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

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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


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: 2005-09-01

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