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MBC in Press, published online ahead of print May 17, 2006
Mol. Biol. Cell 10.1091/mbc.E05-09-0849

A more recent version of this article appeared on August 1, 2006
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Submitted on September 11, 2005
Revised on May 1, 2006
Accepted on May 5, 2006

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

B. S Winters,* B. K. Mohan Raj,* E. E. Robinson, R. A. Foty, and S. A. Corbett

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

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 fibronectin (FN) matrix when grown in monolayer culture (2D). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3D). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression when 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 pharmacologic and siRNA 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.

*These authors contributed equally to this work.

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




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