Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion

Abstract

Activation of proMMP-2 by MT1-MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP-2 bound to MT1-MMP on the cell surface acts as a receptor for proMMP-2. Subsequently, adjacent TIMP-2-free MT1-MMP activates the proMMP-2 in the ternary complex. In this study, we demonstrate that MT1-MMP forms a homophilic complex through the hemopexin-like (PEX) domain that acts as a mechanism to keep MT1-MMP molecules close together to facilitate proMMP-2 activation. Deletion of the PEX domain in MT1-MMP, or swapping the domain with the one derived from MT4-MMP, abolished the ability to activate proMMP-2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1-MMP lacking the catalytic domain (MT1PEX-F) efficiently inhibited complex formation of the full-length enzymes and activation of pro MMP-2. Furthermore, expression of MT1PEX-F inhibited proMMP-2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1-MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue.

Fig. 1. MT1-MMP forms a homophilic complex through the PEX domain. (A) A schematic representation of MT1-MMP mutants used in the experiments. Pro, propeptide; FLAG, FLAG epitope; Myc, c-Myc epitope; CD, catalytic domain; H, hinge; PEX, hemopexin-like domain; TM, transmembrane domain. (B) COS1 cells were transfected with the expression plasmid for MT1-F and/or MT1-Myc. The cells were lysed and subjected to immunoprecipitation using anti-FLAG M2 antibody-conjugated beads as described in Materials and methods. The whole-cell lysates (Whole Cell) and immunoprecipitated materials (Anti-FLAG IP) were subjected to western blotting using an anti-FLAG M2 antibody (Anti-FLAG) or anti-c-Myc antibody (Anti-Myc). (C) COS1 cells were transfected with the expression plasmids for MT1-F, MT1Cat and/or MT1PEX. The cells were lysed and subjected to immunoprecipitation using anti-FLAG beads as above. The samples were analyzed by western blotting using an anti-FLAG M2 antibody (Anti-FLAG), anti-MT1-MMP PEX (Anti-PEX) or anti-MT1Cat (Anti-Cat).

Fig. 2. MT1-MMP directly forms a homophilic complex. (A) A schematic representation of MT1EAΔTM expressed in E.coli. E/A, Glu240 to Ala mutation. (B) Gel filtration analysis of MT1EAΔTM at different concentrations. Three different concentrations of MT1EAΔTM (0.33, 0.66 and 1.65 µM) were subjected to gel-permeation column chromatography on Superdex 200. The inset shows SDS–PAGE analysis of MT1EAΔTM under reducing (Red) and non-reducing (NR) conditions. Bands were visualized by Coomassie Blue. (C) MT1EAΔTM (50 µg/ml) was treated with trypsin (0.1 µg/ml) at 37°C for 1 h to separate the catalytic domain and PEX. Trypsin was inactivated by the addition of 2 mM phenylmethylsulfonyl fluoride to the mixture. The sample was then subjected to gel-permeation column chromatography on Superdex 75. The inset shows the SDS–PAGE analysis of the purified PEX and catalytic domains on the column under reducing and non-reducing conditions; the bands were visualized by Coomassie Blue. (D) Escherichia coli-expressed MT1-MMP PEX and MT4-MMP PEX were subjected to gel-permeation column chromatography on Superdex 75. The inset shows the SDS–PAGE analysis of MT4-MMP PEX under reducing and non-reducing conditions; the bands were visualized by Coomassie Blue.

Fig. 3. Examination of MT1-MT4PEX for the activation of proMMP-2 on the cell surface. (A) A schematic representation of MT1-MT4PEX. MT4-PEX, PEX domain derived from MT4-MMP. (B) Activation of proMMP-2 by MT1-MMPs. COS1 cells were transfected with the expression plasmids for MT1-MMP, MT1-MT4PEX or vector alone (Mock), and these cells were reacted with purified proMMP-2 (0.25 µg/ml) in the culture medium without serum at 37°C for 18 h. The medium was analyzed by zymography for proMMP-2 processing (top panel), and the cell lysate by western blotting using anti-MT1Cat (middle panel, Anti-Cat). Transfected cells were also subjected to surface biotinylation as described in Materials and methods, and analyzed by western blotting using anti-MT1Cat (bottom panel, Surface Biotinylation). (C) Binding of proMMP-2 to the MT1-MT4PEX-expressing cells tested as described in Materials and methods. Samples were analyzed by gelatin zymography. (D) In situ gelatin degradation activities of MT1-MT4PEX-expressing cells were studied by culturing cells on DQ gelatin-coated coverslips as described in Materials and methods. Gelatin degradation activity was visualized as the fluorescent area. A bright field image is also shown. Bars: 50 µm.

Fig. 4. Examination of dimer formation of the MT1-MMP ectodomain on the cell surface. (A) A schematic representation of the mutant MT1-MMP/NGFR chimeras. NGFR-TM/CP, NGFR-derived transmembrane and cytoplasmic domains; TK, tyrosine kinase domain; Y, possible tyrosine residue to be phosphorylated; MT4-PEX, PEX domain derived from MT4-MMP. (B) COS1 cells were transfected with expression plasmids for MT1-F/NGFR, MT1PEX-F/NGFR, MT1Cat-F/NGFR, MT4PEX-F/NGFR or vector alone (Mock). Cell lysates were subjected to western blot analysis using the anti-FLAG M2 monoclonal antibody (Anti-FLAG, upper panel) or anti-phosphotyrosine PY20 monoclonal antibody (Anti-PY). (C) COS1 cells were co-transfected with MT1-F/NGFR and/or MT1PEX-F, MT1Cat-F, or MT1-F. Cells were then subjected to western blotting using anti-FLAG M2 (Anti-FLAG) or PY20 (Anti-PY). The relative intensity of the bands detected by PY20 was analyzed with NIH Image, and is indicated. (D) COS1 cells were co-transfected with MT1-F/NGFR and MT1PEX-F at the indicated amounts of DNA. Cells were then subjected to western blotting using anti-FLAG M2 (Anti-FLAG) or PY20 (Anti-PY). The relative intensity of the bands detected by PY20 is indicated.

Fig. 5. Effect of the constitutively active form of Rac1 (Rac1DA) on the dimer formation of MT1-MMP and its ability to activate proMMP-2. (A) Effect of Rac1DA on the dimer formation of MT1-F/NGFR. COS1 cells were transfected with the expression plasmids for MT1-F/NGFR, MT1PEX-F and/or Rac1DA. Cell lysates were subjected to western blot analysis using PY20 (Anti-PY) and anti-FLAG M2 antibodies (Anti-FLAG). The relative intensity of the bands detected by PY20 was analyzed with NIH Image, normalized by the relative intensity of the bands of MT1-F/NGFR detected by Anti-FLAG M2 antibody, and is indicated. (B) Transfected COS1 cells were stained with PY20 (Anti-PY) as described in Materials and methods. F-actin was also stained with Alexa488-conjugated Phalloidin (Alexa488 Phalloidin). The white arrows indicate the sites where F-actin and PY signals were co-localized. Bars: 10 µm. (C) COS1 cells were transfected with the expression plasmids for MT1-MMP and Rac1DA, and these cells were reacted with purified proMMP-2 (0.25 µg/ml). The medium was analyzed by zymography for proMMP-2 processing (top panel), and the cell lysate was analyzed by western blotting using the anti-MT1PEX antibody (bottom panel). The relative activation of proMMP-2 to the active form was calculated by measuring the intensity of the bands of proMMP-2 with NIH Image, and is indicated. (D) Transfected COS1 cells were stained with anti-FLAG M1 antibody as described in Materials and methods. F-actin was also stained with Alexa488-conjugated phalloidin (Alexa488 Phalloidin). The white arrows indicate the sites where F-actin and FLAG signals were co-localized. Bars: 10 µm.

Fig. 6. Effect of MT1PEX-F on the proMMP-2 activation by MT1-MMP on the cell surface. (A) COS1 cells were co-transfected with the expression plasmids for MT1-MMP and MT1PEX-F at the amounts of DNA indicated. Purified proMMP-2 (0.25 µg/ml) was then reacted with the cells at 37°C for 18 h. MMP-2 in the culture medium was analyzed by gelatin zymography (top panel), and the cell lysate was subjected to western blotting using anti-MT1PEX antibody (middle panel, Whole cell). Transfected cells were also subjected to surface biotinylation as described in Materials and methods and analyzed by western blotting using anti-MT1PEX (bottom panel, Surface Biotinylation). (B) Binding of proMMP-2 to the transfected cells was tested as described in Materials and methods. Samples were analyzed by gelatin zymography. (C) In situ gelatin degradation activity of transfected cells was studied by culturing cells on the glass slide coated with Alexa488-labeled gelatin as described in Materials and methods. Gelatin degradation activity was visualized as dark, non-fluorescent areas. Bars: 50 µm.

Fig. 7. Effect of MT1PEX-F on the activation of proMMP-2 and the Matrigel invasion activity of HT1080 cells. (A) HT1080 cells were transfected with the stable expression plasmid for MT1PEX-F and an empty vector, and the whole population of hygromycin-resistant cells was pooled and reseeded. After 24 h, the medium was exchanged for serum-free DMEM in the presence or absence of BB94 (10 µM) and the cells were further cultured for 24 h. The culture medium and cell lysate were analyzed by gelatin zymography and western blotting using anti-MT1PEX. (B) HT1080 cells were transfected with the expression plasmid for MT1PEX-F or empty vector. Cells were co-immunostained for endogenous MT1-MMP and MT1PEX-F using rabbit anti-MT1Cat antibody and anti-FLAG M1 antibody, respectively. The white arrows indicate the sites where endogenous MT1-MMP (Anti-MT1Cat) and MT1PEX-F (Anti-FLAG M1) signals were co-localized. Bars: 50 µm. (C) HT1080 cells were transfected with the expression plasmid for MT1PEX-F or empty vector together with the plasmid for GFP. Cells were then subjected to a Matrigel invasion assay using the modified Boiden chamber method as described in Materials and methods. TIMP-2 at 0.5 µg/ml was added to both the upper and lower chamber. GFP-positive cells on the lower chamber surface were counted under fluorescence microscopy. The total numbers of GFP-positive cells in mock and MT1PEX-F-transfected cells were 1.31 × 10⁴ and 1.12 × 10⁴, respectively. The mean and standard deviation of four in each group are indicated as a graph.