Regulation and function of matrix metalloproteinases during frog metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

REGULATION AND FUNCTION OF THE MATRIX METALLOPROTEINASES (MMPS) DURING TH-INDUCED TISSUE REMODELING. Our earlier expression and function studies in organ cultures have shown that the MMP stromelysin-3 (ST3) is directly regulated by TR at the transcription level and is required for ECM remodeling and TH-induced cell death in the animal intestine. Furthermore, transgenic overexpression of ST3 but not a catalytically inactive mutant in premetamorphic tadpoles is sufficient to cause ECM remodeling and apoptosis in the intestinal epithelium, demonstrating that ST3 is necessary and sufficient for larval epithelial cell death. Aside from ST3, several other MMPs are also regulated by TH during metamorphosis, consistent with the complex nature of the ECM, which would require multiple enzymes for its remodeling and degradation during metamorphosis. To investigate whether different MMPs have different functions during metamorphosis, we have analyzed the expression of several other MMPs. In particular, we have shown that the MMPs GelA and MT1-MMP (a membrane-type MMP) are coordinately regulated during metamorphosis. Using Xenopus embryogenesis as a model, we showed that Xenopus GelA and MT1-MMP were coexpressed during embryogenesis and associated with each other in vivo. Furthermore, we have shown through overexpression of both MMPs in developing embryos that GelA and MT1-MMP has a cooperative role during development, at least in part through the activation of pro-GelA by MT1-MMP. We are now in the process of analyzing the role of these MMPs during metamorphosis by using transgenic overexpression similar to what we have done for ST3.[unreadable] [unreadable] IDENTIFICATION AND CHARACTERIZATION OF AN IN VIVO SUBSTRATE OF ST3. Toward understanding the mechanism by which ST3 affects tissue remodeling, we have recently identified the 37 kd laminin receptor (LR), a cell surface receptor for the ECM protein laminin, as an in vivo substrate of ST3. LR binds to ST3 and can be cleaved by ST3 at two sites in the extracellular domain between the transmembrane domain and laminin binding sequence, separating the cells from laminin. Interestingly, ST3 cleavage sites in LR are conserved in human LR and high levels of LR are known to be present in tumor cells, which are often surrounded by fibroblasts expressing ST3. Thus, LR may be a conserved substrate of ST3 and its cleavage by ST3 may alter cell-ECM interactions, thereby mediating the effects of ST3 on cell fate and behavior during development and pathogenesis. To investigate the functional importance of LR cleavage by ST3 during development, we 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. More importantly, we plan to use the LR cleavage mutants generated here in transgenic studies to investigate the role of LR cleavage by ST3 during metamorphosis.