Pdx-1 or Pdx-1-VP16 protein transduction induces beta-cell gene expression in liver-stem WB cells - PubMed (original) (raw)
Pdx-1 or Pdx-1-VP16 protein transduction induces beta-cell gene expression in liver-stem WB cells
Juliette Cuvelier Delisle et al. BMC Res Notes. 2009.
Abstract
Background: Pancreatic duodenal homeobox-1 (Pdx-1) or Pdx-1-VP16 gene transfer has been shown to induce in vitro rat liver-stem WB cell conversion into pancreatic endocrine precursor cells. High glucose conditions were necessary for further differentiation into functional insulin-producing cells. Pdx-1 has the ability to permeate different cell types due to an inherent protein transduction domain (PTD). In this study, we evaluated liver-to-pancreas conversion of WB cells following Pdx-1 or Pdx-1-VP16 protein transduction.
Findings: WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. beta-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into beta-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and beta-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein.
Conclusion: These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for beta-cell engineering for diabetes cell therapy.
Figures
Figure 1
PTDPdx-1 and TAT fusion protein synthesis and their ability to transduce HepG2 and WB cells. (a) Schematic structure (top panel) and purity (bottom panel) of fusion proteins. PTDPdx-1 and TAT represent protein transduction domains of the Pdx-1 protein and the HIV TAT protein, respectively. His represents hexahistidine tag used to purify proteins by His-tag affinity chromatography. Purified proteins were run on a SDS-PAGE gel (8%) stained with Coomassie blue. Molecular weights are 28 kDa for eGFP, 31 kDa for PTDPdx-1-eGFP and TAT-eGFP. (b) Observation by confocal microscopy of eGFP fluorescence in HepG2 and WB cells treated for 24 hours with 15 μM PTDPdx-1-eGFP, TAT-eGFP, or eGFP protein lacking PTD. Treated cells were observed by confocal microscopy without being fixed in order to exclude artifactual protein uptake [24]. CM-Dil was used to visualize cytoplasmic membrane (red staining). Scale bars = 50 μm.
Figure 2
Synthesis and biological activity of Pdx-1, TAT-Pdx-1, Pdx-1-VP16 and TAT-Pdx-1-VP16 fusion proteins. (a) Schematic structure (top panel) and purity (bottom panel) of Pdx-1, TAT-Pdx-1, Pdx-1-VP16 and TAT-Pdx-1-VP16 fusion proteins. PTDPdx-1 represents protein transduction domain of the Pdx-1 protein, which is naturally present in the four Pdx-1(-VP16) fusion proteins. TAT represents protein transduction domain of the VIH TAT protein. VP16 is the activation domain of Herpes simplex virus I. His represents hexahistidine tag used to purify proteins by His-tag affinity chromatography. Purified proteins were run on a SDS-PAGE gel (8%) stained with Coomassie blue. Molecular weights are 45 kDa for Pdx-1 and TAT-Pdx-1, 60 kDa for Pdx-1-VP16 and TAT-Pdx-1-VP16. (b) Insulin promoter activity after Pdx-1(-VP16) protein transduction. Twelve hours after transient transfection of insulin promoter luciferase plasmid, HepG2 cells were treated with 5 μM Pdx-1(-VP16) fusion proteins for 36 hours. Then luciferase activity was measured. Data are expressed as mean + SEM (Control n = 14, Pdx-1 n = 13, TAT-Pdx-1 n = 7, Pdx-1-VP16 n = 12, TAT-Pdx-1-VP16 n = 19). Insulin promoter activity of controls was arbitrarily set at 1. Statistical comparisons were performed using a non parametric Mann-Withney test, *: p < 0.05, ***: p < 0.001.
Figure 3
Gene expression profile of WB-cells treated with Pdx-1(-VP16) or TAT-Pdx-1(-VP16) proteins in high glucose culture. RT-PCR analysis was performed after two weeks of protein treatment. cDNA from rat pancreas served as control for pancreatic gene expression, and cDNA from rat liver served as positive control for albumin expression (*). Each sample is represented by a single column and corresponds to an independent biological repeat of the experiment (LG (n = 5), HG (n = 5), Pdx-1 (n = 4), TAT-Pdx-1 (n = 4), Pdx-1-VP16 (n = 5), TAT-Pdx-1-VP16 (n = 5)). Each gene is represented by a single row of coloured boxes. Black colouring represents RT-PCR positive samples, whereas white colouring represents negative samples. LG: Low Glucose control WB cells; HG: High Glucose control WB cells. (See additional data 3 for original electrophoresis gel images).
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