cAMP activates the IL-5 promoter synergistically with phorbol ester through the signaling pathway involving protein kinase A in mouse thymoma line EL-4.
Expression of the IL-5 gene in T cells is induced in response to Ag stimulation; however, functional analysis of the IL-5 gene has been limited by lack of an appropriate transfection assay to facilitate measurement of the IL-5 promoter activity in response to T cell activation signals. Here, we describe a transient transfection system with which the IL-5 promoter activity can be assayed quantitatively. Using mouse thymoma line EL-4 cells, which produce several lymphokines including IL-2, IL-3, IL-4, IL-10, and GM-CSF in response to PMA, the effect of cAMP on IL-5 production was examined. These cells produce a low level of IL-5 when stimulated with PMA alone; however, N6, O2-dibutyryl cAMP (Bt2cAMP), in combination with PMA, augmented by more than tenfold the IL-5 production at the mRNA and the protein levels. Likewise, a transient transfection assay revealed that Bt2cAMP activated the IL-5 promoter more than tenfold, in a PMA-dependent manner, thereby indicating that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. Activation of the IL-5 promoter in response to Bt2cAMP and PMA depends on the region spanning from nucleotide position -1,200 to +33 relative to the transcription initiation site. Action of cAMP on the IL-5 promoter is mimicked by cotransfection of the expression plasmid containing cDNA encoding the catalytic subunit of protein kinase A, hence, cAMP probably exerts its action through the signaling pathway that involves protein kinase A. In contrast, Bt2cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene in EL-4 cells is positively regulated by cAMP in a manner opposite that for the IL-2 gene.
Lee
H J
HJ
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Koyano-Nakagawa
N
N
Naito
Y
Y
Nishida
J
J
Arai
N
N
Arai
K
K
Yokota
T
T
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004357:Drug Synergism; D005786:Gene Expression Regulation / Q000187:drug effects*; D007376:Interleukin-2 / Q000096:biosynthesis; D015848:Interleukin-5 / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013945:Thymoma / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D007376:Interleukin-2; D015848:Interleukin-5; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate