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Characterization of Heparanase-induced Phosphatidylinositol 3-Kinase-AKT Activation and Its Integrin Dependence
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Staffan Johansson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 17, 12366-12375 p.Article in journal (Refereed) Published
Abstract [en]

Heparanase functions as a heparan sulfate-degrading enzyme and as a ligand for an unidentified signaling receptor(s). Here, several reactions involved in the activation of the PI3K-AKT pathway by latent heparanase were characterized. Protein suppression using specific siRNAs revealed that heparanase-induced phosphorylation of AKT at Ser-473 was RICTOR-mTOR-dependent, whereas ILK and PAK1/2 were dispensable. p110 alpha was the PI3K catalytic isoform preferred by heparanase for AKT activation and cell proliferation because the p110 alpha inhibitor YM024 blocked these processes. Heparanase-induced AKT phosphorylation was low in mouse embryonic fibroblast cells expressing a RAS interaction-defective p110 alpha compared with wild type cells, indicating that RAS has an important role in the PI3K-AKT activation. The response to heparanase was also inefficient in suspension cultures of several cell lines, suggesting a requirement of integrins in this pathway. Adhesion via either alpha V beta 3 or alpha 5 beta 1 promoted heparanase-induced AKT phosphorylation, and a stronger effect was seen when both integrins were engaged. Simultaneous inhibition of FAK and PYK2 using a chemical inhibitor, or suppression of their expression, inhibited heparanase-induced AKT activation and cell proliferation. Stimulation of cells with heparanase enhanced their resistance against oxidative stress-or growth factor starvation-induced apoptosis. These results demonstrate that there is an intimate crosstalk between the heparanase receptor(s) and integrins during induction of the prosurvival PI3K-AKT pathway by heparanase.

Place, publisher, year, edition, pages
2013. Vol. 288, no 17, 12366-12375 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-200681DOI: 10.1074/jbc.M112.435172ISI: 000318157600067OAI: oai:DiVA.org:uu-200681DiVA: diva2:624769
Available from: 2013-06-03 Created: 2013-06-03 Last updated: 2016-01-17
In thesis
1. Adhesion Dependent Signals: Cell Survival, Receptor Crosstalk and Mechanostimulation
Open this publication in new window or tab >>Adhesion Dependent Signals: Cell Survival, Receptor Crosstalk and Mechanostimulation
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The integrin family of cell surface receptors is evolutionary conserved and found in all multicellular animals. In humans 8-alpha and 18-beta integrins are non-covalently associated into 24 dimers. Integrins mediate cell-extracellular matrix and cell-cell interactions and participate in cell signalling. This ideally places integrins to regulate vital processes such as cell adhesion, migration, differentiation and cytoskeleton dynamics. Integrins also play a fundamental role in regulating cell survival and anoikis. In this thesis molecular mechanisms employed by integrins to induce signal transduction, independently or through crosstalk with other receptors, were characterised.

Rictor-mTOR (mTORC2) was required for Akt Ser473 phosphorylation in response to β1 integrin-mediated adhesion as well as EGF-, PDGF- or LPA-stimulation of MCF7 cells. ILK and PAK were dispensable for Akt Ser473 phosphorylation upon β1 integrin-engagement or EGF-stimulation. PAK was needed when this phosphorylation was induced by PDGF or LPA. β1 integrin-promoted cell survival during serum starvation conditions was mTORC2 dependent, indicating the importance of Akt Ser473 phosphorylation.

mTORC2 was also required for Akt Ser473 phosphorylation induced upon heparanase treatment of cells. Heparanase preferred PI3K catalytic subunit p110α for the upstream lipid phosphorylation required for Akt activation. Interaction between this subunit and Ras was needed for optimal Akt phosphorylation upon heparanase exposure. Cell adhesion strongly promoted heparanase signalling, which was more efficient in β1 integrin-expressing fibroblasts compared to cells lacking this subunit. The cooperative signalling between integrins and heparanase involved FAK and PYK2 since simultaneous silencing of these kinases suppressed heparanase-triggered Akt activation. Furthermore, the resistance of cells to apoptosis induced by H2O2 or serum starvation was promoted by heparanase. 

Integrin stimulation by adhesion or cyclic stretching showed divergent downstream signalling responses. Cell attachment on integrin-specific ligands lead to robust phosphorylation of several intracellular integrin-effectors, e.g. p130CAS, FAK, Akt and ERK 1/2. However, mechanical cell stretching only triggered prominent phosphorylation of ERK 1/2. Signalling induced at early stages of integrin-mediated cell adhesion occurred independently of intracellular contraction. Reactive oxygen species (ROS) generated during adhesion and cell stretching influenced integrin signalling. Inhibition of mitochondrial ROS production blocked adhesion-induced Akt phosphorylation. In contrast, stretch-induced ERK 1/2 phosphorylation was elevated when extracellular ROS was scavenged. These results indicate that the two types of integrin stimuli generate signals by different mechanisms.   

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 868
Integrins, Signal transduction, Protein kinase B, Akt, PI3K, Heparanase, ROS, Mechanosignalling
National Category
Cell and Molecular Biology
Research subject
Molecular Cellbiology; Biology with specialization in Molecular Cell Biology; Cell Research
urn:nbn:se:uu:diva-195712 (URN)978-91-554-8604-4 (ISBN)
Public defence
2013-04-12, BMC C4:305, Institutionen för medicinsk biokemi och mikrobiolog, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2013-03-20 Created: 2013-02-26 Last updated: 2014-01-27Bibliographically approved

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