Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation
Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation
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DOI：
10.1046/j.1523-1755.2002.00333.x
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发表时间：
2002-05-01
影响因子：
19.6
通讯作者：
Sisic, Z
中科院分区：
文献类型：
--
作者：
Strutz, F;Zeisberg, M;Sisic, Z
背景。上皮间质转化（EMT）在胚胎发育和肿瘤发生中发挥重要作用，并在纤维发生过程中的器官重塑中得到描述。在肾脏中，通过转化生长因子 (TGF)-β1 和表皮生长因子 (EGF) 的共孵育，可以在培养的近端肾小管上皮中有效诱导 EMT。最近，我们还在具有明显间质纤维化的人肾脏中观察到碱性成纤维细胞生长因子-2（FGF-2）蛋白和mRNA的过度表达。本研究的目的是比较 FGF-2 作为肾小管上皮细胞 EMT 促进剂与 EGF 和 TGF-β1 的作用。我们分析了三种细胞因子对EMT四个不同方面的形态发生影响：细胞运动、细胞标志物的表达和调节、细胞外基质（ECM）蛋白的合成和分泌以及基质降解。方法。通过迁移测定研究细胞运动性，并通过免疫荧光和免疫印迹分析细胞分化标记物。此外，通过荧光素酶报告基因构建体和稳定转染分析了上皮粘附分子 E-钙粘蛋白和成纤维细胞特异性蛋白 1 (FSP1) 的调节。 I 型和 IV 型胶原以及纤连蛋白的 ELISA 用于 ECM 合成，酶谱用于分析基质降解。结果。在两个肾小管细胞系中，FGF-2 诱导细胞穿过肾小管基底膜的运动。所有三种细胞因子均诱导波形蛋白和 FSP1 的表达，但通过免疫荧光检测，只有 FGF-2 和 TGF-β1 降低细胞角蛋白的表达。这些效应在远端肾小管上皮细胞齿中最为明显，并通过免疫印迹分析得到证实。 TGF-β1 加 FGF-2 使 E-钙粘蛋白的表达降低 61.5 +/- 3.3%，细胞角蛋白的表达降低 91 +/- 0.5%。相反，间充质标记物α-平滑肌肌动蛋白（SMA）和FSP1被FGF-2分别诱导2.2+/-0.1倍和6.8+/-0.9倍。有趣的是，间充质标记物 OB-钙粘蛋白的从头表达仅由 FGF-2 和 EGF 诱导，而不由 TGF-β1 诱导。所有tin-cc细胞因子都会刺激FSP1并降低E-钙粘蛋白启动子活性。 FGF-2 还诱导细胞内纤连蛋白合成，但不诱导分泌，后者仅由 TGF-β1 刺激。最后，酶谱分析表明，FGF-2 诱导 MMP-2 活性增加 2.6 +/- 0.5 倍，MMP-9 活性增加 2.4 +/- 0.1 倍，提供了基底膜崩解和转化上皮细胞迁移至间质的机制。结论。 FGF-2 通过刺激微环境蛋白酶对基于器官的上皮单位的解聚至关重要，从而对 EMT 机制做出重要贡献。此外，上皮和间质标记蛋白的表达似乎在启动子水平上受到影响。
Background. Epithelial-mesenchymal transformation (EMT) plays an important role in embryonic development and tumorigenesis and has been described in organ remodeling during fibrogenesis. in the kidney, EMT can be induced efficiently in cultured proximal tubular epithelium by coincubation of transforming growth factor (TGF)-beta1 and epidermal growth factor (EGF). Recently, we also have observed overexpression of basic fibroblast growth factor-2 (FGF-2) protein and mRNA in human kidneys with marked interstitial fibrosis. The aims of the present study were to compare the effects of FGF-2 as a facilitator of EMT in tubular epithelial cells with EGF and TGF-beta1. We analyzed the morphogenic effects of the three cytokines on four different aspects of EMT: cell motility, expression and regulation of cellular markers, synthesis and secretion of extracellular matrix (ECM) proteins as well as matrix degradation.Methods. Cell motility was studied by a migration assay and cell differentiation markers were analyzed by immunofluorescence and immunoblots. In addition, regulation of the epithelial adhesion molecule E-cadherin and fibroblast-specific protein 1 (FSP1) were analyzed by luciferase reporter constructs and stable transfections. ELISAs for collagen types I and IV and fibronectin were used for ECM synthesis, and zymograms were utilized for analysis of matrix degradation.Results. FGF-2 induced cell motility across a tubular basement membrane in two tubular cell lines. All three cytokines induced the expression of vimentin and FSP1, but only FGF-2 and TGF-beta1 reduced cytokeratin expression by immunofluorescence. These effects were most demonstrable in the distal tubular epithelial cell tine and were confirmed by immunoblot analyses. Expression of E-cadherin was reduced by 61.5 +/- 3.3% and expression of cytokeratin by 91 +/- 0.5% by TGF-beta1 plus FGF-2. Conversely, the mesenchymal markers alpha-smooth muscle actin (SMA) and FSP1 were induced with FGF-2 by 2.2 +/- 0.1-fold and 6.8 +/- 0.9-fold, respectively. Interestingly, de novo expression of the mesenchymal marker OB-cadherin was induced only by FGF-2 and EGF but not by TGF-beta1. All tin-cc cytokines stimulated FSP1 and decreased E-cadherin promoter activity. FGF-2 also induced intracellular fibronectin synthesis but not secretion, the latter of which was stimulated exclusively by TGF-beta1. Finally, zymographic analyses demonstrated that FGF-2 induced MMP-2 activity by 2.6 +/- 0.5-fold and MMP-9 activity by 2.4 +/- 0.1-fold, providing a mechanism for basement membrane disintegration and migratory access of transforming epithelium to the interstitium.Conclusions. FGF-2 makes an important contribution to the mechanisms of EMT by stimulating microenvironmental proteases essential for disaggregation of organ-based epithelial units. Furthermore, the expression of epithelial and mesenchymal marker proteins seems to be affected at the promoter level.