Naturally occurring mutations in the human HNF4alpha gene impair the function of the transcription factor to a varying degree

The hepatocyte nuclear factor (HNF)4alpha, a member of the nuclear receptor superfamily, regulates genes that play a critical role in embryogenesis and metabolism. Recent studies have shown that mutations in the human HNF4alpha gene cause a rare form of type 2 diabetes, maturity onset diabetes of the young (MODY1). To investigate the properties of these naturally occurring HNF4alpha mutations we analysed five MODY1 mutations (R154X, R127W, V255M, Q268X and E276Q) and one other mutation (D69A), which we found in HepG2 hepatoma cells. Activation of reporter genes in transfection assays and DNA binding studies showed that the MODY1-associated mutations result in a variable reduction in function, whereas the D69A mutation showed an increased activity on some promoters. None of the MODY mutants acted in a dominant negative manner, thus excluding inactivation of the wild-type factor as a critical event in MODY development. A MODY3-associated mutation in the HNF1alpha gene, a well-known target gene of HNF4alpha, results in a dramatic loss of the HNF4 binding site in the promoter, indicating that mutations in the HNF4alpha gene might cause MODY through impaired HNF1alpha gene function. Based on these data we propose a two-hit model for MODY development.     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.     

Functional properties of the R154X HNF-4alpha protein generated by a mutation associated with maturity-onset diabetes of the young, type 1

Mutations in the hepatocyte nuclear factor 4alpha (HNF-4alpha) gene are associated with one form of maturity-onset diabetes of the young (MODY1). The R154X mutation generates a protein lacking the E-domain which is required for normal HNF-4alpha functions. Since pancreatic beta-cell dysfunction is a feature of MODY1 patients, we compared the functional properties of the R154X mutant in insulin-secreting pancreatic beta-cells and non-beta-cells. The R154X mutation did not affect nuclear localisation in beta-cells and non-beta-cells. However, it did lead to a greater impairment of HNF-4a function in beta-cells compared to non-beta-cells, including a complete loss of transactivation activity and a dominant-negative behaviour. .     

Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4alpha (HNF4alpha) gene

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4alpha1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4alpha gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4alpha protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4alpha P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4alpha P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6alpha. p53 bound HNF6alpha in vivo and in vitro and prevented HNF6alpha from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6alpha in vivo. However, since the R249S p53 mutant also bound HNF6alpha, binding HNF6alpha is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4alpha expression in response to cellular stress are discussed.     

HNF-4alpha: from MODY to late-onset type 2 diabetes

Maturity-onset diabetes of the young (MODY) is a rare subtype of type 2 diabetes that is characterized by autosomal-dominant inheritance and can be caused by mutations in hepatocyte nuclear factor 4alpha (HNF-4alpha). Odom and colleagues have combined chromatin immunoprecipitation with promoter microarrays to identify numerous promoters occupied by HNF-4alpha in the human liver and islet, suggesting a very broad role for HNF-4alpha in glucose homeostasis. This notion is supported by recent genetic studies linking HNF-4alpha to the much more common late-onset type 2 diabetes.     

Hepatocyte nuclear factor-4alpha is a central transactivator of the mouse Ntcp gene

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1alpha and retinoid X receptor-alpha:retinoic acid receptor-alpha binding sites in the mouse 5'-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4alpha, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse (mNtcp). A 1.1-kb 5'-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1alpha, HNF-4alpha cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4alpha consensus site at -345/-326 and -335/-316 bp, respectively. p-475bpmNtcpLUC is not transactivated by HNF-1alpha but shows a 50-fold enhanced activity upon cotransfection with HNF-4alpha. Gel mobility shift assays demonstrated a complex of the HNF-4alpha-element formed with liver nuclear extracts that was blocked by an HNF-4alpha specific antibody. HNF-4alpha binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4alpha-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4alpha via a conserved distal cis-element independently of HNF-1alpha.