Transcriptional regulation of 5-aminolevulinate synthase by phenobarbital and cAMP-dependent protein kinase.
5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital 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-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.
Varone
C L
CL
Departamento de Química Biológica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Giono
L E
LE
Ochoa
A
A
Zakin
M M
MM
Cánepa
E T
ET
D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000818:Animals; D020134:Catalytic Domain; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004357:Drug Synergism; D004790:Enzyme Induction / Q000187:drug effects*; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology; D009154:Mutation / Q000235:genetics; D010634:Phenobarbital / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D007527:Isoenzymes; D012333:RNA, Messenger; D005576:Colforsin; D006427:Hemin; D000242:Cyclic AMP; D000624:5-Aminolevulinate Synthetase; D017868:Cyclic AMP-Dependent Protein Kinases; D010634:Phenobarbital