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PDX-1 Protein Is Internalized by Lipid Raft-Dependent Macropinocytosis

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Abstract:

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell–surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4-64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4-64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.

Keywords: Endocytosis; Lipid raft macropinocytosis; PDX-1; Protein transduction domain; Protein transduction system

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000005783982648

Affiliations: 1: *Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan, 2: Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan 3: Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan 4: Department of Experimental Therapeutics, Translational Research Center, Kyoto University Graduate School of Medicine, 54 kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan 5: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan 6: §Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, ¶PRESTO, JST, Kawaguchi, Saitama, Japan

Publication date: September 1, 2005

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.

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