Development Of The Pancreas To The Discovery Of Transcription Factor Protein Transduction Domain And Application Research

Protein transduction domains(PTDs) are small cationic peptides.Many PTD-fused full-length functional proteins have been demonstrated to transduce into cells and tissues.Such technology could eliminate the size restrictions,enabling previously unavailable large molecules to modulate in vivo biology and alleviate disease.Pax4,a paired-box transcription factor,is a key regulator of pancreatic islet cell growth and differentiation.Here,we report for the first time that the Pax4 protein can permeate into various cell types including pancreatic islets. The Paired Domain of Pax4 serves as a novel protein transduction domain (PTD).The Paired-EGFP protein can transduce in a dose- and timedependent manner.The cellular uptake of Pax4 PTD can be completely blocked by heparin,whereas cytochalasin D and amiloride Were partially effective in blocking the Pax4 protein entry,which suggested important roles of cell surface glycosaminoglycans and macropinocytosis during the protein transduction.Transduced intact Pax4 protein functions similarly to the endogenous Pax4.It inhibits the Pax6 mediated transactivation and protects Min6 cells against TNFα-induced apoptosis.Pax4 protein protects rat islet cells against apoptosis through the activation of bcl-xL and c-myc gene expression.In this study,we show that the purified Pax4 protein can be transduced into cells and analyzed the molecular mechanism of Paired PTD transduction. These data suggest that Pax4 protein transduction could be a safe and valuable strategy for protecting islet cell growth in culture from apoptosis. Transcription factors provide the genetic instructions that drive pancreatic development.PDX-1,NeuroD,Nkx6.1,Pax 4 and MafA are reported to function in regulating the expression of the insulin gene and several other islet-specific genes in matureβ-cells.Many studies have been shown to overexpress pancreatic transcription factors using virus-mediated approaches, but it would be difficult to apply the virus-mediated approach to clinical medicine.PTDs are known to allow various proteins to be efficiently delivered into cells through the plasma membrane,and thus the protein delivery system is very promising at this point.MafA,a basic-leucine zipper(bZIP) transcription factor,is expressed only inβ-cell and functions as a potent activator of insulin gene transcription.Here we reported for the first time that the MafA protein is capable of penetrating the cell membrane through the Basic sequence.The MafAb-EGFP protein can transduce into various living cells(HEK293,IEC6,PLC/PRF/5).The cellular uptake of MafA PTD can be completely blocked by heparin,whereas cytochalasin D and amiloride were partially effective in blocking the protein entry,similar with the Pax4 PTD and most of other PTDs.Furthermore, transcription activation of MafA protein was still preserved after internalized into cells.MafA protein can transduce into IEC6 cells.The cells could be induced to differentiate into insulin-producing cells.MafA protein remains functionally active in vivo.The STZ induced diabetic ICR mice return to normal glycemia, 24h after the injection of MafA protein.MafA can induce insulin gene expression in small intestine cells.Quantitative RT-PCR revealed a 4.1- and 9.8-fold increase in insulin 1 and insulin 2 transcripts.The insulin-positive cells are observed at the intervillus epithelia in the MafA-treated intestine.In this study,we show that the purified MafA protein can be transduced into cells and analyzed the molecular mechanism of Basic PTD transduction. The application of MafA protein with transduction activity might become useful and convenient tool for diabetes mellitus treatment.