SYNERGISTIC INDUCTION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR GENE-EXPRESSION BY GLUCOCORTICOIDS AND CYCLIC-NUCLEOTIDES IN RAT HTC HEPATOMA-CELLS

We have reported previously that tissue-type plasminogen activator (tP A) gene expression is regulated by glucocorticoids and cyclic nucleoti des in HTC rat hepatoma cells. Incubation of HTC cells with the synthe tic glucocorticoid dexamethasone (Dex) transiently increases tPA messe nger RNA accumulation 2-fold, whereas incubation with 8-bromo-cAMP (cA MP) alone results in a sustained P-fold increase. Nuclear run-on studi es indicate that these effects occur at the level of gene transcriptio n. In combination, however, Dex and cAMP act synergistically to induce tPA messenger RNA levels 10- to 15-fold; this synergistic induction i s at least in part transcriptional. We now report that this synergisti c induction of tPA gene transcription requires concomitant protein syn thesis. Furthermore, the action of Dex must precede that of cAMP, and the action of Dex requires ongoing protein synthesis, whereas the acti on of cAMP has no such requirement. To further investigate the mechani sm of the synergistic induction of tPA gene transcription, we cloned t he tPA promoter from an HTC genomic library. We established the start site of transcription in HTC cells by primer extension and determined the nucleotide sequence of 2.3 kilobasepairs (kb) of the 5'-flanking r egion, including 1.7 kb of sequence not previously reported. A 2.3-kb segment of the rat tPA promoter has been ligated to a chloramphenicol acetyltransferase reporter gene and its hormonal regulation evaluated in transient and stable transfection studies in HTC cells. Although th is promoter length is sufficient to mediate the P-fold induction in ge ne expression seen with cAMP alone, it is not sufficient to recapitula te the synergistic induction of endogenous tPA gene transcription seen with Dex plus cAMP in combination. We have ruled out relief of transc riptional arrest as the mechanism of the synergistic induction. Theref ore, we suggest that sequences lying outside the most proximal 2.3 kb of tPA promoter mediate the synergistic interaction of Dex and cAMP.