Strategies for Cloning New MMPs and TIMPs

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play important roles in the remodeling of connective tissues associated with normal mammalian development and growth, and in the degradative processes accompanying diseases such as rheumatoid arthritis, pulmonary emphysema or tumor cell invasion and metastasis (1 ). Because of the importance of these proteins in both normal and pathological conditions, over the last years many groups have tried to clone the diverse MMPs mediating these matrix remodeling events as well as the different TIMPs able to balance their proteolytic activities. The first evidence for the occurrence of MMPs was reported about 35 yr ago by Gross and Lapiere who described the presence of diffusible collagenolytic factors in tissue cultures of bullfrog tadpoles (2 ). Some years after this finding, several groups independently reported the existence of naturally occurring metalloproteinase inhibitors known as TIMPs and active against most members of the MMP family (3 ). The utilization of standard biochemical methods allowed the isolation of the first MMP and TIMP family members and their subsequent physico-chemical characterization. However, these studies were seriously hampered by the small amount of proteases and inhibitors usually found in normal conditions. The observation that these proteins were much more abundant in a series of pathological conditions such as inflammatory or tumor processes or during extracellular matrix remodeling events, facilitated the identification of additional members of both families and the molecular cloning of the first MMPs and TIMPs. More recently, the advent of more powerful molecular biology techniques and improved cloning strategies has made it possible to identify a large number of novel MMP and TIMP family members. To date, 18 distinct MMPs have been identified, cloned, and characterized in vertebrates (4 ). In addition, MMPs have been also cloned from embryonic sea urchin (5 ), green alga (6 ) and soybean leaves (7 ). The complexity of the TIMP family has also expanded during the last years and a total of 4 distinct inhibitors with ability to control the proteolytic activity of MMPs have been cloned and characterized at the molecular level (8 ). These new additions to the growing list of MMPs and TIMPs have provided much more complexity to the field but have also opened new views on the role of these proteins in normal and pathological processes. Thus, evidence is accumulating that MMPs are not exclusively involved in the proteolytic degradation of extracellular matrix components, playing also direct roles in essential cellular processes such as differentiation, proliferation, angiogenesis and apoptosis (9 ). Similarly, TIMPs appear to have additional roles other than their direct inhibition of MMP proteolytic activity, and a number of reports have described their involvement in cell growth (10 ). The delineation of expanding roles for these proteins in a wide variety of biological processes has also reinforced previous observations indicating that misregulation of these proteases and inhibitors can have important pathological consequences. Nevertheless, it seems clear that most of this progress has been only possible by the cloning of an unexpected large number of these proteins. This chapter will give an overview of the different strategies used for cloning MMPs and TIMPs and their application to the identification and characterization of putative yet unknown members of these protein families that play essential roles in both normal and pathological conditions.