Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.
Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.
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DOI：
10.1091/mbc.e05-09-0849
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发表时间：
2006-08
影响因子：
3.3
通讯作者：
B. S. Winters;B. K. Raj;E. Robinson;R. Foty;S. Corbett
中科院分区：
文献类型：
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作者：
B. S. Winters;B. K. Raj;E. Robinson;R. Foty;S. Corbett
致癌性转化与纤连蛋白（FN）基质组装减少有关。例如，HT-1080纤维肉瘤和MAT-LyLu细胞系在单层培养（二维[2D]系统）中生长时均不能组装FN基质。在这项研究中，我们表明，这些细胞重新获得组装FN矩阵的能力时，他们生长为聚集体（三维[3D]系统）。与单层培养中生长的细胞相比，3D中FN基质组装与Raf-1蛋白表达降低相关。这种效应与定量RT-PCR测定的Raf-1 mRNA水平降低有关，而与内源性Raf-1的蛋白酶体介导的降解无关。有趣的是，Raf-1启动子-报告基因构建体的瞬时表达表明Raf-1启动子在3D中的活性增加，这表明向3D培养的过渡可以调节Raf-1 mRNA的稳定性。最后，为了证实Raf-1表达降低导致FN基质组装增加，我们使用了Raf-1的药理学和小干扰RNA敲低。这恢复了2D培养中细胞组装FN基质的能力。此外，Raf-1的过表达阻止了3D培养的细胞的FN基质组装，导致聚集体致密化降低。这项工作为细胞微环境如何影响Raf-1表达以调节3D中的细胞-FN相互作用提供了新的见解。
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.