PDX-1 protein is internalized by lipid raft-dependent macropinocytosis

Hirofumi Noguchi, Shinichi Matsumoto, Teru Okitsu, Yasuhiro Iwanaga, Yukihide Yonekawa, Hideo Nagata, Masayuki Matsushita, Fan Yan Wei, Hideki Matsui, Kohtaro Minami, Susumu Seino, Yumi Masui, Shiroh Futaki, Koichi Tanaka

Research output: Contribution to journalArticle

43 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)637-645
Number of pages9
JournalCell Transplantation
Volume14
Issue number9
DOIs
Publication statusPublished - 2005
Externally publishedYes

Keywords

  • Endocytosis
  • Lipid raft macropinocytosis
  • PDX-1
  • Protein transduction domain
  • Protein transduction system

ASJC Scopus subject areas

  • Biomedical Engineering
  • Cell Biology
  • Transplantation

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    Noguchi, H., Matsumoto, S., Okitsu, T., Iwanaga, Y., Yonekawa, Y., Nagata, H., Matsushita, M., Wei, F. Y., Matsui, H., Minami, K., Seino, S., Masui, Y., Futaki, S., & Tanaka, K. (2005). PDX-1 protein is internalized by lipid raft-dependent macropinocytosis. Cell Transplantation, 14(9), 637-645. https://doi.org/10.3727/000000005783982648