Deroanne C F, Hajitou A, Calberg-Bacq C M, Nusgens B V, Lapière C M
Laboratories of Connective Tissues Biology, University of Liège, Sart-Tilman, Belgium.
Cancer Res. 1997 Dec 15;57(24):5590-7.
The infection of normal mouse mammary EF43 cells by a retroviral vector carrying either Fgf-3 (EF43.Fgf-3) or Fgf-4 (EF43.Fgf-4) cDNA resulted in the transformation of cells displaying different tumorigenic potentials in nude mice (A. Hajitou and C-M. Calberg-Bacq, Int. J. Cancer, 63: 702-709, 1995). EF43.Fgf-4 produced rapidly developing tumors at all sites of inoculation, whereas EF43.Fgf-3 produced slowly growing tumors only in the mammary fat pad. Cells infected with the vector carrying the selection gene alone (EF43.C) were not tumorigenic. The angiogenic properties of these cells were tested in an in vitro angiogenesis model using human umbilical vein endothelial cells (HUVECs) cultured at the surface of a type I collagen gel and their capacity to form tube-like structures on invasion of the gel. Only the conditioned medium (CM) of EF43.Fgf-4 induced an angiogenic morphotype in HUVECs. In parallel, the mRNA expression of matrix metalloproteinase 1 and c-ETS-1 was increased in the HUVECs displaying a differentiated phenotype, whereas the tissue inhibitor of matrix metalloproteinase 1 mRNA level was decreased. Recombinant human fibroblast growth factor 4 (FGF-4) did not induce an angiogenic phenotype in HUVECs by itself. By Western blot analysis, a high expression of vascular endothelial growth factor (VEGF) was detected in the EF43.Fgf-4 CM. This result was confirmed by Northern blot analysis of total RNA extracted from the three cell types; the steady-state level of VEGF mRNA was low and equivalent in EF43.C and EF43.Fgf-3, whereas it was strongly increased in EF43.Fgf-4. Culturing EF43 cells carrying only the selection gene with increasing concentrations of recombinant human FGF-4 resulted in a dose-dependent stimulation of VEGF. The induction of the angiogenic morphotype and the parallel modulations of the biosynthetic phenotype in HUVECs were completely suppressed by adding a neutralizing antibody directed against VEGF to EF43.Fgf-4 CM. Furthermore, inhibition of protein kinase C by bisindoylmaleimide suppressed the angiogenic phenotype induced by the CM of EF43.Fgf-4. Our results point to an indirect angiogenic activity of FGF-4 through the autocrine induction of VEGF secretion by EF43.Fgf-4 cells, an original signaling pathway that might be significant in tumor progression and metastasis.
携带Fgf-3（EF43.Fgf-3）或Fgf-4（EF43.Fgf-4）cDNA的逆转录病毒载体感染正常小鼠乳腺EF43细胞，导致细胞发生转化，在裸鼠中表现出不同的致瘤潜力（A. Hajitou和C-M. Calberg-Bacq，《国际癌症杂志》，63：702 - 709，1995）。EF43.Fgf-4在所有接种部位均产生快速生长的肿瘤，而EF43.Fgf-3仅在乳腺脂肪垫中产生缓慢生长的肿瘤。仅感染携带选择基因的载体（EF43.C）的细胞不具有致瘤性。在体外血管生成模型中测试了这些细胞的血管生成特性，该模型使用培养在I型胶原凝胶表面的人脐静脉内皮细胞（HUVECs）以及它们在侵入凝胶时形成管状结构的能力。只有EF43.Fgf-4的条件培养基（CM）在HUVECs中诱导出血管生成形态。同时，在表现出分化表型的HUVECs中，基质金属蛋白酶1和c-ETS-1的mRNA表达增加，而基质金属蛋白酶1组织抑制剂mRNA水平降低。重组人成纤维细胞生长因子4（FGF-4）本身不会在HUVECs中诱导血管生成表型。通过蛋白质印迹分析，在EF43.Fgf-4 CM中检测到血管内皮生长因子（VEGF）的高表达。从这三种细胞类型中提取的总RNA的Northern印迹分析证实了这一结果；VEGF mRNA的稳态水平在EF43.C和EF43.Fgf-3中较低且相当，而在EF43.Fgf-4中则强烈增加。用浓度递增的重组人FGF-4培养仅携带选择基因的EF43细胞，导致VEGF受到剂量依赖性刺激。通过向EF43.Fgf-4 CM中添加针对VEGF的中和抗体，完全抑制了HUVECs中血管生成形态的诱导以及生物合成表型的平行调节。此外，双吲哚马来酰亚胺对蛋白激酶C的抑制作用抑制了EF43.Fgf-4 CM诱导的血管生成表型。我们的结果表明FGF-4具有间接血管生成活性，通过EF43.Fgf-4细胞自分泌诱导VEGF分泌，这是一种可能在肿瘤进展和转移中起重要作用的原始信号通路。
