Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor.
The mouse heme oxygenase-1 (HO-1) gene, ho-1, contains two inducible enhancers, E1 and E2. Of several cell lines tested, induction of an E1/luciferase fusion construct, pE1-luc, by CdCl(2) is most pronounced in MCF-7 cells. In these cells, E1, but not E2, is necessary and sufficient for ho-1 gene activation. Exposure of MCF-7 cells to 10 micrometer CdCl(2) stimulates phosphorylation of ERK, JNK, and p38 mitogen-activated protein kinases, implicating one or more of these signaling pathways in ho-1 gene induction. SB203580, an inhibitor of p38, diminishes cadmium-stimulated pE1-luc expression and HO-1 mRNA levels by up to 70-80%. PD098059, an ERK pathway inhibitor, does not affect HO-1 mRNA induction at the highest concentration (40 micrometer) tested. Similarly, co-expression of a dominant-negative mutant of p38alpha, but not of ERK1, ERK2, JNK1, or JNK2, reduces basal and cadmium-induced pE1-luc activity. E1 contains binding sites for the activator protein-1 (Fos/Jun), Cap'n'Collar/basic leucine zipper (CNC-bZIP), and CCAAT/enhancer-binding protein (C/EBP) families of transcription factors. A dominant-negative mutant of Nrf2 (a CNC-bZIP member), but not of c-Jun or C/EBPbeta, inhibits pE1-luc activation by cadmium. Induction of the endogenous ho-1 gene is also inhibited by the Nrf2 mutant. Mutations of E1 that inhibit cadmium inducibility also suppress the trans-activation and DNA binding activities of Nrf2, and SB203580, but not PD098059, attenuates Nrf2-mediated trans-activation of pE1-luc. Taken together, these results indicate that cadmium induces ho-1 gene expression via sequential activation of the p38 kinase pathway and Nrf2.
Alam
J
J
Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, Louisiana 70121, USA. jalam@ochsner.org
Wicks
C
C
Stewart
D
D
Gong
P
P
Touchard
C
C
Otterbein
S
S
Choi
A M
AM
Burow
M E
ME
Tou
J
J
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms; D002104:Cadmium / Q000494:pharmacology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D006801:Humans; D007739:L Cells; D008565:Membrane Proteins; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D048056:Mitogen-Activated Protein Kinase 9; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D048051:p38 Mitogen-Activated Protein Kinases
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D002104:Cadmium; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; D048056:Mitogen-Activated Protein Kinase 9; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one