Foerg Christina, Ziegler Urs, Fernandez-Carneado Jimena, Giralt Ernest, Merkle Hans P
Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Pharm Res. 2007 Apr;24(4):628-42. doi: 10.1007/s11095-006-9212-1. Epub 2007 Mar 3.
Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a gamma-zein related sequence.
Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-gamma/TNF-alphaa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis.
We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-gamma/TNF-alphaa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement.
Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery.
生物大分子进入细胞受到细胞膜屏障功能的限制。将此类分子与能够转运穿过细胞膜的细胞穿透肽（CPP）相连，为生物医学研究开辟了新视野。在此，我们研究了人降钙素衍生的分支CPP hCT(9 - 32)-br和γ-玉米醇溶蛋白相关序列SAP的细胞内化情况。
通过共聚焦激光扫描显微镜（CLSM）和荧光激活细胞分选（FACS），利用适当的对照，对增殖期和汇合期的MDCK细胞进行荧光标记CPP的内化实验。在模拟炎症上皮病变的炎症性、IFN-γ/TNF-α诱导的汇合期MDCK模型中进一步阐述内化情况。通过下拉实验和蛋白质印迹分析监测增殖期和汇合期MDCK细胞中活性形式的Rho-GTP酶（Rho-A和Rac-1）的活性。
我们观察到肽通过一种提示脂筏和网格蛋白包被小窝均参与的过程，显著地被内吞进入增殖期的MDCK细胞。然而，在汇合期的MDCK细胞中，我们注意到内吞作用大量但非化合物特异性的减缓。这与Rho-GTP酶对内吞作用的下调相对应，先前已确定Rho-GTP酶与内吞运输密切相关。事实上，我们发现内吞内化与活性Rho-A相关；反之，发现MDCK细胞密度、细胞分化程度和内吞减缓与活性Rac-1相关。据我们所知，这是第一项揭示先前观察到的CPPs在分化限制下进入上皮细胞模型内化情况的研究。在模拟炎症上皮病变的炎症性、IFN-γ/TNF-α诱导的汇合期MDCK模型中，CPP内化以细胞因子浓度依赖性方式增强，最大增强率高达90%。我们认为细胞因子诱导汇合期MDCK细胞中脂筏的重新分布导致了这种增强。
我们的研究结果强调了分化细胞模型在CPP内化研究中的重要性，并指出炎症上皮病变作为CPPs用于细胞递送应用的潜在合适环境。
