K-Ras regulates the steady-state expression of matrix metalloproteinase 2 in fibroblasts
K-Ras regulates the steady-state expression of matrix metalloproteinase 2 in fibroblasts
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DOI：
10.1074/jbc.m301931200
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发表时间：
2003-08-22
影响因子：
4.8
通讯作者：
Wolfman, A
中科院分区：
文献类型：
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作者：
Liao, JH;Wolfman, JC;Wolfman, A
成纤维细胞组成性地表达基质金属蛋白酶2 (MMP-2)，其特异性地切割IV型胶原，这是基底膜的主要结构成分。血清停药没有改变MMP-2的表达水平，这表明MMP-2的表达受到一系列影响MMP-2启动子的稳态条件的调节。显性阴性Ras蛋白的表达显著抑制了MMP-2的转录，从而提示稳态Ras功能在调控MMP-2表达中的作用。Kirsten-Ras （K-Ras）敲除成纤维细胞表达的MMP-2基础水平检测不到，而N-Ras敲除成纤维细胞表达的MMP-2组成水平与野生型对照成纤维细胞相似。通过MMP-2启动子荧光素酶报告试验，我们发现K-Ras敲除的成纤维细胞中，c-K(B)-Ras的短暂表达部分恢复了MMP-2的转录，而cK(A)-Ras则没有。一种磷酸肌醇-3 （PI-3）激酶特异性抑制剂（LY294002）降低了野生型成纤维细胞中MMP-2的基础水平。通过过度表达PI-3激酶调控域（p85）阻断PI-3激酶信号传导也可下调稳态MMP-2水平。与野生型成纤维细胞相比，不表达AKT1的成纤维细胞也表达了较少的MMP-2。这些数据表明，c-K(B)-Ras通过PI-3激酶：akt依赖性信号通路调节稳态MMP-2表达。由于大多数MMP-2检测是使用血清饥饿成纤维细胞的条件培养基进行的，这些数据也强调了我们之前的观察结果，即Ras蛋白在没有急性有丝分裂刺激的情况下具有功能。此外，这是第一次证明一个特定的稳态函数可归因于K(B)-Ras。
Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression. Kirsten-Ras (K-Ras) knockout fibroblasts express undetectable basal levels of MMP-2, whereas N-Ras knockout fibroblasts expressed constitutive levels of MMP-2 similar to those observed in wild-type control fibroblasts. Using an MMP-2 promoter-luciferase reporter assay, we demonstrated that the transcription of MMP-2 in K-Ras knockout fibroblasts was partially restored by transient expression of c-K(B)-Ras but not cK( A)-Ras. A phosphoinositide-3 (PI-3) kinase-specific inhibitor (LY294002) decreased the basal level of MMP-2 in wild-type fibroblasts. Blocking PI-3 kinase signaling by overexpression of the regulatory domain of PI-3 kinase (p85) also down-regulated the steady-state MMP-2 levels. Fibroblasts that fail to express AKT1 also expressed decreased amounts of MMP-2 compared with wild-type fibroblasts. These data suggest that steady-state MMP-2 expression is regulated by c-K( B)-Ras through a PI-3 kinase: AKT-dependent signaling pathway. Because the majority of the MMP-2 assays were performed using conditioned media from serum-starved fibroblasts, these data also highlight our previous observations that Ras proteins have functions in the absence of acute mitogenic stimulations. In addition, this is the first demonstration of a specific steady-state function attributable to K(B)-Ras.