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Enhancement of insulin responsiveness by nitric oxide-mediated inactivation of protein-tyrosine phosphatases.

ABSTRACT: NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesized that NO-induced inactivation of endogenous protein-tyrosine phosphatases (PTPs) would enhance insulin receptor-mediated signaling. To test this hypothesis, we devised a new method of the PTP labeling using a cysteine sulfhydryl-reacted probe. Under the acidic conditions employed in this study, the probe recognized the reduced and active forms but not the S-nitrosylated and inactive forms of endogenous PTPs. Our data suggest that phosphatases SHP-1, SHP-2, and PTP1B, but not TC-PTP, are likely S-nitrosylated at the active site cysteine residue concomitantly with a burst of NO production in signaling response to insulin stimulation. These results were further confirmed by phosphatase activity assays. We investigated further the role of NO as a regulator of insulin signaling by RNA interference that ablates endogenous eNOS expression in endothelial MS-1 cells. We have shown that eNOS-dependent NO production is essential for the activation of insulin signaling. Our findings demonstrate that NO mediates enhancement of insulin responsiveness via the inhibition of insulin receptor phosphatases.

SUBMITTER: Hsu MF

PROVIDER: S-EPMC2832942 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Enhancement of insulin responsiveness by nitric oxide-mediated inactivation of protein-tyrosine phosphatases.

Hsu Ming-Fo MF Meng Tzu-Ching TC

The Journal of biological chemistry 20100111 11

NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesi ...[more]

PMID: 20064934

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Project description:In the last decade, immune therapies against human cancers have emerged as a very effective therapeutic strategy in the treatment of various cancers, some of which are resistant to current therapies. Although the clinical responses achieved with many therapeutic strategies were significant in a subset of patients, another subset remained unresponsive initially, or became resistant to further therapies. Hence, there is a need to develop novel approaches to treat those unresponsive patients. Several investigations have been reported to explain the underlying mechanisms of immune resistance, including the anti-proliferative and anti-apoptotic pathways and, in addition, the increased expression of the transcription factor Yin-Yang 1 (YY1) and the programmed death ligand 1 (PD-L1). We have reported that YY1 leads to immune resistance through increasing HIF-1? accumulation and PD-L1 expression. These mechanisms inhibit the ability of the cytotoxic T-lymphocytes to mediate their cytotoxic functions via the inhibitory signal delivered by the PD-L1 on tumor cells to the PD-1 receptor on cytotoxic T-cells. Thus, means to override these resistance mechanisms are needed to sensitize the tumor cells to both cell killing and inhibition of tumor progression. Treatment with nitric oxide (NO) donors has been shown to sensitize many types of tumors to chemotherapy, immunotherapy, and radiotherapy. Treatment of cancer cell lines with NO donors has resulted in the inhibition of cancer cell activities via, in part, the inhibition of YY1 and PD-L1. The NO-mediated inhibition of YY1 was the result of both the inhibition of the upstream NF-?B pathway as well as the S-nitrosylation of YY1, leading to both the downregulation of YY1 expression as well as the inhibition of YY1-DNA binding activity, respectively. Also, treatment with NO donors induced the inhibition of YY1 and resulted in the inhibition of PD-L1 expression. Based on the above findings, we propose that treatment of tumor cells with the combination of NO donors, at optimal noncytotoxic doses, and anti-tumor cytotoxic effector cells or other conventional therapies will result in a synergistic anticancer activity and tumor regression.

Project description:IntroductionThe atypical Sts phosphatases negatively regulate signaling pathways in diverse immune cell types, with two of their molecular targets being the related kinases Syk and Zap-70. Mice lacking Sts expression (Sts-/- ) are resistant to infection by the live vaccine strain (LVS) of Francisella tularensis. Although the mechanisms underlying the enhanced resistance of Sts-/- mice have not been definitively established, Sts-/- bone marrow-derived monocytes (BMMs) demonstrate greater clearance of intracellular LVS following ex vivo infection, relative to wild type cells. To determine how the Sts proteins regulate monocyte bactericidal properties, we analyzed responses of infected cells.MethodsMonocyte bacterial clearance was assayed using ex vivo coculture infections followed by colony-forming unit analysis of intracellular bacteria. Levels of gene expression were quantified by quantitative reverse-transcription polymerase chain reaction, levels of Nos2 protein levels were quantified by Western blot analysis, and levels of nitric oxide (NO) were quantified directly using the Griess reagent. We characterized monocyte cytokine production via enzyme-linked immunosorbent assay.ResultsWe demonstrate that Sts-/- monocyte cultures produce elevated levels of interferon-γ (IFNγ) after infection, relative to wild type cultures. Sts-/- monocytes also demonstrate heightened responsiveness to IFNγ. Specifically, Sts-/- monocytes produce elevated levels of antimicrobial NO following IFNγ stimulation, and this NO plays an important role in LVS restriction. Additional IFNγ-stimulated genes, including Ip10 and members of the Gbp gene family, also display heightened upregulation in Sts-/- cells. Both Sts-1 and Sts-2 contribute to the regulation of NO production, as evidenced by the responses of monocytes lacking each phosphatase individually. Finally, we demonstrate that the elevated production of IFNγ-induced NO in Sts-/- monocytes is abrogated following chemical inhibition of Syk kinase.ConclusionOur results indicate a novel role for the Sts enzymes in regulating monocyte antibacterial responses downstream of IFNγ.

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Enhancement of insulin responsiveness by nitric oxide-mediated inactivation of protein-tyrosine phosphatases.

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Enhancement of insulin responsiveness by nitric oxide-mediated inactivation of protein-tyrosine phosphatases.

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