title: PDX-1 protein is internalized by lipid raft-dependent macropinocytosis.
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.
authors.affiliation: Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, Sakyo-ku, Japan. noguchih@kuhp.kyoto-u.ac.jp
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Noguchi
authors.forename: Shinichi
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Teru
authors.initials: T
authors.lastname: Okitsu
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Iwanaga
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Yonekawa
authors.forename: Hideo
authors.initials: H
authors.lastname: Nagata
authors.forename: Masayuki
authors.initials: M
authors.lastname: Matsushita
authors.forename: Fan-Yan
authors.initials: FY
authors.lastname: Wei
authors.forename: Hideki
authors.initials: H
authors.lastname: Matsui
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Minami
authors.forename: Susumu
authors.initials: S
authors.lastname: Seino
authors.forename: Yumi
authors.initials: Y
authors.lastname: Masui
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D001692:Biological Transport; D022461:Caveolins / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D010873:Pinocytosis; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D022461:Caveolins; D002966:Clathrin; D018398:Homeodomain Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein