Pancreatic Development In The Discovery And Function Of Related Genes Neurod/beta2 Protein Transduction Domain

;Protein transduction domains (PTDs), such as HIV Tat PTD, have been widely used as delivery tools into living cells. NeuroD (neurogenic differentiation 1), or named BETA2 (β -cell E-box transactivator 2), belonging to basic helix-loop-helix family, playcentral roles during the pancreas development.Here we reported for the first time that the helix-loop-helix (HLH) domain of basic helix-loop-helix (bHLH) family was a novel type of PTD, and the intact motif was required for translocation. Efficient internalization had been obtained with HLH domains derived from bHLH protein, NeuroD/Beta2, in various cell types including human liver tumor derived PLC cells, hamster ovary derived CHO cells, human cervix derived HeLa cells, human lung derived LLC cells, and primary rat islets, and the transduction was dose- and time-dependent. Confocal experiment showed that the internalized HLH-EGFP localized at cytoplasm and nuclear. Cellular uptake of HLH PTD was barely or slightly inhibited by the metabolic, phagocytosis, clathrin- or caveolar-endocytosis formation inhibitors, but significantly and substantially reduced by heparin and macropinocytosis inhibitor, which suggested important roles of cell surface glycosaminoglycans and macropinocytosis during the protein transduction. Furthermore, luciferase assay showed that addition of NeuroD-EGFP protein gave 1.8 fold transactivation, significantly higher than that of pCMV-EGFP control (paired-T test, P<0.05, n=3).Our results demonstrated a new motif of PTD different from previous models as cationic residues cluster or amphipathic helix. The HLH domain is also the character of the bHLH family, which implies a large number of PTDs could be discovered in this family to fit diffferent purposes, and some of them could be directly recruited to penetrate cell membrane according to their crucial roles in deveploment such as NeuroD and Ngn3.Type I diabetes, also known as insulin-dependent diabetes mellitus, is the result of insulin deficiency caused by the autoimmune destruction of the insulin-producing pancreatic β-cells. And it have been reported that pancreas duct, fetal liver, ES or MSC cells could differentiate into insulin producing cells by virus-mediated expression of pancreas development related genes, such as Pdx-1, Ngn3,NeuroD in vitro or in vivo. However, potential immunogenisis, virus toxicity and mutant induced ability restricted further application of the virus gene delivery system.Here we explored whether liver progenitor cell, such as HepG2 and liver oval cells, could differentiate into insulin-producing cells by using protein transduction of the pancreatic duodenal homeobox 1 (PDX1) and/or NeuroD proteins. Confocal experiment showed that PDX1-EGFP and NeuroD-EGFP can efficiently get into HepG2 and liver oval cells. RT-PCR test indicated that protein treated liver oval cells activated multiple beta-cell genes, and Real-time PCR test showed that the treated cells produced and stored considerable amounts of insulin. Cytoimmunofluorescence and western-blot experiments demonstrated that endogenetic insulin, PDX-1 and NeuroD could significantly express in protein treated cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes. Furthermore, after the intraperitoneal injection of NeuroD protein, the STZ induced diabetic ICR mice returned to normal glycemia, and it remained for 4 days, repeated injection could cause the similar results again. Body weight and quantity of insulin mRNA in the liver were assayed and proved the improvement of hyper-glycemic complication after protein administration. This approach offers a much more safe, efficiency and easy-operated method for IDDM treatment.