GENOME-WIDE IDENTIFICATION OF XENOPUS MATRIX METALLOPROTEINASES. Earlier expression studies by us and others have found that most of the MMPs that have been analyzed are regulated either directly or indirectly by TH in at least some organs/tissues to varying degrees. Thus, it is of interest to determine whether other MMPs are also regulated by TH and whether different MMPs have different functions during metamorphosis in different organs/tissues. Toward this goal, we have carried out a genome-wide analysis of MMP genes in both X. laevis and the closely related species X. tropicalis through a bioinformatic approach by making use of the genome sequence information for X. tropicalis and cDNA sequences available for X. laevis and tropicalis genes from the NIH Frog Initiatives Program. We examined X. laevis and X. tropicalis ESTs and genomic sequences for MMPs and obtained likely homologs for 20 out of the 25 MMPs known in higher vertebrates. Four of the five missing MMPs, i.e. MMPs 8, 10, 12 and 27, were all encoded on human chromosome 1 and the other missing MMP, MMP22 (a chicken MMP), was also absent in human genome. In addition, we discovered several novel MMPs which appear to be derived from unique duplications over evolution and are present in the genomes of both Xenopus species. Our results suggest that MMP genes undergo dynamic changes over evolution. It will be of interest in the future to investigate whether MMP expression and functions during vertebrate development are conserved. The sequence information reported here should facilitate such an endeavor in the near future. DIFFERENTIAL REGULATION OF CELL TYPE SPECIFIC APOPTOSIS BY STROMELYSIN-3. The MMP stromelysin-3 (ST3) is induced by TH and its expression correlated with cell death during metamorphosis. We have previously shown that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. To investigate the roles of ST3 in other organs and especially on different cell types, we analyzed the effect of transgenic overexpression of ST3 in the tail of premetamorphic tadpoles. We observed for the first time that ST3 expression, in the absence of T3, caused significant muscle cell death in the tail of premetamorphic transgenic tadpoles. On the other hand, only relatively low levels of epidermal cell death were induced by precocious ST3 expression in the tail, contrasting what takes place during natural and T3-induced metamorphosis when ST3 expression is high. This cell type specific apoptotic response to ST3 in the tail suggests distinct mechanisms regulating cell death in different tissues. Furthermore, our analyses of laminin receptor, an in vivo substrate of ST3 in the intestine (see below), suggest that laminin receptor cleavage may be an underlying mechanism for the cell type specific effects of ST3. CHARACTERIZATION OF AN IN VIVO SUBSTRATE OF ST3. Toward understanding the mechanism by which ST3 affects tissue remodeling, we have previously identified the 37 kd laminin receptor (LR), a cell surface receptor for the ECM protein laminin, as an in vivo substrate of ST3. ST3 cleaves LR at two distinct sites that are conserved in mammalian LR. Human ST3 and LR are both associated with tumor development and cancer progression and human LR can also be cleaved by ST3, implicating a role of LR cleavage by ST3 in human cancers. We have now carried out a series of mutational analyses on the two cleavage sites in LR. Our findings revealed that in addition to primary sequence at the cleavage site (positions P3-P3', with the cleavage occurring between P1-P1'), flanking sequences/conformation also influenced the cleavage of LR by ST3. Furthermore, alanine substitution studies led to a surprising finding that surrounding sequence and/or conformation dictated the site of cleavage in LR by ST3. These results thus have important implications in our understanding of substrate recognition and cleavage by ST3 and argue for the importance of studying ST3 cleavage in the context of full-length substrates. Furthermore, the LR cleavage mutants generated here will also be valuable tools for future studies on the role of LR cleavage by ST3 in vertebrate development and cancer progression.
