Mariarosaria Santillo

Role of the small GTPase Rab7 in the late endocytic pathway.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen‐1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.

DNA Damage, Homology-Directed Repair, and DNA Methylation

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Oxidative stress and the pathogenesis of scleroderma : the Murrell's hypothesis revisited

Systemic sclerosis (SSc, scleroderma) is a devastating, immune-mediated, multisystem disorder characterized by microvasculature damage, circulating autoantibodies, and fibroblast activation, leading to massive fibrosis of skin, vessels, muscles, and visceral organs. Scleroderma causes disability and death as the result of end-stage organ failure. At present, no specific diagnostic nor therapeutic tools are available to handle the disease. In spite of significant effort, the etiology and pathogenesis of SSc remain obscure and, consequently, the disease outcome is unpredictable. Several years ago, Murrell suggested a unifying hypothesis linking the pathogenesis of scleroderma to the generation of a large excess of reactive oxygen species. This hypothesis has been substantiated by several reports indicating the presence of an abnormal redox state in patients with scleroderma. This review will summarize the available evidence supporting the link between free radicals and the main pathological features of scleroderma.

Evidence for secretion of cytosolic CuZn superoxide dismutase by Hep G2 cells and human fibroblasts

The role so far ascribed to intracellular CuZn superoxide dismutase is that of an intracellular scavenger of oxygen radicals. However, other functions of cytosolic CuZn superoxide dismutase have been hypothesized. For example, CuZn superoxide dismutase incubated with rat hepatocyte cells in culture inhibits 3-hydroxy-3methylglutaryl CoA reductase, thereby reducing cholesterol synthesis. We recently demonstrated the presence of surface membrane receptors for CuZn superoxide dismutase, suggesting possible autocrine or paracrine activities. The aim of the present study was to investigate whether cytosolic CuZn superoxide dismutase can be secreted by human hepatocarcinoma and fibroblast cells lines. Proteins in human hepatocellular carcinoma (Hep G2) cells and human fibroblasts were biosynthetically labelled with [35S]-cysteine; then cell lysates and media were immunoprecipitated with rabbit polyclonal anti-human CuZn superoxide dismutase antibodies and separated by 12% polyacrylamide gel electrophoresis. Both Hep G2 cells and human fibroblasts produce and secrete CuZn superoxide dismutase which was detectable in cells and medium as a single protein band with the same electrophoretic mobility as human erythrocyte CuZn superoxide dismutase. These data suggest that CuZn superoxide dismutase, an enzyme thus far considered to be located exclusively intracellularly is secreted by at least two cell lines. This is consistent with autocrine or paracrine roles for CuZn superoxide dismutase.

Early and Late Events Induced by PolyQ-expanded Proteins IDENTIFICATION OF A COMMON PATHOGENIC PROPERTY OF POLYQ-EXPANDED PROTEINS

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24–36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

NO‐induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn‐SOD activity and expression via RAS/ERK1/2 pathway

To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn‐superoxide dismutase (Mn‐SOD) expression and activity. In addition, the possible involvement of Ras/extracellular‐regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30‐min sublethal OGD (95% N2 and 5% CO2). Then, after a 24‐h interval, neurons were exposed to 3 h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn‐SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L‐NAME, a well known NOS inhibitor, the increase in Mn‐SOD expression occurring during IPC was reduced and, as a result, IPC‐induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn‐SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn‐SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn‐SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post‐transcriptionally activates Mn‐SOD expression and activity, thus promoting neuroprotection during preconditioning.

HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway

In this study we have investigated the effects of the small GTP‐binding‐protein Ras on the redox signalling of the human neuroblastoma cell line, SK‐N‐BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK‐HaRas) cells than in control cells. The treatment of cells with 4‐(2‐aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK‐HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67phox and Rac to the plasma membrane. These effects depended on the mitogen‐activated protein kinase kinase/extracellular signal‐regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas‐mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67phox and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas‐induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.

Secretion and increase of intracellular CuZn superoxide dismutase content in human neuroblastoma SK-N-BE cells subjected to oxidative stress

CuZn superoxide dismutase (SOD) secretion was detected in media of [35S]cysteine-labeled human neuroblastoma SK-N-BE cells precipitated with antihuman CuZn SOD antibodies. The ability of Fe2+/ascorbate oxidative stress to induce CuZn SOD in SK-N-BE cells was evaluated by Western blot analysis. The results showed that, like human hepatocarcinoma cells and human fibroblasts, SK-N-BE cells secrete CuZn SOD. In addition, the CuZn SOD concentration was higher in cells subjected to oxidative stress than in unstressed cells. The secretion of CuZn SOD and the ability of Fe2+/ascorbate to increase its protein content in SK-N-BE cells indicates that this enzyme protects the brain from damage induced by oxidative stress.