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Studies of PDGF receptor signaling in vitro and in vivo
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelet-derived growth factor receptor (PDGFR) signaling is essential for proliferation, migration and survival of cells of mesenchymal origin; however, its deregulation has been associated with various diseases, including cancer. The aim of this thesis was to clarify the molecular mechanisms of PDGFR signaling regulation. We have studied PDGFR downregulation, identified the E3 ligases and deubiquitinases (DUBs) acting on the receptor, characterized the role of the downstream effector Erk5, as well as elucidated the role of PDGFRβ isoform in tumor growth and angiogenesis.

As Erk5 activation has been associated with tumorigenesis, it is important to delineate the pathway from activated PDGFR to Erk5. Here, we demonstrate not only a complex mechanism for PDGF-induced Erk5 activation that involves Mek5, Mekk2, Mek1/2, PI3K and classical PKCs, but also a novel function for Erk5 by showing that PDGF-BB affects BMP-Smad signaling in an Erk5 pathway-dependent manner, indicating a crosstalk between tyrosine kinase receptor and serine/threonine receptor signaling.

By investigating PDGFRβ downregulation, we demonstrated that ubiquitination of PDGFRβ, mediated by Cbl-b and c-Cbl, is essential for the receptor internalization, signaling, as well as downstream biological responses. Additionally, as ubiquitination is a reversible post-translational modification, we identified USP4 as one of the DUBs acting on PDGFRβ and discovered that USP4 interacts with PDGFRβ, removing both K48- and K63-linked polyubiquitin chains, and increases its stability, in both normal and cancer cells.

Although several studies have highlighted the therapeutic benefit of PDGFR inhibition in cancer treatment, all available PDGFR kinase inhibitors have secondary targets; consequently, the details underlying the importance of PDGFR in tumorigenesis remain unknown. By targeting specifically PDGFRβ in the stroma of various tumor models, we showed that specific inhibition of PDGFRβ signaling suppresses growth of tumors with high levels of PDGF-BB, whereas the multi-target kinase inhibitor imatinib has less effect, indicating the significance of selective targeting of PDGFRβ.

Our data provide new insights into the molecular events underlying PDGFRβ signaling and downregulation, highlight its importance as cancer therapeutic target and lead the way for further discoveries.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 67
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1538
Keywords [en]
PDGF, signaling, cancer, Erk5, kinase, Cbl, ubiquitination, deubiquitinase, pericytes, tumor growth, angiogenesis, low molecular weight inhibitors
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-376248ISBN: 978-91-513-0571-4 (print)OAI: oai:DiVA.org:uu-376248DiVA, id: diva2:1285221
Public defence
2019-03-22, B42, Biomedical Center, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-02-27 Created: 2019-02-03 Last updated: 2019-03-18
List of papers
1. Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling
Open this publication in new window or tab >>Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling
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2016 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 28, no 9, p. 1422-1431Article in journal (Refereed) Published
Abstract [en]

Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase C gamma and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The cooperation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKC zeta interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling. (C) 2016 Published by Elsevier Inc.

Keywords
Erk5, PDGFR beta, Mekk2, MAP kinase, PKC, Smad
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-302674 (URN)10.1016/j.cellsig.2016.06.013 (DOI)000380595500027 ()27339033 (PubMedID)
External cooperation:
Funder
Swedish Cancer Society, 140332
Available from: 2016-09-08 Created: 2016-09-08 Last updated: 2019-02-03Bibliographically approved
2. The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor beta (PDGFR beta) Internalization and Signaling
Open this publication in new window or tab >>The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor beta (PDGFR beta) Internalization and Signaling
2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 22, p. 11608-11618Article in journal (Refereed) Published
Abstract [en]

Protein ubiquitination controls protein stability and subcellular localization of tyrosine kinase receptors, hence affecting signaling both quantitatively and qualitatively. In this report, we demonstrate that, after ligand stimulation, the PDGF beta receptor (PDGFR beta) becomes ubiquitinated in a manner requiring both the c-Cbl and Cbl-b ubiquitin ligases. Simultaneous depletion of c-Cbl and Cbl-b resulted in reduced ligand-induced PDGFR beta clearance from the cell surface because of reduced endocytosis of the receptor. Cbl-b formed a complex with c-Cbl, as well as with the PDGFR beta, in response to PDGF-BB stimulation. We were unable to find a direct interaction between the receptor and c-Cbl, raising the possibility that Cbl-b is necessary for c-Cbl to interact with PDGFR beta. Phosphorylated Tyr-1021 in PDGFR beta was the primary interaction site for Cbl-b, with some contribution from Tyr-1009. Depletion of c-Cbl and Cbl-b led to an increased ligand-induced tyrosine phosphorylation of the receptor. Several tyrosine residues with elevated phosphorylation (i.e. Tyr-579, Tyr-581, Tyr-1009, and Tyr-1021) have previously been shown to interact with Src kinases and PLC gamma. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLC gamma phosphorylation were enhanced, whereas activation of other pathways, such as Erk1/2 MAP kinase and Akt, were not affected. In addition, Stat3 phosphorylation, which has been connected to Src activity, was also elevated in cells lacking c-Cbl and Cbl-b. Functionally, we found that cells depleted of c-Cbl and Cbl-b were more prone to migrate toward PDGF-BB, whereas no reproducible effect on cell proliferation could be observed. In conclusion, internalization as well as signaling via PDGFR beta are controlled by ubiquitination.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-299504 (URN)10.1074/jbc.M115.705814 (DOI)000377264800013 ()27048651 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2016-07-22 Created: 2016-07-22 Last updated: 2019-02-03Bibliographically approved
3. The ubiquitin-specific protease 4 de-ubiquitinates and stabilizes PDGF receptor β
Open this publication in new window or tab >>The ubiquitin-specific protease 4 de-ubiquitinates and stabilizes PDGF receptor β
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-375769 (URN)
Available from: 2019-01-31 Created: 2019-01-31 Last updated: 2019-02-03
4. Specific targeting of PDGFRβ inhibits growth and angiogenesis in tumors with high PDGF-BB expression
Open this publication in new window or tab >>Specific targeting of PDGFRβ inhibits growth and angiogenesis in tumors with high PDGF-BB expression
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-375771 (URN)
Available from: 2019-01-31 Created: 2019-01-31 Last updated: 2019-02-03

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