Membrane-type-1 matrix metalloproteinase in pericellular proteolysis and cell migration

.......................................................................... 9 Introduction.................................................................... 10 Extracellular matrix........................................................... 10 Interstitial matrix....................................................... 10 Basement membrane................................................... 12 Role of extracellular matrix on cellular communication............... 14 Extracellular matrix remodeling............................................ 15 Matrix metalloproteinases.............................................. 15 Collagenases..................................................... 16 Gelatinases....................................................... 17 Stromelysins and other MMPs................................ 19 Membrane-type matrix metalloproteinases.......................... 19 Structural features............................................... 20 Activity and substrate specificity............................ 21 Regulation of MT-MMP activity..................................... 23 Gene expression................................................ 23 Zymogen activation............................................ 25 Metalloproteinase inhibitors................................... 27 MT1-MMP and TIMP-2 in proMMP-2 activation..................29 Cell migration..................................................................... 31 Pericellular proteolysis.................................................. 32 Interplay between proteolysis, adhesion, and signaling... 32 Proteolytic cascades............................................33 Role of MT1-MMP in cell migration................................. 34 Biological roles of MT1-MMP ...............................................34 Angiogenesis............................................................. 35 Bone development....................................................... 36 Cancer.....................................................................38 Aims of the present study...................................................41 Materials and methods...................................................... 42 Growth factors, chemicals, and enzymes............................. 42 Cell culture and treatments............................................. 42 Antibodies................................................................ 43 Northern hybridization and RNAse protection......................43 Expression constructs .................................................. 43 Transfection of cells .................................................... 44 SDS-PAGE and immunoblotting ..................................... 44 Gelatin zymography and reverse zymography...................... 44 Pulse-chase analysis and immunoprecipitation...................... 45 Purification and N-terminal sequencing of MT1-MMP........... 45 Cell surface biotinylation ............................................. 46 Cell surface cross-linking ............................................. 46 In vitro analyses of MT1-MMP cleavage........................... 46 GST-fusion protein purification...................................... 46 Immunofluorescence and internalization assays................... 47 In vitro invasion assay.................................................. 47 Results........................................................................... 48 Differential regulation of MT1-MMP gene expression and proMMP-2 activation (I) .................................... ......48 Calcium ionophores inhibit proMT1-MMP activation, binding of TIMP-2 and proMMP-2, and proMMP-2 activation (I, II) ....................................................49 Inactivating autocatalytic MT1-MMP cleavage produces 43kDa cell surface and 20-kDa soluble fragments (I, II, IV) ...49 Oligomerization through hemopexin and cytoplasmic domains regulates the activity and turnover of MT1MMP (III) ......... .................................................50 MT1-MMP targeting through its cytoplasmic domain is critical for cell invasion through Matrigel (IV) ......... ......50 Endocytosis in clathrin-coated pits regulates cell surface expression of MT1-MMP (V) ......... ..........................51 Discussion.............. .........................................................52 Correlation between MT1-MMP expression and proMMP-2 activation (I) ......... ..............................................52 Role of proMT1-MMP processing/activation in proMMP-2 activation (II) ......... .............................................53 Autocatalytic MT1-MMP inactivation (I, II, IV) ......... .........54 Identification of oligomeric MTI-MMP complexes (III) ...... ...55 ProMMP-2 activation and autocatalytic MT1-MMP inactivation in the oligomeric complexes (II, III, IV).... ......57 MT1-MMP targeting and trafficking during cell migration (IV, V)............................................................ ...60 Perspective......................................................................63 Acknowledgements......... ..................................................64 References.......................................................................65