Wakisaka Naohiro, Murono Shigeyuki, Yoshizaki Tomokazu, Furukawa Mitsuru, Pagano Joseph S
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599-7295, USA.
Cancer Res. 2002 Nov 1;62(21):6337-44.
We have shown that the EBV oncoprotein, latent membrane protein 1 (LMP1), induces a constellation of tumor-invasiveness factors. Fibroblast growth factor (FGF)-2 is angiogenic as well mitogenic. Although FGF-2 does not contain a hydrophobic signal sequence for secretion, FGF-2 is released extracellularly. However, the mechanism by which FGF-2 is released is unclear. Here we show first that LMP1 induces in epithelial cells the expression of FGF-2 mRNA and protein through both LMP1 COOH-terminal activation domains, CTAR 1 and CTAR 2, which can activate nuclear factor (NF)-kappaB signaling and also the p38 mitogen-activated protein kinase pathway. Coexpression of IkappaBalpha (S32A/S36A), which cannot be phosphorylated and prevents NF-kappaB activation, with LMP1 inhibited induction of FGF-2 by LMP1, which suggests that LMP1 induces FGF-2 via NF-kappaB signaling. Moreover, unlike phorbol 12-myristate 13-acetate LMP1 also induced the release of the M(r) 18,000 isoform of FGF-2 protein. Transfection of Ad-AH cells with LMP1 deletion mutants lacking either CTAR 1 or CTAR 2 also induced the release of the protein. Secretion was confirmed in 293 cells, which do not contain detectable endogenous FGF-2 protein, cotransfected with FGF-2 and LMP1. Finally, Na(+)/K(+)-ATPase participates in FGF-2 release, independently of the classical endoplasmic reticulum/Golgi pathway. In this study, the release of M(r) 18,000 FGF-2 protein was partially suppressed by ouabain, which inhibits the activity of Na(+)/K(+)-ATPase alpha1 subunit, but not by Brefeldin A, which inhibits the endoplasmic reticulum/Golgi-dependent secretory pathway. In contrast, the release of M(r) 18,000 FGF-2 protein was almost completely inhibited by IkappaBalpha (S32A/S36A). These results suggest that FGF-2 release is independently mediated by NF-kappaB signaling, not simply a consequence of induction itself. Thus, NF-kappaB signaling is involved in induction of expression and release of FGF-2 by LMP1.
我们已经证明，EB病毒癌蛋白潜伏膜蛋白1（LMP1）可诱导一系列肿瘤侵袭因子。成纤维细胞生长因子（FGF）-2具有血管生成和促有丝分裂作用。尽管FGF-2不含有用于分泌的疏水信号序列，但它能在细胞外释放。然而，FGF-2释放的机制尚不清楚。在此我们首次表明，LMP1通过其两个COOH末端激活域CTAR 1和CTAR 2在上皮细胞中诱导FGF-2 mRNA和蛋白的表达，这两个激活域可激活核因子（NF）-κB信号通路以及p38丝裂原活化蛋白激酶途径。不能被磷酸化并阻止NF-κB激活的IkappaBalpha（S32A/S36A）与LMP1共表达时，可抑制LMP1对FGF-2的诱导，这表明LMP1通过NF-κB信号通路诱导FGF-2。此外，与佛波酯12-肉豆蔻酸酯13-乙酸盐不同，LMP1还诱导了分子量为18,000的FGF-2蛋白异构体的释放。用缺乏CTAR 1或CTAR 2的LMP1缺失突变体转染Ad-AH细胞也诱导了该蛋白的释放。在与FGF-2和LMP1共转染的293细胞（不含有可检测到的内源性FGF-2蛋白）中证实了分泌。最后，Na(+)/K(+)-ATP酶参与FGF-2的释放，独立于经典的内质网/高尔基体途径。在本研究中，哇巴因可部分抑制分子量为18,000的FGF-2蛋白的释放，哇巴因抑制Na(+)/K(+)-ATP酶α1亚基的活性，但布雷菲德菌素A不能抑制，布雷菲德菌素A抑制内质网/高尔基体依赖性分泌途径。相反，IkappaBalpha（S32A/S36A）几乎完全抑制了分子量为18,000的FGF-2蛋白的释放。这些结果表明，FGF-2的释放是由NF-κB信号通路独立介导的，而不仅仅是诱导本身的结果。因此，NF-κB信号通路参与了LMP1诱导FGF-2的表达和释放。
