Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly

• Published in: Molecular biology of the cell Vol. 17; no. 8; pp. 3386 - 3396
• Main Authors: Winters, B S, Raj, B K Mohan, Robinson, E E, Foty, R A, Corbett, S A
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.08.2006
• Subjects: Animals; Cell Culture Techniques; Down-Regulation - genetics; Extracellular Matrix - metabolism; Extracellular Signal-Regulated MAP Kinases - metabolism; Fibronectins - metabolism; Fibronectins - secretion; Focal Adhesion Protein-Tyrosine Kinases - metabolism; Gene Expression Regulation; Humans; Oncogene Protein pp60(v-src) - metabolism; Proteasome Inhibitors; Proto-Oncogene Proteins c-raf - genetics; Proto-Oncogene Proteins c-raf - metabolism; Rats; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Interfering - genetics
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E05-09-0849

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.