Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis

Abstract

Membrane-type matrix metalloproteinase 1 (MT1-MMP) plays a critical role in extracellular matrix remodeling under both physiological and pathological conditions. However, the mechanisms controlling its activity on the cell surface remain poorly understood. In this study, we demonstrate that MT1-MMP is regulated by endocytosis. First, we determined that Con A induces proMMP-2 activation in HT1080 cells by shifting endogenous MT1-MMP from intracellular compartments to cell surface. This phenotype was mimicked by the cytoplasmic truncation mutant MT1 Delta C with more robust pro-MMP-2 activation and cell surface expression than wild-type MT1-MMP in transfected cells. MT1 Delta C was subsequently shown to be resistant to Con A treatment whereas MT1-MMP remains competent, suggesting that Con A regulates MT1-MMP activity through cytoplasmic domain-dependent trafficking. Indeed, MT1-MMP was colocalized with clathrin on the plasma membrane and with endosomal antigen 1 in endosomes. Internalization experiments revealed that MT1-MMP is internalized rapidly in clathrin-coated vesicles whereas MT1 Delta C remains on cell surface. Coexpression of a dominant negative mutant of dynamin, K44A, resulted in elevation of MT1-MMP activity by interfering with the endocytic process. Thus, MT1-MMP is regulated by dynamin-dependent endocytosis in clathrin-coated pits through its cytoplasmic domain.

Figure 1

Con A stimulates pro-MMP-2 activation by increasing cell surface presentation of MT1-MMP. (A) Stimulation of pro-MMP-2 activation by Con A in HT1080 cells. Confluent HT1080 cells were either treated in serum-free DMEM alone (lane 1) or with Con A (50 μg/ml, lane 2) for 24 h. Supernatants were analyzed by gelatin zymography (Upper), and cell lysates were analyzed by Western blotting using anti-MT1-MMP Ab (Ab3) as described (Lower) (15, 21). (B) Time course of Con A-stimulated pro-MMP-2 activation in HT1080 cells. HT1080 cells were stimulated with Con A (50 μg/ml) for 0, 12, 24, and 36 h and analyzed by zymography as described in A. Percentages of pro-MMP-2 activation was calculated as active + intermediate/pro + intermediate + active species as determined by densitometry (18) and plotted against incubation time (x axis). (C) Shift of MT1-MMP from intracellular compartment to cell surface by Con A treatment. HT1080 cells grown on coverslips in 6-well plates were either left untreated (a) or treated with Con A (50 μg/ml, b) for 24 h. Cells were then permabilized, stained with anti-MT1-MMP Ab, and scanned by confocal microscopy as described in Materials and Methods. Note that HT1080 cells are all positive for MT1-MMP staining but with varying degree of intensity as shown in a; and they responded to Con A at varying degree as well as shown in b. (D) Accumulation of MT1-MMP in Con A-treated HT1080 determined by cell surface biotinylation. HT1080 cells cultured in the absence (lane 1) or presence (lane 2) of Con A (50 μg/ml) were biotinylated, lysed, and immunoprecipitated with anti-MT1-MMP Ab (Ab3), fractionated on SDS/PAGE, transferred to poly(vinylidene difluoride) membrane, and blotted with alkaline phosphatase-conjugated streptavidin (22). Note the enhancement of MT1-MMP in Con A-treated cells (lane 2 vs. 1).

Figure 2

Regulation of MT1-MMP activity by its cytosolic domain. (A) Schematic illustrations of MT1-MMP and MT1ΔC. Full-length MT1-MMP is depicted with signal peptide (s), prodomain (pro), furin cleavage site (R), catalytic domain (cat), hinge region (H), hemopexin-like domain (Pexin), transmembrane domain (TM), and cytoplasmic domain (C) as described (21). MT1ΔC contains a truncation of the 20-residue cytoplasmic domain downstream of the transmembrane domain as indicated by the downward arrowhead (21). (B) Cytoplasmic domain negatively regulates MT1-MMP activity. HT1080 (lanes 1–3), MDCK (lanes 4–6), COS (lanes 7–9), Chinese hamster ovary (CHO) (lanes 10–12), SKBr3 (lanes 13–15), SKOV3 (lanes 16–18), and HEK293 (lanes 19–21) cells were transfected with control vector (C, lanes 1, 4, 7, 10, 13, 16, and 19), MT1-MMP (WT, lanes 2, 5, 8, 11, 14, 17, and 20) or MT1ΔC (ΔC, lanes 3, 6, 9, 12, 15, 18, and 21). Cellular activation of pro-MMP-2 was analyzed by zymography as described in Fig. 1. Note that varying degree of activation for pro-MMP-2 between the cell lines were observed, but the ΔC mutant consistently exhibited higher activity than the wt molecule. (C and D) Cellular localizations of transfected MT1-MMP and MT1ΔC in HT1080 (C) and MDCK (D) cells. Serial Z-sections for MT1-MMP (Ca) and MT1ΔC (Cb) were acquired by confocal microscopy and one from each scan was presented to show differences in distribution between the wt and cytosolic domain truncation mutant. (D) Vertical scans were performed on the corresponding cells and shown in a′ and b′. The arrowheads mark the plasma membrane. Note the general lack of intracellular signals for MT1ΔC vs. the prominent intracellular signal for the wt molecule.

Figure 3

Cytoplasmic domain is required for Con A-mediated stimulation of pro-MMP-2 activation. MDCK cells were transfected with control vector (lanes 1 and 2), MT1-MMP (lanes 3 and 4), and MT1-MMPΔC (lanes 5 and 6) for 4 h before being washed three times with PBS and replenished with fresh culture media. Fresh media containing 5% FBS was added to the cells (24 h later) either alone (lanes 1, 3, and 5) or with Con A (50 μg/ml). The conditioned media were harvested and analyzed by zymography (12 h later) (5 μl/lane Upper). The cells were lysed and analyzed by Western blotting using anti-MT1-MMP Ab (Lower). Note that MT1-MMPΔC is smaller in size because of the truncation of the cytoplasmic domain. The weak signals from control-transfected cells may be endogenous MT1-MMP (21).

Figure 4

Colocalization of MT1-MMP, clathrin, and EEA1 and cytoplasmic domain-dependent internalization. (A) MT1-MMP and clathrin colocalization. MDCK cells transfected with MT1-MMP (1 μg) were incubated with rabbit anti-MT1-MMP Ab at 4°C, followed by fixation and staining with mouse anti-clathrin Ab as described in Materials and Methods). MT1-MMP (green, a and d) and clathrin (red, c and f) were detected by confocal microscopy with horizontal (Upper, a-c) and vertical (Lower, d-f) scans. Two pictures were then merged and presented as b and e. Note that the expression of clathrin and MT1-MMP are heterogeneous in nature and a representative field is presented to depict clathrin inside the plasma membrane and MT1-MMP on the other side of the plasma membrane (b and e). (B) MT1-MMP in endosomes. Cells transfected as in A were fixed, permabilized, and stained with anti-MT1-MMP Ab (see Aa) or anti-EEA1 Ab (c). The same secondary Abs were used for the detections as in A. Note that the signal for intracellular MT1-MMP overwhelms its presence on plasma membrane (a). The typical ring-type endosomes are clearly visible as orange color and selectively marked by arrows (a–c). (C) Differential internalization for MT1-MMP and MT1-MMPΔC. HT1080 cells were transfected with MT1-MMP (a and b) or MT1-MMPΔC (c and d). These cells were labeled with anti-MT1-MMP Ab at 4°C for 2 h, 24 h later. The Ab was removed and cells were washed three times with cold PBS before being shifted to 37°C for commencement of the internalization program. Cells were fixed at 0 (a and c) or 40 min (b and d) and stained with FITC-conjugated goat anti-rabbit secondary Ab. Note that the wt MT1-MMP internalized into endosome-like structures in 40 min (b), whereas MT1-MMPΔC failed to be internalized appreciably (d). (D) Clathrin colocalizes with internalized MT1-MMP. HT1080 cells transfected with MT1-MMP were labeled with anti-MT1-MMP Ab and allowed to internalize for 20 min as described in C. Cells were then fixed, permeabilized, and stained with anti-clathrin Ab, followed by secondary Abs conjugated with FITC or rhodamine. MT1-MMP (red, a) and clathrin (green, c) were detected by confocal microscopy. The merged panel (b) depicts colocalization between clathrin and MT1-MMP (arrowheads).

Figure 5

Dynamin K44A stimulates MT1-MMP-dependent pro-MMP-2 activation by blocking endocytosis. (A) K44A increases MT1-MMP activity in MDCK cells. MDCK cells were transfected with control vector alone (lanes 1, 5, and 9) or MT1-MMP expression vector (lanes 2–4, 6–8, and 10–12) plus dynamin (lanes 3, 7, and 11), dynamin K44A (lanes 4, 8, and 12). Cells were washed 24 h later with PBS and replenished with fresh serum-free media supplemented with recombinant MMP-2. After an additional 24-h incubation, conditioned media were harvested and analyzed by zymography (lanes 9–12), and cells were lysed and analyzed for MT1-MMP (lanes 1–4) or dynamin (lanes 5–8). Note that K44A significantly enhances pro-MMP-2 activation without enhancing the amount of MT1-MMP expression or processing (lane 12 vs. 10; 4 vs. 2). (B) Blockade of MT1-MMP endocytosis by dynamin K44A. MT1-MMP was cotransfected with dynamin (a-c) or K44A (d-f) into N2A cells. Cells were fixed and stained with anti-MT1-MMP and anti-dynamin Abs as described in Fig. 4 legend. MT1-MMP (a and d) and dynamin (c and f) were obtained by confocal microscopy. The merged pictures (b and e) depict any colocalization. Note that dynamin K44A, not its wt, increases MT1-MMP expression on plasma membrane (d vs. a).