Heregulin and forskolin-induced cyclin D3 expression in Schwann cells: role of a CCAAT promoter element and CCAAT enhancer binding protein.

Heregulin, a polypeptide growth factor, and forskolin, an adenylyl cyclase activator, synergistically stimulate expression of cyclin D3 and cell division in Schwann cells. Heregulin induces expression in Schwann cells of a luciferase reporter gene linked to the cyclin D3 promoter. Forskolin markedly augments reporter expression in the presence of heregulin. Deletion analysis identified several promoter sites that contribute to high-level reporter expression in heregulin- and forskolin-treated Schwann cells. A promoter fragment that contains 103 bp of 5'-flanking sequence produced significant reporter expression in heregulin- and forskolin-stimulated cells. Deletion of a consensus CCAAT site within this promoter fragment caused a nearly complete loss of reporter expression. Similar results were obtained when CCAAT site mutations were introduced into the promoter. Heregulin and forskolin increased steady-state levels of CCAAT/enhancer binding protein-beta (C/EBPbeta) in Schwann cells. Mobility shift assays identified proteins in Schwann cell nuclear extracts that formed stable complexes with the cyclin D3 CCAAT promoter element and were disrupted by anti-C/EBPbeta antibody. Transfection of Schwann cells with C/EBPbeta cDNA increased cyclin D3 reporter expression. In contrast to these results, mutation of a cAMP response element in the cyclin D3 promoter had only a modest effect on heregulin- and forskolin-stimulated reporter expression. These findings demonstrate that C/EBPbeta plays a key role in the heregulin and cAMP-dependent regulation of cyclin D3 expression in Schwann cells.