A prospero-related homeodomain protein is a novel co-regulator of hepatocyte nuclear factor 4α that regulates the cholesterol 7α-hydroxylase gene

Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7 alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4 alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4 alpha. Prox1 strongly inhibited HNF4 alpha and peroxisome proliferators-activated receptor gamma coactivator-1 alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4 alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.