Spatial Regulation of MT1-MMP in Epithelial Cells

• Main Author: Bird, Sarah Anne
• Published: Imperial College London 2007
• Subjects: 571.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487203

Tube structures are fundamental in multicellular organisms and are found in many epithelial organs. Madine-Darby Canine Kidney (MOCK) epithelial cells are used as a model to study tubulogenesis as they form branching tubules in a 3-D type-l collagen gel upon stimulation with Hepatocyte Growth Factor (HGF). During this process, cells need to degrade the surrounding collagen matrix and they have been shown to primarily utilise the collagenase membrane-type I metalloproteinase (MTI-MMP, MMP-14) to achieve this. The aim of this thesis is to investigate the mechanisms regulating MTl-MMP during this process. We observed that MTlMMP preferentially localised to the apical surface of polarised MOCK cells cultured on type-I collagen. However, upon stimulation with HGF, a significant amount of MTI-MMP was detected on the basal surface. This localisation has a functional impact as cells degrade type-I collagen only upon HGF stimulation in 2-D culture. We hypothesise that MTl-MMP activity is spatially regulated in polarised MOCK cells, enabling enzyme activity to be higher at the growing tip of the tubule than atthe base. We also observed that MOCK cell attachment to collagen is required for MTl-MMP to localise to the basal surface. By analysing domain deletion mutants of MTI-MMP, it became clear that HGF-dependent localisation to the basal surface is catalytic domain/linker-I and hemopexin domain-dependent. Specifically, the MT-Loop C63pYAYlREG170 ), a unique sequence in the catalytic domain of all;ransmembrane-type MT-MMPs, is essential for basal localisation. Further characterisation of the MT-Loop region indicated that it also plays a critical role in the localisation of MTl-MMP in non-epithelial cells. COS-7 cells expressing the MT-Loop deletion mutant showed a significant reduction in collagen and gelatin degradation in 2-0 culture, even though the cell surface expression level and in vitro catalytic activity of this mutant was similar to full-length enzyme. Taken together, my research has revealed a novel mechanism that regulates MTl-MMP and hence tube formation of MOCK cells, which may be applicable to tubulogenesis in vivo.