TAT-MafA Fusion Protein Induces Intestinal Epithelial Cells IEC-6 Into Insulin Positive Cells

MafA, a lately discoveredβ-cell-specific member of the Maf family transcription fact- ors, seems to regulate genes involved inβ-cell functions and development besides insulin.Protein transduction domains (PTDs) are polypeptides containing strong cationic cluster of amino acids, which can carry large molecules into many kinds of mamalian cells. The TAT protein from human immunodeficiency viruses has been shown to enter cells when added exogenously.The facts that pancreas and intestine share the same development background indicate the intestinal cells have potential to transdifferentiate into insulin positive ones. The intestinal crypt cells may provide a useful tool for diabetes gene/cell therapy.In this study, we firstly constructed prokaryotic expression vector pET32aTAT- MafA by fusing TAT with MafA. And found the purified TAT-MafA fusion protein expressed by E.coli can permeate into many kinds of cells, such as HEK293, LEPCs, and IEC-6. There was no obvious transduction efficiency detected among those cell lines. We observed the internalization is time- and dose- dependent. The transduction efficiency reached the highest level when protein concentration was 1μM. The stimulation of transcription of insulin gene promoter shows the TAT mediated MafA retains its own biological activities after transduction. To investigate whether TAT mediated MafA is able to induce IEC-6(rat intestinal epithelial cells) into insulin positive cells, we incubated IEC-6 with TAT-MafA. The expression of insulin and some other important factors in pancreas was detected in the IEC-6 by RT-PCR analysis 24h after incubation. We concluded that TAT-MafA can transdifferentiate intestinal epithelial cells into insulin producing ones. Furthermore the hyperglycemia in STZ induced diabetic mice was alleviated by TAT-MafA after tail vein injection.The fusion protein with high efficiency in transduction and be capable of transdifferentiating intestinal epithelial into insulin positive cells set the foundation for using intestine and protein transducion as a new strategy to treat with diabetes. Unlike traditional gene therapy using viruses or liposomes directly to mediate genes into target organ, the protein transduction technology is promising in the treatment of type 1 diabetes , since it is more convenient, more effective and safer.