Expression of progenitor cell markers during expansion of sorted human pancreatic beta cells.

Functional pancreatic beta cell mass is dynamic and although fully differentiated, beta cells are capable of reentering the cell cycle upon appropriate stimuli. Stimulating regeneration-competent cells in situ is clearly the most desirable way to restore damaged tissue. Regeneration by dedifferentiation and transdifferentiation is a potential source of cells exhibiting a more developmentally immature phenotype and a wide differentiation potential. In this context and to gain a better understanding of the transformation induced in human beta cells during forced in vitro expansion, we focused on identifying differences in gene expression along with phenotypical transformation between proliferating and quiescent human beta cells. FACS-purified beta cells from three different human pancreata were cultured during 3-4 months (8-10 subcultures) on HTB-9 cell matrix with hepatocyte growth factor. Gene expression profiling was performed on cells from each subculture on "in-house" pancreas-specific microarrays consisting of 218 genes and concomitant morphological transformations were studied by immunocytochemistry. Immunocytochemical studies indicated a shift from epithelial to neuroepithelial cell phenotype, including progenitor cell features such as protein gene product 9.5 (PGP 9.5), Reg, vimentin, and neurogenin 3 protein expression. The expression of 49 genes was downregulated, including several markers of endocrine differentiation while 76 were induced by cell expansion including several markers of progenitor cells. Their pattern also argues for the transdifferentiation of beta cells into progenitor cells, demonstrating neuroepithelial features and overexpressing both PBX1, a homeodomain protein that can bind as a heterodimer with PDX1 and could switch the nature of its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our study of the machinery that regulates human beta cell expansion and dedifferentiation may help elucidate some of the critical genes that control the formation of adult pancreatic progenitor cells and hence design targets to modify their expression in view of the production of insulin-secreting cells.

The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4alpha activities and introduces a PKA phosphorylation site in its DNA-binding domain.

HNF4alpha (hepatocyte nuclear factor 4alpha) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic beta-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4alpha gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115-->Ser) HNF4alpha mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4alpha-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic beta-cell lines, this mutation resulted in strong impairments of HNF4alpha transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1alpha, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115-->Glu) mutation mimicking phosphorylation reduced HNF4alpha DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4alpha function.

Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4alpha dimerisation and transcriptional activity.

Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.

Mutations in hepatocyte nuclear factor 4alpha (HNF4alpha) gene associated with diabetes result in greater loss of HNF4alpha function in pancreatic beta-cells than in nonpancreatic beta-cells and in reduced activation of the apolipoprotein CIII promoter in hepatic cells.

Mutations in the HNF4alpha gene have been correlated with maturity-onset diabetes of the young, which is characterized mainly by pancreatic beta-cell dysfunction and is also associated with mild liver abnormalities. HNF4alpha D126Y and D126H mutations were found in a patient with early-onset type 2 diabetes, and the R324H mutation was found in a common type 2 diabetic nephropathic patient. We investigated whether these mutations, which have not yet been functionally characterized, impair HNF4alpha function in three cell models: HEK 293 embryonal kidney cells, HepG2 hepatoma cells, and betaTC3 pancreatic beta-cells. The R324H mutation had no effect on HNF4alpha function with either the HNF1alpha and L-type pyruvate kinase (LPK) promoters, but the D126Y and D126H mutations impaired HNF4alpha transcriptional activities in all tested cell lines. These impairments by D126Y and D126H mutations, which are located in the T box, are not due to a loss of dimerization but to a loss of DNA binding. Interestingly, the strongest functional consequences of these mutations were observed on the HNF1alpha promoter in betaTC3 cells. Given the key role of the transcription factor HNF1alpha in pancreatic beta-cell function, it can be inferred that impairment of HNF4alpha function by these mutations affects metabolic pathways in pancreatic beta-cells and contributes to development of diabetes. Moreover, the HNF4alpha-mediated activation of the apolipoprotein CIII promoter in HepG2 cells was significantly impaired by D126Y and D126H mutations. These results support clinical findings that liver function can also be impaired in diabetic patients having HNF4alpha mutations.