ABSTRACT
Integrins are extracellular matrix (ECM) receptors that are key players in the regulation of tumour cell invasion. The laminin-binding integrin α3ß1 has previously been shown to regulate adhesion and migration of carcinoma cells in part through co-operative signalling with the tetraspanin family of transmembrane proteins. However, the spatial and temporal regulation of crosstalk between these families of transmembrane proteins in intact cells remains poorly understood. Here we have used fluorescence resonance energy transfer (FRET) to demonstrate for the first time that α3ß1 and the tetraspanin CD151 directly associate at the front and retracting rear of polarised migrating breast carcinoma cells in both two-dimentional (2D) and three-dimentional (3D)matrices. Furthermore, localised α3ß1-CD151 binding correlates with lower CD151 homodimerisation in cells migrating on laminin or within matrigel. Loss of α3ß1 integrin leads to increased CD151 homodimer formation, increased activation of Rho GTPase, loss of cell polarity and decreased invasion in 3D ECM. As a result, α3-silenced cells show decreased actin-based membrane protrusion and retraction in both 2D and 3D environments. These data demonstrate that associations between α3ß1 and CD151 occur dynamically within discrete subcellular compartments and act to establish local GTPase signalling to promote tumour cell invasion. These novel findings shed light on the complex crosstalk and switching between receptor complexes in response to different extracellular cues during cell invasion in 3D environments.
Subject(s)
Cell Culture Techniques/methods , Cell Membrane/metabolism , Integrin alpha3beta1/metabolism , Tetraspanin 24/metabolism , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Female , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Integrin alpha3beta1/genetics , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Protein Binding , Protein Multimerization , RNA Interference , Tetraspanin 24/chemistry , Tetraspanin 24/genetics , Transfection , rho GTP-Binding Proteins/metabolismABSTRACT
BACKGROUND: The A subunit of factor XIII (FXIII-A) functions as an intracellular transglutaminase (TG) in the megakaryocyte/platelet lineage, where it probably participates in the cytoskeletal remodeling associated with cell activation. However, so far, the precise role of cellular FXIII (cFXIII) and the functional consequences of its absence in FXIII-A-deficient patients are unknown. OBJECTIVES AND METHODS: In this study, we used platelets from four patients with congenital deficiency of FXIII-A to study the role of cFXIII in platelet functions. RESULTS: We found that FXIII-A represents the only detectable source of TG activity in platelets and that the binding of fibrinogen in response to thrombin receptor agonist peptide (TRAP) stimulation was significantly reduced in platelets from the patients. In agreement with this, in control platelets, monodansyl-cadaverine (MDC), a competitive amino-donor for TGs, inhibited fibrinogen binding induced by TRAP in a dose-dependent manner. Moreover, upon adhesion to fibrinogen, normal platelets incubated with MDC as well as FXIII-A-deficient platelets showed a distinct extension pattern with reduced lamellipodia and increased filopodia formation, suggesting a delay in spreading. CONCLUSIONS: These findings provide evidence for the direct involvement of cFXIII-dependent TG activity in the regulation of platelet functions.
Subject(s)
Blood Platelets/metabolism , Factor XIIIa/physiology , Adult , Base Sequence , Blood Platelets/enzymology , Blotting, Western , Child, Preschool , DNA Primers , Factor XIIIa/genetics , Factor XIIIa/metabolism , Female , Humans , In Vitro Techniques , Mutation , Platelet Activation , Polymerase Chain Reaction , RNA, Messenger/geneticsABSTRACT
BACKGROUND: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. OBJECTIVES: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. METHODS: We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. RESULTS: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved. CONCLUSIONS: The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.
