Transcriptional regulation of 5-aminolevulinate synthase by phenobarbital and cAMP-dependent protein kinase

5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that ca talyzes the first and rate-limiting step of the heme biosynthesis. There ar e two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encod es a housekeeping isozyme that is apparently expressed in all tissues. In t his report we examine the mechanisms by which phenobarbital and cAMP regula te housekeeping ALA-S expression. We have determined that cAMP and phenobar bital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-depe ndent protein kinase (PKA) has been examined. A synergism effect on ALA-S m RNA induction is observed in rat hepatocytes treated with pairs of selectiv e analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-fla nking region is able to provide cAMP and phenobarbital stimulation to chlor amphenicol O-acetyltranferase fusion vectors in transiently transfected Hep G2 cells. ALA-S promoter activity is induced by cotransfection with an expr ession vector containing the catalytic subunit of PKA. Furthermore, cotrans fection with a dominant negative mutant of the PKA regulatory subunit impai rs the cAMP analog-mediated increase, but the phenobarbital-mediated induct ion is not modified. Our data suggest that the transcription factor cAMP-re sponse element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the bas al and phenobarbital or cAMP analog-mediated induction of ALA-S promoter ac tivity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the tran scriptional level, while heme represses it. (C) 1999 Academic Press.