Involvement of serum response element in okadaic acid-induced EGR-1 transcription in human T-cells.
The present work has examined the effects of okadaic acid, an inhibitor of serine/threonine protein phosphatase, PP-1 and PP-2A, on the regulation of EGR-1 gene expression in normal peripheral blood T- and Jurkat cells. The results demonstrate that okadaic acid treatment is associated with a transient induction of EGR-1 gene expression which was detectable by 30 min to 1 h and peaked at 3-6 h. EGR-1 mRNA was superinduced in cells treated with both okadaic acid and the protein synthesis inhibitor cycloheximide. The half-life of EGR-1 mRNA was similar in both control and okadaic acid-treated cells. In contrast, treatment with both okadaic acid and cycloheximide prolonged the half-life of EGR-1 transcripts. Nuclear run-on assays demonstrated that induction of EGR-1 gene expression by okadaic acid is controlled at least in part by a transcriptional mechanism. Transient expression assays with EGR-1 promotor fragments linked to the chloramphenicol acetyltransferase gene demonstrate that okadaic acid-induced EGR-1 transcription is conferred by the 5' most distal CArG box, CC (AT)6GG, in the EGR-1 promoter. Moreover, chloramphenicol acetyltransferase activity was induced by okadaic acid when the 5' most distal CArG element was linked to the heterologous herpes simplex virus thymidine kinase promoter, and not induced with a similar heterologous construct containing a mutated CArG sequence. These studies demonstrate that okadaic acid regulates EGR-1 gene expression at the transcriptional level via the CArG element and suggest that PP-1 and PP-2A play a role in T-cell activation.
Chauhan
D
D
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Kharbanda
S M
SM
Uchiyama
H
H
Sukhatme
V P
VP
Kufe
D W
DW
Anderson
K C
KC
D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis*; D051766:Early Growth Response Protein 1; D004988:Ethers, Cyclic / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000032:analysis / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D004988:Ethers, Cyclic; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D019319:Okadaic Acid; D003513:Cycloheximide; D015500:Chloramphenicol O-Acetyltransferase; D017027:Protein Tyrosine Phosphatases