A nuclear receptor,hepatocyte nuclear factor 4, differently contributes to the human and mouse angiotensinogen promoter activities

Angiotensinogen (AGT), mainly produced in the liver, is the precursor of angiotensin II, an important regulator of blood pressure and electrolyte homeostasis. We previously showed, in hepatoma-derived HepG2 cells that a hepatocyte nuclear factor 4 (HNF4) potentiated human AGT (hAGT) promoter activity and identified its binding sites (termed regions C and J) in the hAGT promoter region. We also showed in transgenic mouse (TgM) that the hAGT is abundantly expressed in the kidney where the level of endogenous mouse AGT (mAGT) expression is low. To elucidate molecular mechanisms of the AGT gene activation in the kidney, we first investigated the HNF4 and AGT expression in the mouse kidney. Northern blot, in situ hybridization and immunohistochemical analyses revealed that the hAGT and HNF4 were both expressed in the proximal tubular (PT) cells of the kidney. We then transfected the hAGT reporter constructs into immortalized mouse PT (mProx) cells and found that regions C and J contributed additively to the HNF4-potentiated hAGT promoter activity. Curiously, no obvious HNF4 binding motif was found in the corresponding region of the mAGT promoter and co-transfected HNF4 failed to activate this promoter in neither HepG2 nor mProx cells. These results suggest that the high-level hAGT expression in the TgM kidney is, at least in part, due to a presence of high-affinity HNF4 binding sites in its promoter.     

The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.     

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.