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.

Activation of c-Jun NH2-terminal kinase (JNK) pathway during islet transplantation and prevention of islet graft loss by intraportal injection of JNK inhibitor

“…Protein transduction systems have been employed for the delivery of full-length proteins into many cell types [38][39][40][41][42][43][44][45][46][47]. A series of protein transduction domains (PTDs, also known as cell-penetrating peptides), such as antennapedia [39,40], VP22 [41], TAT [42][43][44][45][46] and poly-arginine [31,33,47], have been shown to cross biological membranes efficiently and to promote the delivery of peptides and proteins into cells.…”

“…A series of protein transduction domains (PTDs, also known as cell-penetrating peptides), such as antennapedia [39,40], VP22 [41], TAT [42][43][44][45][46] and poly-arginine [31,33,47], have been shown to cross biological membranes efficiently and to promote the delivery of peptides and proteins into cells. These properties make protein transduction potentially useful for islet transplantation.…”

Induction of Pancreatic Stem/Progenitor Cells into Insulin-Producing Cells by Adenoviral-Mediated Gene Transfer Technology

“…Several studies have suggested that adult β-cells might originate diabetes. Replacement of the β-cell mass offers an alternative to standard insulin treatment and may overcome from duct or duct-associated cells (4, 7,13,29,[31][32][33]. Ductal progenitor cells in the pancreas would become the long-term side effects associated with current therapies.…”

Reversal of Streptozotocin-Induced Diabetes in Mice by Cellular Transduction With Recombinant Pancreatic Transcription Factor Pancreatic Duodenal Homeobox-1

“…Given its key role in pancreatic development, islet ␤-cell regeneration, and liver-to-endocrine pancreas transdifferentiation, Pdx1 represents an ideal candidate for protein therapy, especially since its antennapedia-like domain is a PTD, mediating its rapid entry into cells (23)(24)(25). Previous in vitro studies involving pancreatic duct and islet cells (23), as well as embryonic stem cells (26), have demonstrated that Pdx1 cell entry induces insulin gene expression and positive autoregulation of Pdx1 gene expression (23,26).…”