and immunocytochemical analyses confirmed the presence of the CaR message and protein in AT-3 prostate carcinoma cells. Enforced expression of CaR protein by stable transfection in human embryonic kidney (HEK)-293 cells, which normally don't express the receptor, resulted in resistance to SV-induced apoptosis in the presence of elevated Ca2+o. In addition to preventing SV-induced death, elevated Ca2+o also abrogated apoptosis induced by c-Myc overexpression/serum deprivation in rat 1A fibroblasts, and these fibroblasts were shown to express CaR message and protein. Altogether, these observations suggest that Ca2+o can act with the CaR to prevent apoptosis and define a novel mechanism by which calcium ions can regulate cell survival.

Suppression of steady-state, but not stimulus-induced NF-kappaB activity inhibits alphavirus-induced apoptosis.

Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus.

Purification of a multipotent antideath activity from bovine liver and its identification as arginase: nitric oxide-independent inhibition of neuronal apoptosis.

Catalase is an antioxidant enzyme that has been shown to inhibit apoptotic or necrotic neuronal death induced by hydrogen peroxide. We report the purification of a contaminating antiapoptotic activity from a commercial bovine liver catalase preparation by following its ability to inhibit apoptosis when applied extracellularly in multiple death paradigms. The antiapoptotic activity was identified by protein microsequencing as arginase, a urea cycle and nitric oxide synthase-regulating enzyme, and confirmed by demonstrating the presence of antiapoptotic activity in a >97% pure preparation of recombinant arginase. The pluripotency of recombinant arginase was demonstrated by its ability to inhibit apoptosis in multiple paradigms including rat cortical neurons induced to die by glutathione depletion and oxidative stress, by 100 nM staurosporine treatment, or by Sindbis virus infection. The protective effects of arginase in these apoptotic paradigms, in contrast to previous studies on excitotoxic neuronal necrosis, are independent of nitric oxide synthase inhibition. Rather, arginase-induced depletion of arginine leads to inhibition of protein synthesis, resulting in cell survival. Because inhibitors of nitric oxide synthesis and of protein synthesis have been shown to decrease necrotic and apoptotic death, respectively, in animal models of stroke and spinal cord injury, arginine-depleting enzymes, capable of simultaneously inhibiting protein synthesis and nitric oxide generation, may be propitious therapeutic agents for acute neurological diseases. Furthermore, our results suggest caution in attributing the cytoprotective effects of some catalase preparations to catalase.

The epidermal growth factor receptor associates with and recruits phosphatidylinositol 3-kinase to the platelet-derived growth factor beta receptor.

Receptor tyrosine kinases are classified into subfamilies, which are believed to function independently, with heterodimerization occurring only within the same subfamily. In this study, we present evidence suggesting a direct interaction between the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor beta (PDGFbeta) receptor (PDGFbetaR), members of different receptor tyrosine kinase subfamilies. We find that the addition of EGF to COS-7 cells and to human foreskin Hs27 fibroblasts results in a rapid tyrosine phosphorylation of the PDGFbetaR and results in the recruitment of phosphatidylinositol 3-kinase to the PDGFbetaR. In R1hER cells, which overexpress the EGFR, we find ligand-independent tyrosine phosphorylation of the PDGFbetaR and the constitutive binding of a substantial amount of PI-3 kinase activity to it, mimicking the effect of ligand in untransfected cells. In support of the possibility that this may be a direct interaction, we show that the two receptors can be coimmunoprecipitated from untransfected Hs27 fibroblasts and from COS-7 cells. This association can be reconstituted by introducing the two receptors into 293 EBNA cells. The EGFR/PDGFbetaR association is ligand-independent in all cell lines tested. We also demonstrate that the fraction of PDGFbetaR bound to the EGFR in R1hER cells undergoes an EGF-induced mobility shift on Western blots indicative of phosphorylation. Our findings indicate that direct interactions between receptor tyrosine kinases classified under different subfamilies may be more widespread than previously believed.

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration.

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.