Spatial-specific TGF-β1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung
Spatial-specific TGF-β1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung
复制标题
DOI：
10.1016/s0171-9335(99)80040-5
复制
发表时间：
1999-10-01
影响因子：
6.6
通讯作者：
Warburton, D
中科院分区：
文献类型：
--
作者：
Zhao, JS;Sime, PJ;Warburton, D
转化生长因子（TGF）-β的表达和激活在哺乳动物器官形态发生中所起的精确时空作用仍不完全清楚。使用含有工程TGF-β 1 cDNA的复制缺陷型腺病毒载体，我们研究了在培养的胚胎小鼠肺分支形态发生过程中局部过表达潜伏或突变的组成型活性TGF-β 1蛋白的空间效应。通过气管内显微注射重组腺病毒实现外源基因向肺上皮的转移，而将肺浸没在病毒中导致基因转移到胸膜和皮下间充质细胞中，如通过β-半乳糖苷酶的细胞化学染色所揭示的。只有用活性而非潜伏性TGF-β 1基因转染的肺显示活性TGF-β水平升高。通过气管内微量注射，上皮细胞过度表达活性而非潜伏性TGF-β 1可抑制36%的肺分支形态发生。相比之下，用活性或潜伏性TGF-β 1重组病毒浸没的肺没有表现出对分支的抑制作用。采用竞争性聚合酶链反应结合逆转录技术检测肺基因调控。直接呼吸道微量注射过表达活性TGF-β 1的腺病毒导致上皮表面活性蛋白（SP）-C和SP-B mRNA水平的剂量依赖性抑制分别高达76%和70%，而相比之下，纤连蛋白和基质Gla蛋白（MGP）mRNA水平保持稳定。然而，在1x 10（11）pfu/ml活性TGF-β 1病毒存在下，将肺浸没在表达活性TGF-β 1的腺病毒中，显示纤连蛋白和MGP mRNA水平的浓度依赖性诱导分别高达4.3倍和4.7倍。另一方面，通过显微注射或浸没用表达潜伏性TGF-β 1的腺病毒处理的肺未能证明对上皮或间充质基因表达的任何调节作用。我们的结论是，腺病毒介导的过度表达激活TGF-β 1在特定的空间隔室的结果分别在分支形态发生和上皮特异性基因表达的抑制，或在不影响形态发生或上皮特异性基因表达的情况下诱导基质基因表达，这取决于给药途径。此外，缺乏潜在的TGF-β 1过表达的影响，强烈表明，TGF-β激活本身提供了一个重要的基因座的TGF-β信号传导的空间效应，在胚胎肺分支形态发生的精细调节。
The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-beta plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-beta 1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-beta 1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal microinjection of recombinant adenovirus, while submerging lungs in virus resulted iii gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for beta-galactosidase. Only lungs transfected with active, but not latent TGF-beta 1 gene, showed elevated levels of active TGF-beta. Epithelial over-expression of active, but not latent TGF-beta 1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36%. In contrast, lungs submerged with either active or latent TGF-beta 1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-beta 1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76 % and 70 %, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-beta 1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1x10(11) pfu/ml active TGF-beta 1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-beta 1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated overexpression of activated TGF-beta 1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-beta 1 over-expression strongly suggests that TGF-beta activation per se provides an important locus of fine regulation of the spatial effects of TGF-beta signaling during embryonic lung branching morphogenesis.