Activation of the 9E3/cCAF chemokine by phorbol esters occurs via multiple signal transduction pathways that converge to MEK1/ERK2 and activate the Elk1 transcription factor.
Using primary fibroblasts in culture, we have investigated the signal transduction mechanisms by which phorbol esters, a class of tumor promoters, activate the 9E3 gene and its chemokine product the chicken chemotactic and angiogenic factor. This gene is highly stimulated by phorbol 12,13-dibutyrate (PDBu) via three pathways: (i) a small contribution through protein kinase C (the commonly recognized pathway for these tumor promoters), (ii) a contribution involving tyrosine kinases, and (iii) a larger contribution via pathways that can be interrupted by dexamethasone. All three of these pathways converge into the mitogen-activated protein kinases, MEK1/ERK2. Using a luciferase reporter system, we show that although both the AP-1 and PDRIIkB (a NFkappaB-like factor in chickens) response elements are capable of activation in these normal cells, regions of the 9E3 promoter containing them are unresponsive to PDBu stimulation. In contrast, we show for the first time that activation by PDBu occurs through a segment of the promoter containing Elk1 response elements; deletion and mutation of these elements abrogates 9E3/chicken chemotactic and angiogenic factor expression. Electrophoretic mobility shift assays and functional studies using PathDetect systems show that stimulation of the cells by phorbol esters leads to activation of the Elk1 transcription factor, which binds to its element in the 9E3 promoter.
Li
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Department of Biology, University of California, Riverside, California 92521, USA.
Vaingankar
S M
SM
Green
H M
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Martins-Green
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M
D000818:Animals; D030161:Avian Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D019743:Chemokines, CXC / Q000235:genetics; D002642:Chick Embryo; D016207:Cytokines / Q000235:genetics*; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000494:pharmacology; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D015221:Potassium Channels / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D050807:ets-Domain Protein Elk-1
D030161:Avian Proteins; D019743:Chemokines, CXC; D016207:Cytokines; D004268:DNA-Binding Proteins; C071280:EMF-1 protein, Gallus gallus; D016328:NF-kappa B; D015221:Potassium Channels; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D015240:Phorbol 12,13-Dibutyrate; D003907:Dexamethasone; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases