Faculty Bibliography

Oxidative stress induces miR-200c, the predominant microRNA (miRNA) in lung tissues; however, the antioxidant role and biochemistry of such induction have not been clearly defined. Therefore, a lung adenocarcinoma cell line (A549) and a normal lung fibroblast (MRC-5) were used as models to determine the effects of miR-200c expression on lung antioxidant response. Hydrogen peroxide (H2O2) upregulated miR-200c, whose overexpression exacerbated the decrease in cell proliferation, retarded the progression of cells in the G2/M-phase, and increased oxidative stress upon H2O2 stimulation. The expression of three antioxidant proteins, superoxide dismutase (SOD)-2, haem oxygenase (HO)-1, and sirtuin (SIRT) 1, was reduced upon H2O2 stimulation in miR-200c-overexpressed A549 cells. This phenomenon of increased oxidative stress and antioxidant protein downregulation also occurs simultaneously in miR-200c overexpressed MRC-5 cells. Molecular analysis revealed that miR-200c inhibited the gene expression of HO-1 by directly targeting its 3'-untranslated region. The downregulation of SOD2 and SIRT1 by miR-200c was mediated through zinc finger E-box-binding homeobox 2 (ZEB2) and extracellular signal-regulated kinase 5 (ERK5) pathways, respectively, where knockdown of ZEB2 or ERK5 decreased the expression of SOD2 or SIRT1 in A549 cells. LNA anti-miR-200c transfection in A549 cells inhibited the endogenous miR-200c expression, resulting in increased expressions of antioxidant proteins, reduced oxidative stress and recovered cell proliferation upon H2O2 stimulation. These findings indicate that miR-200c fine-tuned the antioxidant response of the lung cells to oxidative stress through several pathways, and thus this study provides novel information concerning the role of miR-200c in modulating redox homeostasis of lung.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commonly pervasive inherited disease in many parts of the world. The complete lack of G6PD activity in a mouse model causes embryonic lethality. The G6PD-deficient Caenorhabditis elegans model also shows embryonic death as indicated by a severe hatching defect. Although increased oxidative stress has been implicated in both cases as the underlying cause, the exact mechanism has not been clearly delineated. In this study with C. elegans, membrane-associated defects, including enhanced permeability, defective polarity and cytokinesis, were found in G6PD-deficient embryos. The membrane-associated abnormalities were accompanied by impaired eggshell structure as evidenced by a transmission electron microscopic study. Such loss of membrane structural integrity was associated with abnormal lipid composition as lipidomic analysis revealed that lysoglycerophospholipids were significantly increased in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism leading to defective embryonic development could be attributed to the increased activity of calcium-independent phospholipase A2 (iPLA) in G6PD-deficient embryos. This notion is further supported by the fact that the suppression of multiple iPLAs by genetic manipulation partially rescued the embryonic defects in G6PD-deficient embryos. In addition, G6PD deficiency induced disruption of redox balance as manifested by diminished NADPH and elevated lipid peroxidation in embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to abnormal embryonic development in G6PD-deficient C. elegans.

G6PD deficiency has been the most pervasive inherited disorder in the world since having been discovered. G6PD has an antioxidant role by functioning as a major NADPH provider to reduce excessive oxidative stress. NADPH can produce reactive oxygen species and reactive nitrogen species mediated by NADPH oxidase (NOX) and nitric oxide synthase (NOS), respectively. Hence, G6PD also has a pro-oxidant role. Research in the past has focused on the enhanced susceptibility of G6PD-deficient cells or individuals to oxidative challenge. The cyto-regulatory role of G6PD has largely been overlooked. By using a metabolomic approach, it is noted that upon oxidant challenge, G6PD-deficient cells will reprogram the GSH metabolism from regeneration to synthesis with exhaustive energy consumption. Recently, new cellular/physiologic roles of G6PD have been discovered. By using a proteomic approach, it has been found that G6PD plays a regulatory role in xenobiotic metabolism possibly via NOX and the redox sensitive Nrf2 signaling pathway to modulate the expression of xenobiotic metabolizing enzymes. Since G6PD is a key regulator responsible for intracellular redox homeostasis, G6PD deficiency can alter redox balance leading to many abnormal cellular effects such as the cellular inflammatory and immune response against viral infection. G6PD may play an important role in embryogenesis as G6PD-knockdown mouse cannot produce offspring and G6PD-deficient C. elegans with defective egg production and hatching. This array of findings indicates that the cellular and physiologic roles of G6PD, other than the classical role as an antioxidant enzyme, deserve further attention.

Cancer is a worldwide health problem leading to a high incidence of morbidity and mortality. Malignant transformation can occur by expression of oncogenes, over-expression and deregulated activation of proto-oncogenes, and inactivation of tumor suppressor genes. These cellular actions occur through stimulation of oncogenic signaling pathways. Nitric oxide (NO) can induce genetic changes in cells and its intracellular generation can lead to tumor formation and progression. It can also promote anti-tumor activities. The pro- and anti-tumor activities of NO are dependent on its intracellular concentration, cell compartmentalization, and cell sensitivity. NO affects a number of oncogenic signaling pathways. This review focuses on two oncogenic signaling pathways: NO-EGFR-Src-FAK and NO-Ras-EGFR-ERK1/2 MAP kinases. In these pathways, low to intermediate concentrations of NO/S-nitrosothiols (RSNOs) stimulate oncogenic signaling, while high concentrations of NO/RSNO stimulate anti-oncogenic signaling. Increasing knowledge on pro- and anti-tumorigenic activities of NO and related reactive species such as RSNOs has fostered the research and synthesis of novel NO-based chemotherapeutic agents. RSNOs, effective as NO donors and trans-nitrosylating agents under appropriate conditions, may operate as potential chemotherapeutic agents.

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel(R)-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

Abstract Aims: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. Results: Ras signaling pathway stimulation by 50 and/or 100 muM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 muM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. Innovation: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. Conclusion: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane. Antioxid. Redox Signal. 18, 221-238.

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.