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.

List of scientific papers

I. Hypoxia followed by re-oxygenation induces oxidation of tyrosine phosphatases. Sandin A, Dagnell M, Gonon A, Pernow J, Stangl V, Aspenström P, Kappert K, Östman A. Cell Signal. 2011 May; 23(5): 820-6.
https://doi.org/10.1016/j.cellsig.2011.01.004

II. Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases. ten Freyhaus H, Dagnell M, Leuchs M, Vantler M, Berghausen EM, Caglayan E, Weissmann N, Dahal BK, Schermuly RT, Ostman A, Kappert K, Rosenkranz S. Am J Respir Crit Care Med. 2011 Apr 15; 183(8): 1092-102.
https://doi.org/10.1164/rccm.200911-1663OC

III. Mitochondrial reactive oxygen species produced by p66Shc regulate PDGF signaling through protein tyrosine phosphatase oxidation. Jeroen Frijhoff, Markus Dagnell, Elena Beltrami, Marco Giorgio, Arne Östman. [Submitted]

IV. Thioredoxin-mediated selective activation of oxidized PTP1B modulates PDGFβ-receptor tyrosine kinase signaling. Markus Dagnell, Jeroen Frijhoff, Irina Pader, Martin Augsten, Benoit Boivin, Pankaj K. Mandal, Nicholas K. Tonks, Carina Hellberg, Marcus Conrad, Elias S.J. Arnér, Arne Östman. [Submitted]