Probing the recognition specificity of αVβ1 integrin and syndecan-4 using force spectroscopy
Introduction
The dynamic nature of cell spreading and adhesion to the surface, a fine coupling of cells to the substrate requires understanding their interaction surrounding environments. Cell attachments start with a formation of contacts with receptors present on a cell surface. At steady-state conditions, these contacts are converted to focal adhesions (FAs) that from one side are connected to actin filaments located inside the cell and on the other end to specific binding sites present in ECM proteins (Frantz et al., 2010; Humphries, 2006). FAs contain integrins as main players triggering the recruitment of large protein assemblies (Hynes, 1987; Ruoslahti, 1996). These molecules, composed of α and β subunits (Hynes, 1987), are not the only players. Many ECM proteins also contain binding sites to various non-integrin receptors. Among them, syndecans have been found to be present within the region of the focal adhesion area (Morgan et al., 2007; Theocharis et al., 2016). Both receptor types through actin filaments participate in force transduction, spatial control of signaling complex assembly, and regulation of cytoskeletal dynamics (Lin et al., 2015; Morgan et al., 2007). Although cell adhesion requires the presence of their dual engagement, it is not clear how they interact. Over the past two decades, researchers have postulated a synergistic link between integrins and syndecans due to bridging complexes consisting of α5β1 and syndecan-4 (Bass et al., 2009, 2007; Humphries, 2006; Morgan et al., 2007). Such complexes can form a tri-molecular complex in the interaction with ECM proteins or other molecules as it has been demonstrated for binding of α5β1 and SDC4 to endothelial surface molecule Thy-1 (Fiore et al., 2014). Evidence gathered so far had shed light on the molecular basis of the integrin/syndecan interaction and its functional relevance, in particular for signaling pathways (Morgan et al., 2007; Schürpf and Springer, 2011), but a little is known whether their similar role in cell adhesion is reflected as similar interaction potentials.
Alterations in adhesive properties of cells frequently accompany the development of numerous diseases (Daley and Yamada, 2013; Lityńska et al., 2002). Altered expression and functional status of integrins and syndecans are often associated with such pathological processes as metastasis (Burvenich et al., 2008; Lal et al., 2015; Seguin et al., 2015). Such integrins as α2β1 or α5β1 or members of αV integrin family regulate the angiogenesis (Grzesiak and Bouvet, 2006) or cancer invasion (Wei et al., 2007). Several integrins such as α5β1 (Mierke et al., 2010) or αVβ3 (Mierke, 2013) facilitate cancer invasion by influencing cell stiffness and triggering cytoskeletal remodeling, which enable the cell to generate and transmit contractile forces to overcome the steric three dimensional (3D) hindrance of ECMs. Syndecans facilitate integrin-related adhesive interactions in a synergistic way (Humphries, 2006; Hynes, 1987; Morgan et al., 2007; Woods and Couchman, 1994) by possessing structurally distinct chondroitin (CS) and heparin sulfate (HS) chains (Couchman, 2010). Exemplarily, syndecan-4 has often been reported to couple to β1 and β3 integrins present in focal adhesions (Echtermeyer et al., 1999).
Our focus is bladder cancer as it is a 9th most common cancer worldwide possessing the highest incidence and mortality rates (Antoni et al., 2017). One of the main problems in its treatment is the inability to effectively prevent relapse and progression of high-grade non-muscle invasive bladder cancer, which occur in 80 % and 45 % of patients, respectively (Carrion and Seigne, 2002). Bladder is an organ, which is characterized by a large degree of mechanical flexibility that may vary from 9.6 kPa (187 mL, 8.6 mmHg) to 106.9 kPa (327 mL, 27.6 mmHg) depending of the filling volume (Nenadic et al., 2013). This indicates a large deformability of individual bladder cells that has become a driving force for the studies of the mechanical properties of these cells. Deformability of living bladder cancer cells was shown to be significantly larger as compared to the reference, non-malignant cells (Lekka et al., 1999). Surprisingly, there was no correlation between cancer cell deformability and invasiveness that regardless of the state of cancer progression - softer cells indicate a malignant phenotype (Ramos et al., 2014). Bladder is characterized by the largest functional flexibility reflected in greater adaptability of cells to altered microenvironment (Roccabianca and Bush, 2016). Mechanical properties of bladder cancer cells have already been studied by AFM revealing large rigidity of non-malignant cells as compared to cancerous ones that appeared to be more deformable (Lekka et al., 1999; Ramos et al., 2014). Changes in deformability have been shown to be related to actin cytoskeleton organization, thus, simultaneously, they are related to surface adhesive properties, as it has been recently shown that the microenvironment stiffness influences both the formation of focal adhesion and related expression of β1 integrins (Lekka et al., 2019). Immunohistochemical studies have shown that normal human urothelium expresses integrins built of such subunits as α3, αV, β1, and β4. Like for integrins, syndecan expression has also been shown to be characterized by a progressive transformation from the normal urothelium to invasive states (Marzioni et al., 2009; Syrigos et al., 1999). The biological functions of integrins and syndecans reside in their ability to interact with various ligands, by which they modulate cell-ECM interaction. A direct consequence of their specific binding is to regulate cell differentiation and are involved in epithelial-mesenchymal transition and carcinogenesis (Barbouri et al., 2014; Freire-de-Lima, 2014; Iozzo and Sanderson, 2011). Despite that, the molecular basis of their biological function is still poorly defined.
In our research, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVβ1 and syndecan-4 in the interaction with vitronectin, which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. We demonstrated that, despite different unbinding kinetics, the access to both receptors are needed for cell growth and proliferation. A study on cellular response to the accessibility of specific binding sites reveals changes characteristic for bladder cancer cells.
Section snippets
Recombinant proteins
Syndecan-4 (SDC-4, 25−40 kDa, SDS-PAGE, reducing conditions, human recombinant protein from R&D Systems) consist of a core protein (198 AA) with a signal sequence (18 AA), an extracellular domain (127 AA), a transmembrane region (25 AA), and a cytoplasmic tail (28 AA). Integrin αVβ1 (120−170 kDa, SDS-PAGE, reducing conditions, human recombinant protein from R&D Systems) consists of non-covalently linked αV (Phe31-Val992) and β1 (Gln21-Asp728) subunits. Vitronectin (VN, Asp20-Leu478, 70−80 kDa
Cell-dependent variability in the expression of integrins and syndecans
As we are particularly focused on bladder cancer, in our first steps, we defined the expression of integrin subunits and of syndecans in the studied bladder cancer cell lines. Each bladder cancer cell line was characterized by a different stage of cancer progression. Cells were originating from non-malignant cell cancer of ureter (HCV29), carcinoma of grade 2 (5637), carcinoma of grade 3 (HT1376), and transitional cell carcinoma of grade 4 (T24). Protein bands of integrin subunits and syndecans
Conclusions
Our results demonstrate that it is insufficient to consider only integrins as the main players of cell-surface interactions. Syndecans should be considered as their presence influences cell adhesion and proliferation. We demonstrated that αVβ1 and SDC-4 are key players of the interaction with vitronectin in bladder cancer cells. Although these surface receptors share a similar role, the energy landscapes of single molecular complexes reveal higher (integrins) and lower (syndecans) energy
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Science Centre (Poland) project no UMO-2014/15/B/ST4/04737. K.H. and A.P. acknowledge the Ministry of Science and Higher Education in Poland for financial support. The JPK purchase has been realized under the project co-funded by the Małopolska Regional Operational Program, Measure 5.1 – Krakow Metropolitan Area as an important hub of the European Research Area for 2007-2013, project no MRPO.05.01.00-12-013/15. The authors are thankful to Mrs. Joanna
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