Departments of Biochemistry and Medicine, Dartmouth Medical School, Hanover, New Hampshire 03756 Received November 15, 1990; Revised Manuscript Received February 5, 1991 abstract: Collagenase, the only enzyme active at neutral pH that initiatescollagen degradation, is a major gene product of fibroblasts that have been stimulated with a variety of agents, including phorbol esters. To study mechanisms controlling collagenase gene expression, we transiently transfected rabbit synovial fibroblasts with chimeric constructs containing up to 1.2 kb of the rabbit collagenase 5'-flanking DNA linked to the chloramphenicol acetyltransferase gene (CAT). Our data indicate that the magnitude of the phorbol response is directly linkedto the size of the promoter fragment and that the smallest piece of promoter DNA conferring phorbol inducibility is 127 bp. Deletional and mutational analysis of this fragment revealed that the AP-1 sequence alone is insufficient for phorbol inducibility and the presence of at least two additional sequences (a PEA3-like element and a sequence that includes 5'-TTCA-3') is required. In addition, a substantial increase in responsiveness is seen when a fragment containing 182 bp of 5'-flanking DNA is transfected, implicating a 36 bpregion located between-182 and-149 as an enhancer. We conclude (1) that the AP-1 sequence is necessary but insufficient for expression of collagenase in adult fibroblasts,(2) that phorbol inducibility depends on cooperation among several sequence elements within the collagenase promoter, and (3) that regulation of this promoter is more complex than previously described.(Collagen is the body’s most abundant protein and isa major component of the extracellular matrix. Collagen degradation is initiated by the enzyme collagenase (Jeffrey, 1986) and occurs in a number of normal and disease processes including wound healing, uterine resorption, tumor invasion, and ar-thritis. The same collagenase gene product is expressedby a variety of celltypes including endothelial cells (Herron et al., 1986), keratinocytes (Lin et al., 1987), macrophages (Campbell et al., 1987), chondrocytes (Lin et al., 1988; Stephenson et al., 1987), and fibroblasts (Goldberg et al., 1986; Whitham et al., 1986; Brinckerhoff et al., 1987). Collagenase is a major gene product of fibroblasts, and, indeed, increased production of collagenase by synovial fibroblasts lining the joint is largely responsible for the extensive destruction of connective tissue seen in rheumatoid arthritis (Harris, 1985).