Integrin-extracellular matrix (ECM) interactions in two-dimensional (2D) culture systems are widely studied (Goldstein and DiMilla, 2002. J Biomed. Mater. Res. 59, 665-675; Koo et al., 2002. J. Cell Sci. 115, 1423-1433). Less understood is the role of the ECM in promoting intercellular cohesion in three-dimensional (3D) environments. We have demonstrated that the alpha5beta1-integrin mediates strong intercellular cohesion of 3D cellular aggregates (Robinson et al., 2003. J. Cell Sci. 116, 377-386). To further investigate the mechanism of alpha5beta1-mediated cohesivity, we used a series of chimeric alpha5beta1-integrin-expressing cells cultured as multilayer cellular aggregates. In these cell lines, the alpha5 subunit cytoplasmic domain distal to the GFFKR sequence was truncated, replaced with that of the integrin alpha4, the integrin alpha2, or maintained intact. Using these cells, alpha5beta1-integrin-mediated cell aggregation, compaction and cohesion were determined and correlated with FN matrix assembly. The data presented demonstrate that cells cultured in the absence of external mechanical support can assemble a FN matrix that promotes integrin-mediated aggregate compaction and cohesion. Further, inhibition of FN matrix assembly blocks the intercellular associations required for compaction, resulting in cell dispersal. These results demonstrate that FN matrix assembly contributes significantly to tissue cohesion and represents an alternative mechanism for regulating tissue architecture.

Integrins and cadherins are considered to have distinct and opposing functions. Integrins are traditionally cited for their role in cell-substratum interactions, whereas cadherins are thought to mediate strong intercellular cohesion. Together, these adhesion systems play crucial roles in a wide variety of cellular and developmental processes including cell migration, morphology, differentiation and proliferation. In this manuscript we present evidence that integrins possess the ability to mediate strong intercellular cohesion when cells are grown as 3D aggregates. Much of the data elucidating the role of integrins as mediators of cell-extracellular matrix (ECM) interactions have been generated using conventional cell culture techniques in which cells are plated onto ECM-coated 2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We hypothesized that, within this meshwork, integrin-ECM interactions may impart cohesivity to an aggregate of cells by linking adjacent cells together. To test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to express α5β1 integrin and found that these cells formed compact, spherical aggregates. We measured aggregate cohesivity using tissue surface tensiometry, a novel technique that quantifies cell-cell cohesivity of spheroids under physiological conditions. We determined that α5β1 integrin is capable of conferring strong cohesivity (σ=8.22±0.68 dynes/cm) to aggregates of α5-integrin-transfected cells. This cohesion was found to be independent of cadherin expression and was significantly greater than the cohesivity conferred onto CHO-B2 cells transfected with N-cadherin (σ=3.14±0.20 dynes/cm, P≤0.0001), a more traditional cell-cell cohesion system. Fibronectin-null CHO cells that express α5β1 integrin but do not secrete endogenous fibronectin do not form aggregates in fibronectin-depleted medium. Addition of increasing amounts of exogenous dimeric fibronectin to these cells resulted in a dose-dependent compaction. However, compaction failed to occur in the presence of fibronectin monomers. These data indicate that fibronectin is required for α5β1-mediated compaction and that the dimeric structure of fibronectin is essential for this process. Additionally, aggregate formation of the α5 integrin transfectants was inhibited by an RGD peptide thus confirming α5β1 integrin specificity. Collectively, these data confirm our hypothesis that α5β1 integrin acts through fibronectin to link adjacent cells together, thus promoting strong intercellular cohesion in 3D cellular aggregates.

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D. INTRODUCTIONFibronectin (FN) is a multifunctional, adhesive glycoprotein that has wide tissue distribution and is essential for normal development and tissue repair (Hynes, 1990;Schwarzbauer, 1991;Sottile and Hocking, 2002). Cells secrete FN as a disulfide-bonded dimer that binds principally to integrin cell surface receptors. Integrin-FN interactions allow unfolding of the soluble protein and its assembly into a detergentinsoluble fibrillar matrix that can modulate cell morphology, growth, and tissue architecture (Schwarzbauer and Sechler, 1999;Wierzbicka-Patynowski and Schwarzbauer, 2003). FN matrix assembly can also regulate the subsequent deposition and organization of other extracellular matrix molecules, including fibrinogen, collagen-1, and thrombospondin-1 (Sottile and Hocking, 2002). As a consequence, FN fibrillogenesis initiates the formation of a dynamic protein meshwork that provides important structural and environmental cues required for normal cell behavior.One hallmark of malignant transformation in vitro is the loss of FN matrix assembly in two-dimensional (2D) culture. For example, transformed cells frequently show decreased FN synthesis, loss of FN receptor expression, or both (Olden and Yamada, 1977;Plantefaber and Hynes, 1989), and in many cases, the loss of surface FN assayed in these cells correlates with malignant transformation, in vivo. Similarly, oncogenic cells can demonstrate loss of normal integrin function, despite adequate receptor expression. For example, human HT-1080 fibrosarcoma cells express the FN-binding integrin ␣5␤1 and adhere to FN-coated substrates, but they lack the ability to assemble a FN matrix e...