Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation.
Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage beta-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet beta-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.
Tang
Dong-Qi
DQ
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0275, USA.
Cao
Li-Zhen
LZ
Chou
Wayne
W
Shun
Lu
L
Farag
Christine
C
Atkinson
Mark A
MA
Li
Shi-Wu
SW
Chang
Lung-Ji
LJ
Yang
Li-Jun
LJ
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers; D004702:Endocrine Glands / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D017405:Herpes Simplex Virus Protein Vmw65 / Q000502:physiology*; D018398:Homeodomain Proteins / Q000502:physiology*; D008099:Liver / Q000166:cytology*; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D051761:Paired Box Transcription Factors / Q000502:physiology*; D051381:Rats; D015534:Trans-Activators / Q000502:physiology*
D017931:DNA Primers; D017405:Herpes Simplex Virus Protein Vmw65; D018398:Homeodomain Proteins; D051761:Paired Box Transcription Factors; C491708:Pax4 protein, rat; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein