Abstract
Hepatocyte nuclear factor (HNF)-1beta, a homeodomain-containing transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues. Recent genetic studies have identified two HNF-1beta mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes. Despite the defects being in the same gene, diverse severities of disease are observed in the affected subjects. To investigate the molecular mechanism by which mutations might cause various phenotypic features, wild type and mutant proteins were transiently expressed in insulin-producing (MIN6) and hepatic (HepG2) cells. Luciferase reporter assay showed that both mutations resulted in a marked reduction of transactivation activity. Because their dimerization activity was found to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with wild type, both of the mutants significantly decreased wild type activity in HepG2 cells. In contrast, although A263fsinsGG functioned similarly in MIN6 cells, R177X failed to affect wild type activity in this cell line. Immunohistochemical analysis of the mutants suggests that this functional divergence might be generated by the modification of nuclear localization. These results suggest that HNF-1beta mutations may impair pancreatic beta-cell function by loss-of-function and dominant-negative mechanisms.
Highlights
Hepatocyte nuclear factor (HNF)-1, a homeodomaincontaining transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues
Transactivation Activity of Wild Type and Mutant hepatocyte nuclear factor (HNF)-1 Proteins—To investigate the molecular mechanism by which HNF-1 mutations cause impaired glucose tolerance, transactivation activities of R177X and A263fsinsGG were analyzed in liver cell and pancreatic -cell lines by luciferase reporter assay using the promoter of the human gene for GLUT2, which mediates facilitative glucose transport in these tissues
The deleted reporter construct still represented a significant increase of activity mediated by wild type HNF-1, suggesting that GLUT2 gene expression could be indirectly regulated by other transcription factors whose expression is up-regulated by HNF-1
Summary
Hepatocyte nuclear factor (HNF)-1, a homeodomaincontaining transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues. Recent genetic studies have identified two HNF-1 mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes. HNF-1␣ forms a homodimer or heterodimer with structurally related HNF-1 (9 –11), and they function together to regulate gene expression in liver, pancreas, and some other tissues In this context, the HNF-1 gene was screened for mutations in subjects with early onset type 2 diabetes/MODY, and two mutations of R177X and A263fsinsGG were found in two Japanese families [12, 13]. The molecular mechanisms by which mutations in the HNF-1 gene might cause diabetes and generate the diverse phenotypic features were addressed by functional analysis of the mutant proteins
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