title: 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.
authors.affiliation: Department of Surgery, Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Winters
authors.forename: B K Mohan
authors.initials: BK
authors.lastname: Raj
authors.forename: E E
authors.initials: EE
authors.lastname: Robinson
authors.forename: R A
authors.initials: RA
authors.lastname: Foty
authors.forename: S A
authors.initials: SA
authors.lastname: Corbett
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D015536:Down-Regulation / Q000235:genetics; D005109:Extracellular Matrix / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D061988:Proteasome Inhibitors; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats
chemical_list: D005353:Fibronectins; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases