Yusong Yang

Antioxidant Role of Glutathione S-Transferases: Protection Against Oxidant Toxicity and Regulation of Stress-Mediated Apoptosis

It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione peroxidase activity and that these enzymes can also detoxify lipid peroxidation end products such as 4-hydroxynonenal (4-HNE). In this article, recent studies suggesting that the Alpha class GSTs provide a formidable defense against oxidative stress are critically evaluated and the role of these enzymes in the regulation of oxidative stress-mediated signaling is reviewed. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that lipid peroxidation products, particularly hydroperoxides and 4-HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the Alpha class GSTs through the regulation of the intracellular concentrations of 4-HNE.

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.

Cells preconditioned with mild, transient UVA irradiation acquire resistance to oxidative stress and UVA-induced apoptosis: role of 4-hydroxynonenal in UVA-mediated signaling for apoptosis.

Because 4-hydroxynonenal (4-HNE) has been suggested to be involved in oxidative stress-mediated apoptosis (Cheng, J. Z., Sharma, R., Yang, Y., Singhal, S. S., Sharma, A., Saini, M. K., Singh, S. V., Zimniak, P., Awasthi, S., and Awasthi, Y. C. (2001) J. Biol. Chem. 276, 41213–41223) and UVA irradiation also causes lipid peroxidation, we have examined the role of 4-HNE in UVA-mediated apoptosis. K562 cells irradiated with UVA (3.0 milliwatts/cm2) for 5, 15, and 30 min showed a time dependent increase in 4-HNE levels. As judged by the activation of caspases, apoptosis was observed only in cells irradiated for 30 min. Within 2 h of recovery in normal medium, 4-HNE levels in 5 and 15 min UVA, irradiated cells returned to the basal or even lower levels but in cells irradiated for 30 min, 4-HNE levels remained consistently higher. The cells irradiated with UVA for 5 min and allowed to recover for 2 h in normal medium (UVA-preconditioned cells) showed a remarkable induction of hGST5.8, which catalyzes conjugation of 4-HNE to glutathione (GSH), and RLIP76 (Ral BP-1), which mediates the transport of the conjugate, GS-HNE. In cells irradiated with UVA for 30 min the induction of RLIP76 or hGST5.8 was not observed. The preconditioned cells transported GS-HNE into the medium at a rate about 2-fold higher than the controls and the transport was inhibited (65%) by coating the cells with anti-RLIP76 IgG. Upon treatment with xanthine/xanthine oxidase (XA/XO), 4-HNE, or prolonged UVA exposure, the control cells showed a sustained activation of c-Jun N-terminal kinase (JNK) and apoptosis. However, in the UVA-preconditioned cells, apoptosis was not observed, and JNK activation was inhibited. This resistance of preconditioned cells to XA/XO-, 4-HNE-, or UVA-induced apoptosis could be abrogated when these cells were coated with anti-RLIP76 IgG to block the efflux of GS-HNE. These studies strongly suggest a role of 4-HNE in UVA-mediated apoptosis.

Endothelial glutathione-S-transferase A4-4 protects against oxidative stress and modulates iNOS expression through NF-κB translocation

Our recent work in endothelial cells and human atherosclerotic plaque showed that overexpression of glutathione-S-tranferases (GSTs) in endothelium protects against oxidative damage from aldehydes such as 4-HNE. Nuclear factor (NF)-kappaB plays a crucial role during inflammation and immune responses by regulating the expression of inducible genes such as inducible nitric oxide synthase (iNOS). 4-HNE induces apoptosis and affects NF-kappaB mediated gene expression, but conflicting results on 4-HNEs effect on NF-kappaB have been reported. We compared the effect of 4-HNE on iNOS and the NF-kappaB pathway in control mouse pancreatic islet endothelial (MS1) cells and those transfected with mGSTA4, a alpha-class GST with highest activity toward 4-HNE. When treated with 4-HNE, mGSTA4-transfected cells showed significant upregulation of iNOS and nitric oxide (NO) through (NF)-kappaB (p65) translocation in comparison with wild-type or vector-transfected cells. Immunohistochemical studies of early human plaques showed lower 4-HNE content and upregulation of iNOS, which we take to indicate that GSTA4-4 induction acts as an enzymatic defense against high levels of 4-HNE, since 4-HNE accumulated in more advanced plaques, when detoxification and exocytotic mechanisms are likely to be overwhelmed. These studies suggest that GSTA4-4 may play an important defensive role against atherogenesis through detoxification of 4-HNE and upregulation of iNOS.

Energy Dependent Transport of Xenobiotics and Its Relevance to Multidrug Resistance

Transport mechanisms for the exclusion of toxic xenobiotics and their metabolites from cellular environment are crucial for living organisms. Accumulation of these toxins may affect a number of regulatory and other functions, ultimately leading to cell death. This trafficking of toxins and their metabolites is an energy dependent, primary active process, involving the hydrolysis of nucleotide triphosphates (ATP or GTP), while transferring substrate molecules across the cell membrane, against a concentration gradient of the substrate. Therefore, specific membrane associated proteins, known as efflux pumps, are required to remove these undesirable molecules from the cellular environment. These transport proteins have diverse structural characteristics with molecular weights ranging from 28 kDa to 190 kDa and a broad substrate specificity ranging from anionic to weakly cationic compounds. While these transport mechanisms constitute an important part of the cellular defense machinery, they also pose a formidable threat to the efficacy of chemotherapy against pathogenic bacteria and cancer cells. In cancer cells, the over expression of these proteins may confer a multidrug resistance (MDR) phenotype. This problem of MDR in cancer cells has so far been attributed to the two major families of efflux pumps, P-glycoprotein (Pgp) and multidrug resistance associated proteins (MRP), which belong to the ATP-binding cassette (ABC) super family. However, the existence of these pumps has not been able to explain all types of acquired MDR. Therefore, the importance of transport mechanisms other than these ABC-transporters cannot be ruled out. One such transporter is DNP-SG ATPase, whose identity has recently been established with RLIP76, a Ral binding GTPase activating protein known to be involved in the Ras-Rho-Ral mediated signaling mechanism. In the present article, we review the comparative functional, structural, and molecular characteristics of some transporters and discuss their role in xenobiotic transport and multidrug resistance.

Glutathione-S-transferase protects against oxidative injury of endothelial cell tight junctions.

Oxidative damage of endothelial tight junction permeability is involved in the pathophysiology of a variety of vascular diseases. The authors studied the role of the antioxidant enzyme, human glutathione-S-transferase A4-4 (hGSTA4-4), in regulating expression of major molecules of tight junction in vascular endothelial cells under oxidative stress induced by H(2)O(2). A vascular endothelial cell line, mouse pancreatic endothelial cells (MS1), was transduced with recombinant adenoviral vector containing hGSTA4-4 gene. hGSTA4-4 induced expression of tight junction proteins occludin and zonula occludens (ZO)-1 under oxidative stress. Increased hGSTA4-4 expression correlated with increased transepithelial electrical resistance and decreased tyrosine phosphorylation of occludin and ZO-1 following exposure to H(2)O(2). In addition, morphologic dissociation of occludin, ZO-1, and F-actin during oxidative stress was reduced in hGSTA4-4-expressing cells. To explore a genetic approach for vascular diseases associated with disruption of tight junction proteins, we introduced the same viral vector to blood vessels of mice, rats, and rabbits ex vivo and found strong expression of hGSTA4-4 in endothelial cells. These results demonstrate that oxidative stress mediated disruption of tight junctions in endothelial cells may be attenuated by hGSTA4-4 expression.