Transcriptional Induction of Collagenase-1 in Differentiated Monocyte-like (U937) Cells is Regulated by AP-1 and an Upstream C/EBP-β Site

Abstract

In this report, we demonstrate that the AP-1 site and a distal promoter element regulate transcriptional induction of collagenase-1 during monocytic differentiation. Chloramphenicol acetyltransferase expression constructs containing regions of the human collagenase-1 promoter were stably or transiently transfected into U937 cells, and reporter activity was assessed at various times after the onset of phorbol 12-myristate 13-acetate (PMA)-mediated differentiation. Rapid and strong induction of promoter activity was lost in constructs with a mutant AP-1 element; however, at 16-96 h post-PMA, the mutant collagenase-1 promoter displayed AP-1 independent PMA-mediated transactivation. The AP-1 mutant constructs also showed delayed transcriptional activation in PMA-treated fibroblasts. Western and supershift analyses indicated that functional Jun and Fos proteins were present in nuclear extracts of PMA-differentiated U937 cells. Promoter deletion constructs demonstrated the potential role of distal promoter sequences in regulating collagenase-1 transcription. In particular, Western, supershift, and promoter deletion analyses suggested a role for CCAAT/enhancer-binding protein-beta (C/EBP-beta) binding site between -2010 and -1954 in regulating transcription of collagenase-1 in monocytic cells. Our findings suggest that distinct regulatory elements, acting somewhat independently of each other, control expression of collagenase-1. In addition, our data suggests that the rapid PMA-mediated induction of collagenase-1 transcription is controlled by a mechanism distinct from that regulating the sustained expression of this proteinase in activated macrophages.