| The human keratinocyte line SCC-4 is a model system in which to explore the underlying mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TODD) interferes with the action of the retinoid signaling pathway. Retinoid induction of tissue transglutaminase (TGM2) mRNA was suppressed 60–70% by 10 nM TCDD without affecting the stability of the message and thus appears to result from altered transcription. The possibility that retinoids added to the culture medium were depleted due to metabolic inactivation by P450 enzymes was shown to be unlikely. Stable transfection of a 5 kb promoter sequence flanking the tissue transglutaminase gene demonstrated 2 to 4-fold reporter activity from retinoic acid treatment. However, the cis-acting sequences that may mediate the TCDD suppression were found to lie outside this region and are therefore separate from the site that confers retinoid responsiveness. Microarray screening located additional retinoid-responsive genes that were not sensitve to TCDD indicating TGM2 is uniquely affected in SCC-4 cells. Stimulation of the RARα signaling pathway induced TGM2 as well as two other genes identified from microarray analysis but only TGM2 was sensitive to TCDD suppression. Therefore, TCDD suppression does not occur by targeting a specific retinoid pathway or RAR isoform. It also appears unlikely that the suppressive action of TCDD occurs by direct interference with retinoid action or at retinoid response elements. In conclusion, these findings and the observation that TGM2 expression requires 48 hours of retinoid treatment suggests that maximal activation of the TGM2 promoter is indirect and mediated by a gene product directly induced by retinoids. |
