Stimulation of the P-450 side chain cleavage enzyme (CYP11A1) promoter through ras- and Ets-2-signaling pathways.
Expression of the ovine P-450 side-chain cleavage enzyme gene (CYP11A1) is stimulated by epidermal growth factor (EGF) through a pathway that involves c-Jun in JEG-3 placental cells. Growth factor signaling involves ras-dependent and ras-independent signaling pathways, which in turn regulate gene transcription through related but distinct mitogen-activated protein kinase pathways (MAPKs) including the extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs). We investigated the intracellular signaling pathways governing EGF induction of the CYP11A1 promoter. EGF stimulation of the CYP11A1 promoter (4-fold) was reduced 60% by a dominant negative mutant of ras (N17), and 30-40% by antisense ras. EGF induced both ERK and SAPK activity in JEG-3 cells. EGF-induced CYP11A1 promoter activity was reduced 60% by the MEK1 inhibitor PD098059 and 50% by a dominant negative mutant of the ERK-specific regulator MEK1. In contrast, dominant negative mutants of the SAPK-specific activator, SEK1, induced a further increase in EGF-induced CYP11A1 promoter activity. Constitutively active mutants of ras (V12 or L61) increased CYP11A1 promoter activity 6- to 8-fold. Deletion of the EGF response element (EGF-RE) between -92 and -77 bp reduced ras induction by 60%; however, a residual 3-fold induction remained through the proximal -77 bp. Mutation of the EGF-RE AP-1-like sequence in the context of the native promoter reduced CYP11A1 promoter activation by ras 60%. The EGF-RE sequence was sufficient for 6-fold activation by ras in the context of an heterologous thymidine kinase promoter. Candidate transcription factor targets (c-Jun, c-Ets-2) for the ras-signaling cascade were examined for their effects on CYP11A1 promoter activity. Overexpression of c-Jun induced the CYP11A1 promoter through the EGF-RE; however, c-Ets-2 activation of the CYP11A1 promoter (12-fold) required the proximal ras-responsive promoter sequences that are distinct from the EGF/MEK/c-Jun-responsive element. Induction of the CYP11A1 promoter by EGF involves a ras/MEK1/AP-1-dependent pathway that is distinct from induction by ras/c-Ets-2.
Pestell
R G
RG
Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Albanese
C
C
Watanabe
G
G
Lee
R J
RJ
Lastowiecki
P
P
Zon
L
L
Ostrowski
M
M
Jameson
J L
JL
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D002822:Choriocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004268:DNA-Binding Proteins; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation; D016758:Genes, jun; D011905:Genes, ras; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D020928:Mitogen-Activated Protein Kinases; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D017384:Sequence Deletion; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D002786:Cholesterol Side-Chain Cleavage Enzyme; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human