Functional characterization of novel IL-2 transcriptional inhibitors.
IL-2-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited beta-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription.
Baine
Y
Y
Department of Immunology, Sanofi Winthrop Pharmaceuticals Research Division, Collegeville, PA 19426, USA.
Stankunas
B M
BM
Miller
P
P
Hobbs
C
C
Tiberio
L
L
Koch
J
J
Yoon
K
K
Sawutz
D
D
Surowy
C
C
D001483:Base Sequence; D001577:Benzophenones / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000235:genetics*; D007959:Lymphocyte Culture Test, Mixed; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011720:Pyrazoles / Q000494:pharmacology*; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects
D001577:Benzophenones; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007376:Interleukin-2; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011720:Pyrazoles; D011799:Quinazolines; D014157:Transcription Factors; C092959:WIN 53071; C092960:WIN 61058; D002118:Calcium