The role of HNF1 alpha, HNF3 gamma, and cyclic AMP in glucose-6-phosphatase gene activation

The gene for glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis, is expressed in a tissue-specific manner in the liver and kidney. To understand the molecular mechanisms regulating liver-specific expression of the G6Pase gene, we characterized G6Pase promoter activity by transient expression assays. The G6Pase promoter is active in HepG2 hepatoma cells? but inactive in JEG3 choriocarcinoma or 3T3 cells. DNA elements essential for optimal and liver-specific expression of the G6Pase gene were contained within nucleotides -234 to +3. Deletion analysis revealed that the G6Pase promoter contained three activation elements (AEs) at nucleotides -234 to -212 (AE-I), -146 to -125 (AE-II), and -124 to -71 (AE-III). AE-I contains binding sites for hepatocyte nuclear factors (HNF) 1 and it. Electromobility shift and cotransfection assays demonstrated that HNF1 alpha, but not HNF4, bound to its cognate site and transactivated G6Pase gene expression. The G6Pase promoter contained five HNF3 motifs, 1 (-180/-174), 2 (-139/-133), 3 (-91/-85), 3 (-81/-75), and 5 (-72/-66), and all five sites bound HNF3 gamma with high affinity. Transient expression and cotransfection assays showed that HNF3 site I is not required for basal promoter activity but is essential for HNF3 gamma-activated transcription from the G6Pase promoter. We further showed that HNF3 sites 3! 3, and 5 were essential for basal G6Pase promoter activity and transactivation by HNF3 gamma. AE-H contains, in addition to a HNF3 motif, a cAMP-response element (CRE) and a C/EBP half-site. The G6Pase(-146/-116) DNA containing AE-II formed multiple protein-DNA complexes with HepG2 nuclear extracts, including HNF3 gamma. CRE-binding protein (CREB), C/EBP alpha, and C/EBP beta. We showed that AE-LI mediated transcription activation of the G6Pase gene by cAMP.