Cyclic AMP-mediated augmentation of thrombomodulin gene expression: cell type-dependent usage of control regions

Abstract

We reported that a cell surface thrombin receptor, thrombomodulin (TM), was regulated by cyclic AMP in fibroblasts and in parietal endoderm-like cells derived from F9 embryonal carcinoma cells. In this paper, the genetic basis for augmentation of TM expression by cyclic AMP was studied in F9 and BALB/3T3 cells. Transient expression assays were performed with plasmid constructs containing various 5' flanking sequences of the TM gene and a reporter gene, chloramphenicol acetyltransferase (CAT). Two regulatory DNA regions, the proximal (-411 to -50) and the distal (-1026 to -850), were located. Interplay of the two regions was suggested using a heterologous thymidine kinase promoter in differentiated F9 cells. Both proximal and distal regions contributed to cyclic AMP-dependent augmentation of CAT expression in differentiated F9 cells, whereas only the proximal region was functional in BALB/3T3 cells. The two cell types responded differently also to a protein synthesis inhibitor, cycloheximide, with respect to TM message accumulation. In BALB/3T3 cells TM message accumulation was refractory to the inhibitor in contrast to that of differentiated F9 cells, which was only partially so. We propose that there are at least two separate genomic DNA regions that regulate cyclic AMP-dependent TM gene expression and that their functions are cell type dependent.