In order to investigate the role of type IV collagenase in tumor invasion and metastases, we have focused on the multilevel regulation of this enzyme. We have studied the transcriptional regulation of the 72 kDa collagenase IV enzyme in both normal and human tumor cell lines as well as human tumor tissues. These studies have shown that in contrast with other members of the collagenase enzyme family, the 72 kDa type IV collagenase mRNA levels are increased in response to TGFbeta1, are unaffected by the tumor promoting phorbol esters, and show elevated levels in colorectal tumor tissues when compared with adjacent normal mucosa tissues. Transfection of human tumor cells with expression constructs containing the 72 kDa type IV collagenase gene resulted in phenotypic changes, such as slowed cell growth rates and marked cell vacuolization. These findings suggest that uncomplexed 72 kDa type IV collagenase is not readily secreted by these transformed cells. We have identified a cellular activation mechanism which is cell surface associated and specific for the 72 kDa type IV collagenase enzyme, and which can be induced by pretreatment with phorbol esters or concanavalin A. This cellular activation mechanism does not affect other members of the collagenase gene family. Further characterization and purification of components of this activation mechanism are ongoing. We have also examined the autoproteolytic breakdown of the 72 kDa type IV collagenase following removal of TIMP-2 from the complex. The enzyme has a characteristic pattern of autoproteolytic digestion generating specific 42 kDa and 37.5 kDa gelatin binding fragments, one (37.5 kDa) of which retains proteolytic activity. Finally, we have prepared antibodies and cDNA probes to other members of the collagenase family and have initiated a screening project to examine human lung tumor tissues for the relative mRNA levels and protein expression of the 72 kDa type IV collagenase, stromelysin-1, stromelysin-2, stromelysin-3, PUMP-1 and the 92 kDa type IV collagenase.
