The negative regulation of the rat aldehyde dehydrogenase 3 gene by glucocorticoids: involvement of a single imperfect palindromic glucocorticoid responsive element.

Glucocorticoids repressed the polycyclic aromatic hydrocarbon-dependent induction of Class 3 aldehyde dehydrogenase (ALDH3) enzyme activity and mRNA levels in isolated rat hepatocytes by more than 50 to 80%, with a concentration-dependence consistent with the involvement of the glucocorticoid receptor (GR). No consistent effect on the low basal transcription rate was observed. This effect of glucocorticoids (GC) on polycyclic aromatic hydrocarbon induction was effectively antagonized at the mRNA and protein level by the GR antagonist RU38486. The response was cycloheximide-sensitive, because the protein synthesis inhibitor caused a GC-dependent superinduction of ALDH3 mRNA levels. This suggests that the effects of GC on this gene are complex and both positive and negative gene regulation is possible. The GC-response was recapitulated in HepG2 cells using transient transfection experiments with CAT reporter constructs containing 3.5 kb of 5'-flanking region from ALDH3. This ligand-dependent response was also observed when a chimeric GR (GR DNA-binding domain and peroxisome proliferator-activated receptor ligand-binding domain) was used in place of GR in the presence of the peroxisome proliferator, nafenopin. A putative palindromic glucocorticoid-responsive element exists between -930 and -910 base pairs relative to the transcription start site. If this element was either deleted or mutated, the negative GC-response was completely lost, which suggests that this sequence is responsible, in part, for the negative regulation of the gene. Electrophoretic mobility shift analysis demonstrated that this palindromic glucocorticoid-responsive element is capable of forming a specific DNA-protein complex with human glucocorticoid receptor. In conclusion, the negative regulation of ALDH3 in rat liver is probably mediated through direct GR binding to its canonical responsive element.