2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN MODULATES EXPRESSION OF THE PROSTAGLANDIN G H SYNTHASE-2 GENE IN RAT THYMOCYTES/

As an approach to understanding the molecular mechanism(s) of thymic g ene expression mediated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we investigated the effect of TCDD on expression of prostaglandin G/H synthase-2 (PGHS-2) in rat thymocytes by reverse transcription-polyme rase chain reaction. Incubation of thymocytes with increasing doses of TCDD resulted in inhibition of PGHS-2 gene expression in a concentrat ion-dependent manner, with an IC50 of 10 nM. In contrast, TCDD had no appreciable effect on expression of glyceraldehyde phosphate dehydroge nase. Because the xenobiotic-responsive element is conserved in the PG HS-2 promoter from several animal species, it seems likely that inhibi tion of PGHS-2 expression by TCDD may occur at the level of transcript ion. To test this hypothesis in cultured thymocytes, we characterized the Ah receptor in the thymoma cell line WEHI 7.1. Reverse transcripti on-polymerase chain reaction experiments indicated that TCDD inhibited PGHS-2 expression in this cell line. Sucrose density gradient centrif ugation experiments indicated that WEHI 7.1 cytosol exhibited 9 to 10S ligand-binding activity characteristic of the Ah receptor. The viabil ity of WEHI 7.1 cells incubated with TCDD was comparable to that of co ntrol cells, whereas dexamethasone induced toxicity in a concentration -dependent manner. Transient transfection experiments using PGHS-2 pro moter fragments ligated into a chloramphenicol acetyltransferase repor ter plasmid suggested that TCDD inhibits PGHS-2 transcription, and del etion of the xenobiotic-responsive element failed to exhibit this repr ession. These results demonstrate that TCDD is a potent inhibitor of P GHS-2 gene expression, and they represent the first mechanistic eviden ce for TCDD-dependent inhibition of transcription in thymocytes.