Differentiation restricted endocytosis of cell penetrating peptides in MDCK cells corresponds with activities of Rho-GTPases
Differentiation restricted endocytosis of cell penetrating peptides in MDCK cells corresponds with activities of Rho-GTPases
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DOI：
10.1007/s11095-006-9212-1
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发表时间：
2007-04-01
影响因子：
3.7
通讯作者：
Merkle, Hans P.
中科院分区：
文献类型：
--
作者：
Foerg, Christina;Ziegler, Urs;Merkle, Hans P.
生物大分子进入细胞受到细胞膜屏障功能的限制。将这些分子与可以移位细胞膜的细胞穿透肽（CPP）结合，为生物医学研究开辟了新的视野。在这里，我们研究了 hCT(9-32)-br（一种人降钙素衍生的分支 CPP）和 SAP（一种 γ-玉米醇溶蛋白相关序列）的细胞内化。通过共焦激光扫描显微镜 (CLSM) 和荧光激活细胞分选 (FACS) 使用适当的对照，对增殖和融合的 MDCK 细胞进行荧光标记的 CPP 的内化。在模拟炎症上皮病理的炎症性 IFN-γ/TNF-α 诱导汇合 MDCK 模型中进一步阐述了内化。通过 Pull-down 测定和蛋白质印迹分析监测增殖和汇合 MDCK 细胞中活性形式 Rho-GTP 酶（Rho-A 和 Rac-1）的活性。我们通过表明脂筏和网格蛋白包被的凹坑的过程观察到肽显着内吞到增殖的 MDCK 中。然而，在汇合的 MDCK 中，我们注意到内吞作用出现了巨大但非复合特异性的减慢。这与 Rho-GTP 酶内吞作用的下调相对应，此前已发现 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 有关；反之亦然，MDCK 细胞密度、细胞分化程度和内吞减慢被发现与活性 Rac-1 相关。据我们所知，这是第一项阐明先前观察到的 CPP 分化限制内化到上皮细胞模型的研究。在模拟炎症上皮病理的炎症 IFN-γ/TNF-α 诱导汇合 MDCK 模型中，CPP 内化以细胞因子浓度依赖性方式增强，导致最大增强率高达 90%。我们建议细胞因子诱导汇合 MDCK 中脂筏的重新分布来引起这种增强。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，并指出炎症上皮病理学是应用 CPP 进行细胞递送的潜在利基。
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-alpha a 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-alpha a 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.