Redox-regulation of PTPs; mechanisms and impact on PDGFR signaling

Protein tyrosine phosphatases (PTPs) are reversibly oxidized upon activation of platelet-derived growth factor receptor beta (PDGFβR). Dys-regulation of the PDGFβR signaling pathway is associated with several diseases, including cancers and cardiovascular disease, and is thus a known driver of disease progression. Ligand dependent PDGFβR phosphorylation stimulates cell proliferation and migration. The aim of this thesis was to elucidate redox-regulatory mechanisms of protein tyrosine phosphatases impacting on PDGFβR signaling. In Paper I, we analysed effects of mitochondria-derived ROS on PTP oxidation in models of hypoxia and hypoxia/re-oxygenation (H/R) in vitro and in vivo. We found an increase in PTP oxidation of multiple PTPs, including SHP-2, PTP1B and DEP-1, after exposure of NIH3T3 fibroblasts to H/R. An increase in total PTP oxidation and SHP-2 was also seen in rat cardiomyoblasts after H/R. Furthermore, H/R induced a delay of PDGFR dephosphorylation and also an antioxidant sensitive activation of downstream effectors ERK1/2. In addition, H/R enhanced PDGF-dependent cytoskeletal re-arrangements, which could be abolished by antioxidant treatment. Finally, we found an increase in total PTP oxidation and SHP2 oxidation in tissue extracts from an ex-vivo model of rat heart ischemia-reperfusion. In paper II, we studied expression and activity of PDGFβR pathway components in human pulmonary artery smooth muscle cells (hPASMC) subjected to hypoxia. We show that hypoxia- induced HIF-1α in hPASMC, both in vivo and in vitro, negatively regulate expression of PDGFβR associated PTPs, including PTP1B, DEP-1, TC-PTP and SHP2. The negatively regulation of these PDGFβR-associated PTPs occurred together with an enhanced PDGF receptor activation and an increase in both proliferation and migration of hPASMC. In paper III, we found that p66Shc dependent mitochondrial derived ROS contribute to inactivation of the PDGFβR associated PTPs PTP1B and SHP-2 upon ligand stimulation. In addition, deletion of p66Shc reduced downstream intracellular signaling after PDGF-BB stimulation. Furthermore, p66Shc KO cells displayed a decrease in migratory response to PDGF-BB treatment. In the final study paper IV, we studied the reactivation of oxidized PTPs and its impact on PDGFβR signaling. We showed that cells lacking expression of thioredoxin reductase 1 (TrxR1) displayed an increase in oxidation of PTP1B but not of SHP-2. Furthermore, in vivo oxidized PTP1B was re-activated by addition of Trx system components to cell lysates, whereas SHP-2 was not re-activated. Oxidized recombinant PTP1B was also re-activated by treatment with Trx system components while SHP-2 remained largely unaffected. Intriguingly, the Trx related protein TRP14 also reactivated PTP1B but not SHP-2. Furthermore, PDGFβR phosphorylation and signaling was enhanced in Txnrd1-/- fibroblasts leading to an enhanced proliferative response after PDGF-BB stimulation

12/15-lipoxygenase–derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H2O2, as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF β-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX–derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H2O2 in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation

Knockout of Mitochondrial Thioredoxin Reductase Stabilizes Prolyl Hydroxylase 2 and Inhibits Tumor Growth and Tumor-Derived Angiogenesis

Aims: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial H2O2 abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In the present study we investigated the impact of targeted disruption of Txnrd2 on tumor growth. Results: Tumor cells with a Txnrd2-deficiency failed to activate HIF-1α signaling; it rather caused PHD2 accumulation, HIF-1α degradation and decreased VEGF levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. Innovation: Our data provide compelling evidence for a yet unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular HIF-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. Conclusion: The present work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-HIF-1α axis which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems

Control of protein function through oxidation and reduction of persulfidated states

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness