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Vol. 17, Issue 8, 3386-3396, August 2006

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

Department of Surgery, Robert Wood Johnson Medical School, New Brunswick, NJ 08903

Submitted September 11, 2005; Revised May 1, 2006; Accepted May 5, 2006
Monitoring Editor: Jean Schwarzbauer

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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

* These authors contributed equally to this work.

Address correspondence to: Siobhan A. Corbett ( corbetsi{at}umdnj.edu)

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