Three-dimensional Culture Regulates Raf-1 Expression to Modulate Fibronectin Matrix Assembly

Abstract

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...

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