Redox regulation of ERK1/2 activation induced by sphingosine 1-phosphate in fibroblasts: involvement of NADPH oxidase and platelet-derived growth factor receptor.

BACKGROUND: Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite synthesized after stimulation with growth factors or cytokines. S1P extracellular effects are mediated through specific Gi-protein coupled receptors (GPCRs). Recently, we demonstrated in NIH3T3 fibroblasts stimulated by platelet-derived growth factor (PDGF) or S1P the NADPH oxidase activation and the H(2)O(2) intracellular level increase trough the Gi protein involvement. METHODS: NIH3T3 fibroblast cell cultures were used. Western blot and quantitative analyses by Chemidoc-Quantity-One software were performed. H(2)O(2) level was assayed by fluorescence spectrophotometric analysis, and cell proliferation by counted manually or ELISA kit. RESULTS: This study demonstrates, in NIH 3T3 fibroblasts, a novel redox regulated mechanism of S1P-induced activation of ERK 1/2 related to NADPH oxidase activity and intracellular H(2)O(2) level increase with PDGF receptor tyrosine kinase involvement through a transactivation mechanism. This event is mediated by S1P(1) and S1P(3) receptors by Gi proteins and can contribute to S1P mitogenic signaling. CONCLUSION: These results can be related to mechanisms of cross-talk previously identified between receptor tyrosine kinase, including PDGFreceptor, and several GPCR ligands. GENERAL SIGNIFICANCE: The redox-sensitive ERK1/2 and PDGFr tyrosine kinase activity could be targets for therapies in diseases in which deregulation of intracellular oxidative status and the consequent alteration of S1P and/or PDGF signaling pathway are involved.

Sphingosine 1-phosphate stimulation of NADPH oxidase activity: relationship with platelet-derived growth factor receptor and c-Src kinase.

This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H2O2 production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H2O2 production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H2O2 increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H2O2 production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H2O2 production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.

Protective effect of new S-acylglutathione derivatives against amyloid-induced oxidative stress.

Recent data support the role of oxidative stress in the pathogenesis of Alzheimer disease (AD). In particular, glutathione (GSH) metabolism is altered and its levels are decreased in affected brain regions and peripheral cells from AD patients and in experimental models of AD. In the past decade, interest in the protective effects of various antioxidants aimed at increasing intracellular GSH content has been growing. Because much experimental evidence suggests a possible protective role of unsaturated fatty acids in age-related diseases, we designed the synthesis of new S-acylglutathione (acyl-SG) thioesters. S-Lauroylglutathione (lauroyl-SG) and S-palmitoleoylglutathione (palmitoleoyl-SG) were easily internalized into the cells and they significantly reduced Abeta42-induced oxidative stress in human neurotypic SH-SY5Y cells. In particular, acyl-SG thioesters can prevent the impairment of intracellular ROS scavengers, intracellular ROS accumulation, lipid peroxidation, and apoptotic pathway activation. Palmitoleoyl-SG seemed more effective in cellular protection against Abeta-induced oxidative damage than lauroyl-SG, suggesting a valuable role for the monounsaturated fatty acid. In this study, we demonstrate that acyl-SG derivatives completely avoid the sharp lipoperoxidation in primary fibroblasts from familial AD patients occurring after exposure to Abeta42 aggregates. Hence, we put forward these derivatives as new antioxidant compounds which could be excellent candidates for therapeutic treatment of AD and other oxidative stress-related diseases.

Apoptosis and Bax, Bcl-2, Mcl-1 expression in neutrophils of Crohn's disease patients.

BACKGROUND: The etiology of Crohn's disease (CD) remains unknown, and the defective function of neutrophils appears to be associated with this pathology. Neutrophils undergo spontaneous apoptosis which, if not tightly regulated, can induce the development of chronic inflammatory disease. The Bcl-2 protein family is also involved in the regulation of neutrophil apoptosis. METHODS: This study investigated the apoptosis and expression of some regulatory factors in CD patient and control polymorphonuclear neutrophils (PMN) in suspension and in adhesion on fibronectin, an extracellular matrix protein. These 2 conditions mimic circulating neutrophils before they are recruited at the intestinal levels, and their adhesion to tissue. RESULTS: Apoptosis in CD patient PMN was delayed in suspension and accelerated in adhesion, which is the opposite of what happens in controls. Higher levels of Bax, Bcl-2, and Mcl-1 proteins were registered in freshly isolated CD patient PMN, in contrast to controls, in which Bcl-2 protein was undetectable. Among the studied pro- and antiapoptotic factors, Bax levels seem to be mainly related to the difference in apoptosis between PMN of CD patients and controls. CONCLUSIONS: For the first time it has been demonstrated by direct experimental evidence that apoptosis in CD patient PMN is regulated differently from that of control PMN. Abnormal expression of regulating apoptosis proteins is shown in CD patient PMN. These data suggest that the defective functionality of neutrophils can be the early event responsible for the altered mucosal immune response in CD, and that neutrophil apoptosis may offer a new target for specific drugs and therapy tools.

Redox regulation of platelet-derived-growth-factor-receptor: role of NADPH-oxidase and c-Src tyrosine kinase.

This study identifies some early events contributing to the redox regulation of platelet-derived growth factor receptor (PDGFr) activation and its signalling in NIH3T3 fibroblasts. We demonstrate for the first time that the redox regulation of PDGFr tyrosine autophosphorylation and its signalling are related to NADPH oxidase activity through protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K) activation and H2O2 production. This event is also essential for complete PDGF-induced activation of c-Src kinase by Tyr416 phosphorylation, and the involvement of c-Src kinase on H2O2-induced PDGFr tyrosine phosphorylation is demonstrated, suggesting a role of this kinase on the redox regulation of PDGFr activation. Finally, it has been determined that not only PI3K activity, but also PKC activity, are related to NADPH oxidase activation due to PDGF stimulation in NIH3T3 cells, as it occurs in non-phagocyte cells. Therefore, we suggest a redox circuit whereby, upon PDGF stimulation, PKC, PI3K and NADPH oxidase activity contribute to complete c-Src kinase activation, thus promoting maximal phosphorylation and activation of PDGFr tyrosine phosphorylation.

Redox state and O2*- production in neutrophils of Crohn's disease patients.

The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohn's disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2*-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-alpha)- or phorbol 12-myristate 13-acetate (PMA)-induced O2*- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and beta-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-alpha cannot be caused by excessive reactive oxygen species (ROS) production induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony-stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2*- production and adhesion, caused, in part, by an anomalous response to TNF-alpha, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the chronic inflammation and relapses that characterize this pathology. A possible role of GM-CSF in inducing O2*- production and in restoring the defensive role of neutrophils in CD patients is suggested.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells.

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

Oxidative state and IL-6 production in intestinal myofibroblasts of Crohn's disease patients.

BACKGROUND: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohn's disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. METHODS: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N-acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography (HPLC) methods. Interleukin (IL)-6 production was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL-6 production or is stimulated by tumor necrosis factor alpha (TNFα) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL-6 synthesis, were also increased when there is oxidative stress in ISEMFs. CONCLUSIONS: This study shows for the first time in ISEMFs of CD patients an increased production of IL-6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology.

Involvement of the tyrosine phosphorylation on GSH transport in NIH3T3 fibroblasts.

This study demonstrates the involvement of phosphotyrosine phosphatases on the activity and regulation of GSH ATP-dependent transport system that we have previously identified in NIH3T3 fibroblasts. This is shown by the fact that increases of the initial rate of GSH uptake were measured in NIH3T3 overexpressing a synthetic gene coding for a low-Mr-phosphotyrosine protein phosphatase (LMW-PTP), while decreases were obtained in NIH3T3 overexpressing the phosphatase inactive mutant (LMW-C12SPTP), with respect to NIH3T3neo. Moreover, these results have been confirmed by experiments performed in the same cells by vanadate, and H2O2 treatment on both GSH transport and mediated passive transport of glucose. A possible regulation of this transport system by platelet-derived growth factor receptor (PDGFr) with tyrosine kinase activity is also demonstrated. Moreover, these data show a relationship among GSH, PDGFr and phosphotyrosine phosphatase activity, and suggest a role of GSH transport systems on the cell proliferation process.