title: Recombinant MafA protein containing its own protein transduction domain stimulates insulin gene expression in IEC-6 cells.
abstract: OBJECTIVE
  MafA, a basic leucine zipper (bZIP) transcription factor, functions as a potent activator of insulin gene transcription in β-cell. In this paper, we aimed to investigate whether the entire MafA protein has the self-delivery activity, and that the arginine- and lysine-rich sequence in MafA bZIP domain is an efficient protein transduction domain (PTD).
  
  
  METHODS
  Entire MafA protein internalization was evaluated with Western blot and immunofluorescence. The distribution of the PTD-EGFP (enhanced green fluorescence protein) was examined by fluorescent microscope observation. Luciferase reporter assay was used to investigate the effect of the transduced MafA protein on insulin 2 promoter activity. Additionally, we conducted RT-PCR to detect the expression of insulin mRNA in MafA treated IEC-6 cells.
  
  
  RESULTS
  The arginine- and lysine-rich peptide of MafA serves as a novel PTD. PTD-EGFP can permeate into various cell types including Min6 (a β-cell line), and transduce in a dose- and time-dependent manner. The cellular uptake of MafA PTD can be completely blocked by heparin, whereas cytochalasin D and amiloride were partially effective in blocking the PTD-EGFP protein entry. Transduced intact MafA protein behaves in the same way as the endogenous MafA, stimulating the transcription of insulin promoter and further inducing insulin expression in treated non-β-cell line (IEC-6).
  
  
  CONCLUSIONS
  These results indicate that the MafA PTD could serve as a therapeutic delivery vehicle, and further suggest that MafA protein transduction could be a valuable strategy for enhancing insulin gene transcription without requiring gene transfer technology.
authors.affiliation: FuJian Provincial Key Laboratory of Transplant Biology, Fuzhou General Hospital, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
authors.forename: Hailan
authors.initials: H
authors.lastname: Luo
authors.forename: Huiling
authors.initials: H
authors.lastname: Wu
authors.forename: Michael S
authors.initials: MS
authors.lastname: Lan
authors.forename: Jianming
authors.initials: J
authors.lastname: Tan
authors.forename: Daru
authors.initials: D
authors.lastname: Lu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D007668:Kidney / Q000166:cytology; D051473:Maf Transcription Factors, Large / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D007328:Insulin; C485159:MAFA protein, human; D051473:Maf Transcription Factors, Large; D012333:RNA, Messenger; D011994:Recombinant Proteins