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Adhesion Dependent Signals: Cell Survival, Receptor Crosstalk and Mechanostimulation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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
Keyword [en]
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: urn:nbn:se:uu:diva-195712ISBN: 978-91-554-8604-4OAI: oai:DiVA.org:uu-195712DiVA: diva2:608340
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
List of papers
1. Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival
Open this publication in new window or tab >>Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival
2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 2, e32081- p.Article in journal (Refereed) Published
Abstract [en]

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the "hydrophobic motif" of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells.

National Category
Basic Medicine
urn:nbn:se:uu:diva-170264 (URN)10.1371/journal.pone.0032081 (DOI)000302875500073 ()22384145 (PubMedID)
Available from: 2012-03-09 Created: 2012-03-09 Last updated: 2013-03-22Bibliographically approved
2. Characterization of Heparanase-induced Phosphatidylinositol 3-Kinase-AKT Activation and Its Integrin Dependence
Open this publication in new window or tab >>Characterization of Heparanase-induced Phosphatidylinositol 3-Kinase-AKT Activation and Its Integrin Dependence
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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.

National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-200681 (URN)10.1074/jbc.M112.435172 (DOI)000318157600067 ()
Available from: 2013-06-03 Created: 2013-06-03 Last updated: 2016-01-17
3. The role of mechanical force and ROS in integrin-dependent signals
Open this publication in new window or tab >>The role of mechanical force and ROS in integrin-dependent signals
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, e64897Article in journal (Refereed) Published
Abstract [en]

Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced byintegrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition ofROCK and myosin II activity, i.e. the reactions occurred independently ofintracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching ofthe adherent cells induced a robust phosphorylation only of ERK1/2 and thephosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via alpha 5 beta 1 or alpha v beta 3 integrins were detected. The importance ofmitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signalsinduced by cyclic cell stretching, it abolished the activation of AKT and reduced theactin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composedof separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on theintegrin signals induced by cell attachment and mechanical stretching.

integrin, ROS, mechanosignaling
National Category
Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
urn:nbn:se:uu:diva-170266 (URN)10.1371/journal.pone.0064897 (DOI)000321394700069 ()23738008 (PubMedID)
Swedish Cancer SocietySwedish Research Council
Available from: 2012-03-12 Created: 2012-03-09 Last updated: 2016-08-09Bibliographically approved

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