Original contributionRedox regulation of the signaling pathways leading to eNOS phosphorylation
Introduction
Nitric oxide (NO) is a potent multifunctional biological messenger with many regulatory and immunomodulatory activities. For example, NO mediates interferon-γ-dependent immune suppression of T cell proliferation and of antibody synthesis by CD5+ B cells [1], inhibits proliferation of T cells [2], [3], [4] and nuclear factor κB activation in glial cells [5], and plays an immunosuppressive role during development of experimental graft-vs-host disease [1], [3]. In addition to an inhibitory or proapoptotic role, NO has also been shown to enhance cellular survival [6], [7], [8], [9]. We have previously reported that NO protects human natural killer (NK) cells from activation-induced cell death [10] and that, under certain oxidation–reduction (redox) conditions, protects porcine endothelial cells (PAEC) from being lysed by interleukin (IL)-2 stimulated human NK cells [11]. In the cardiovascular system, production of NO is crucial to maintaining homeostasis [12] and vascular tone, through its role in vasodilation, antiatherogenesis, inhibition of leukocyte–endothelial cell interactions, smooth muscle proliferation, and platelet aggregation [13].
NO is synthesized by a family of enzymes known as NO synthases (NOS). Neuronal NOS (nNOS) and endothelial NOS (eNOS) are produced constitutively, whereas inducible NOS (iNOS) can be induced in a number of cell types by treatment with cytokines or other agents [14]. eNOS is constitutively expressed in endothelial cells and is activated under conditions of Ca2+ influx [15], [16]. eNOS, but not iNOS or nNOS, was associated with NO production in the two systems (NK cells [10] and PAEC [11]) in which we observed a cytoprotective effect of NO, suggesting a role for eNOS in these protective processes. eNOS activation is induced by a number of stimulants, such as shear stress, estrogen, or cytokines, resulting in phosphorylation of human eNOS on Ser 1177 [17], [18] (corresponding to Ser in the 1179 position in the highly homologous porcine eNOS [19], [20]) through a pathway involving phosphatidylinositol 3-kinase (PI3-K), and the Ser/Thr kinase, Akt. It has recently been demonstrated that oxidative stress also induces eNOS phosphorylation through the PI3-K/Akt pathway [18]. However, it is unclear by what mechanism oxidative stress activates this signaling pathway.
Intracellular oxidative stress is predominantly controlled by the natural and ubiquitous thiol-reducing systems, the thioredoxin (TRX)/peroxiredoxin system or glutathione (reduced type glutathione: GSH)/glutathione-dependent peroxidase system. These systems control the intracellular redox state, regulating cell growth and cell death by regulating the activation of transcription factors, the sensitivity of cells to cytokines, and components of the apoptosis pathways. These systems function through oxidation or reduction of protein cysteine sulfhydryl (–SH) groups. Oxidation of SH on protein cysteine residues can cause conformational changes, particularly when the cysteines form either intermolecular or intramolecular disulfide bonds, and can be very important in modulation of signaling pathways [21].
Xenotransplantation of nonhuman organs into humans is being considered as a means to overcome the worldwide shortage of human organs, with pigs being the likely source animals [22]. Whereas the early stages of immunological rejection may be reduced by a variety of means, including genetic engineering of source animals, the later stages remain a serious problem [23]. Because vascular endothelial cells of vascularized xenografts are the primary targets of a recipient's immune response, and because we had observed that NO may provide a means to protect such target cells [11], we focused on the regulation of NO synthesis in PAEC. We now report that phosphorylation of Akt and eNOS is exquisitely redox-sensitive and that regulation through protein thiols is a key component of eNOS phosphorylation signaling in PAEC. We show that intracellular and, especially, cell membrane surface free thiols regulate Akt/eNOS phosphorylation signaling induced by oxidative stress via a pathway that does not involve intracellular glutathione.
Section snippets
Antibodies and other reagents
Anti-phospho-eNOS (Ser 1177) polyclonal Ab, anti-phospho-Akt (Ser 473) polyclonal Ab, and anti-Akt polyclonal Ab were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). According to the Cell Signaling data sheet, the antibody to phospho-eNOS (Ser 1177) was raised against bovine aortic endothelial cells (BAEC) and detects two closely migrating bands by Western analysis, and the faster migrating band corresponds to phosphorylated eNOS and is more intense when lysates of BAEC are
Hydrogen peroxide induces phosphorylation of Akt and eNOS
It has been shown that Akt activates eNOS through phosphorylation of eNOS on Ser 1177 (corresponding to Ser 1179 in porcine eNOS) [17], [18], [19], [20]. Upstream of this, PI3-K phosphorylation activates this kinase to phosphorylate Akt [20], [27]. Oxidative stress in the form of hydrogen peroxide also activates Akt [27]. A more recent report has demonstrated that H2O2 activates eNOS through its effect on Akt [18]. To examine the upstream mechanisms by which oxidative stress induces eNOS
Discussion
Because of the severe shortage of human organs for xenotransplantation, there is a current interest in developing xenotransplantation of vascularized porcine organs into humans. Hence, there is a need to understand mechanisms of survival or destruction of porcine endothelial cells, the first targets of immune attack. Having previously shown that the combination of nitric oxide and thiol deprivation protects porcine endothelial cells from lysis by IL-2-activated human NK cells [11], we decided
Acknowledgments
We thank Dr. Andrew Byrnes and Dr. Onesmo Mpanju, both of the FDA Center for Biologics Evaluation and Research, for their critical reading of our manuscript, and Dr. Emily Shacter, FDA Center for Drug Evaluation and Research, for her advice.
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