Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases - PubMed (original) (raw)

Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases

I Yana et al. Mol Biol Cell. 2000 Jul.

Free PMC article

Abstract

Membrane type-1 matrix metalloproteinase (MT1-MMP) is the prototypical member of a subgroup of membrane-anchored proteinases that belong to the matrix metalloproteinase family. Although synthesized as a zymogen, MT1-MMP plays an essential role in extracellular matrix remodeling after an undefined process that unmasks its catalytic domain. We now report the existence of a proprotein convertase-MT1-MMP axis that regulates the processing and functional activity of the metalloproteinase. Two sets of basic motifs in the propeptide region of MT1-MMP are identified that potentially can be recognized by the proprotein convertase family of subtilisin-like proteases. Processing of proMT1-MMP as well as the expression of its proteolytic activity were blocked by mutating these recognition motifs or by inhibiting the proprotein convertases furin and PC6 with the serpin-based inhibitor alpha(1) antitrypsin Portland. Furthermore, both furin-dependent and furin-independent MT1-MMP processing pathways are identified that require tethering of the metalloproteinase to the cell surface. These findings demonstrate the existence of a proprotein convertase-MT1-MMP axis that can regulate extracellular matrix remodeling.

PubMed Disclaimer

Figures

Figure 1

A scheme of MT1-MMP mutants. MT1-MMP domains are depicted as shaded boxes. Amino acid substitutions were inserted either into the 108RRKR and 86KAMRRPR domains (each bracketed) alone as 108RRKR → AAAA (MT1-MMP/A4) or 86KAMRRPR → KAMARPR (MT1-MMP/AXXR) or into both domains as an MT1-MMP/AXXR-A4 mutant. Additional mutations included a 240E → A substitution in the catalytic domain to create an enzymically inactive mutant of MT1-MMP, a soluble transmembrane-deletion mutant with the C terminus truncated at S538 (ΔMT1-MMP), a soluble mutant with an RRKR → AAAA substitution (ΔMT1-MMP/A4), or an epitope-tagged variant of MT1-MMP with FLAG inserted at the C terminus of the wild-type proteinase (MT1-MMP/FLAG).

Figure 2

Processing of proMT1-MMP in COS-1 and HT-1080 cells. (A) A scheme of MT1-MMP epitopes. Wild-type MT1-MMP and MT1-MMP containing a FLAG epitope inserted in the prodomain were detected with antibodies directed against a 14-amino acid residue (R160 to K173) in the catalytic domain, an 18-amino acid residue (R437 to K454) in the hemopexin domain, or a FLAG epitope inserted between residues D49 and L50 in the prodomain. (B) Western blot analysis of FLAG/MT1-MMP expression in COS-1 cells. COS-1 cells were transiently transfected with MT1-MMP or FLAG/MT1-MMP expression vectors. Triton X-114 extracts were then analyzed by immunoblotting with either hemopexin domain–specific polyclonal antisera (lanes 1 and 2) or anti-FLAG mAb (lanes 3 and 4). Although anti-MT1-MMP polyclonal antisera recognized both wild-type MT1-MMP (lane 1) and FLAG/MT1-MMP (lane 2), the anti-FLAG mAb recognized the ∼63-kDa pro form of FLAG/MT1-MMP alone (lane 4). The anti-FLAG mAb did not react with wild-type MT1-MMP (lane 3). (C) Western blot analysis of MT1-MMP in transiently transfected COS-1 cells or HT-1080 cells. Triton X-114 extracts of COS-1 cells transfected with control (lane 1) or wild-type MT1-MMP expression vectors and incubated in the absence or presence of 5 μM BB-94 (lanes 2 and 3, respectively) were compared with extracts of HT-1080 cells (lane 4) by immunoblotting with MT1-MMP hemopexin domain–specific polyclonal antisera. The three arrowheads indicate the positions of the putative ∼63-kDa pro form, ∼60-kDa mature form, and ∼45-kDa truncated form of MT1-MMP. COS-1 cell extracts pre-pared from cells transiently transfected with control (lanes 5 and 7) or MT1-MMP (lanes 6 and 8) expression vectors were analyzed by immunoblotting in a tandem manner with hemopexin domain–specific polyclonal antisera or anti-catalytic domain mAb, respectively.

Figure 3

Processing of membrane-anchored and soluble forms of MT1-MMP. (A) Western blot analysis of MT1-MMP variants harboring mutations in the 108RRKR and/or 86KAMRRPR basic motifs. COS-1 cells were transfected with control (lane 1), ΔMT1-MMP (lanes 2–5), or full-length MT1-MMP (lanes 6–10) expression vectors, and cell-free supernatants (lanes 1–5) or Triton X-114 extracts (lanes 6–10) were analyzed by Western blot analysis with hemopexin domain–specific polyclonal antisera. In lanes 2 and 3, cell-free supernatants of COS-1 cells expressing ΔMT1-MMP or ΔMT1-MMP/A4 were compared, whereas in lanes 4 and 5, the ability of α1PDX to inhibit ΔMT1-MMP processing was assessed. The upper bands in lanes 3 and 5 (marked with open arrowheads) are the pro forms of ΔMT1-MMP. In the case of full-length MT1-MMP (lanes 6–10), COS-1 cells expressing the MT1-MMP/A4 mutant (lane 7) or the MT1-MMP/AXXR-A4 mutant (lane 10) displayed a diminished ability to process the proenzyme relative to cells expressing wild-type MT1-MMP (lanes 6 or 8). The MT1-MMP/AXXR mutant (lane 9) was processed comparably to the MT1-MMP control (lane 8). The closed arrowheads mark the positions of pro, mature, and truncated MT1-MMP. (B) Progelatinase A activation and surface display of MT1-MMP in transiently transfected COS-1 cells. For zymography, COS-1 cells were transiently transfected with control (lane 1), MT1-MMP (lanes 2 and 5), MT1-MMP/A4 (lane 3), MT1-MMP/AXXR-A4 (lane 4), or MT1-MMP/AXXR (lane 6) expression vectors. The COS-1 cells were subsequently incubated with progelatinase A for 16 h, and processing was assessed by zymography (lanes 1–6). The upper and lower arrowheads mark the positions of the pro and fully processed forms of gelatinase A, respectively. In lanes 7–10, control-, MT1-MMP–, or MT1-MMP mutant–transfected COS-1 cells were surface biotinyl-ated, and Triton X-114 extracts were immunoblotted with hemopexin domain–specific polyclonal antisera. (C) Effect of α1PDX on MT1-MMP processing and activity. COS-1 cells were cotransfected with wild-type MT1-MMP and a control expression vector (lanes 1 and 3), MT1-MMPE → A and a control expression vector (lane 4), wild-type MT1-MMP and α1PDX (lane 2), or MT1-MMPE → A and α1PDX (lane 5) expression vectors, and Triton X-114 extracts were analyzed by immunoblotting. Progelatinase A activation was monitored by zymography after incubation with COS-1 cells transfected with the control expression vector (lane 6), with MT1-MMP and a control expression vector (lane 7), or with MT1-MMP and α1PDX expression vectors (lane 8). Cell surface biotinylation followed by Western blot analysis demonstrated that trafficking of proMT1-MMP (lane 10) was not affected by the α1PDX expression vector (lane 11).

Figure 4

Furin-dependent processing of MT1-MMP in LoVo and COS-1 cells. (A) Furin-dependent processing in LoVo cells. LoVo cells were transfected with a control expression vector alone (lane 1), ΔMT1-MMP and a control expression vector (lane 2), or ΔMT1-MMP and furin expression vectors (lane 3). The cell-free supernatants were then examined for soluble forms of ΔMT1-MMP by Western blotting. The two arrowheads to the right of lane 3 mark the positions of proΔMT1-MMP and processed ΔMT1-MMP. In lanes 4–7, LoVo cells were transfected with a control expression vector (lane 4), epitope-tagged MT1-MMP and a control expression vector (lane 5), epitope-tagged MT1-MMP and a furin expression vector (lane 6), or epitope-tagged MT1-MMP, furin, and α1PDX expression vectors (lane 7). Immunoblots were performed on Triton X-114 extracts as described above. (B) Furin-dependent processing of proMT1-MMP by soluble furin. COS-1 cells cotransfected with MT1-MMP and either a control expression vector (lane 1) or a furin expression vector (lane 2) displayed similar profiles of MT1-MMP products as assessed by immunoblotting. Processing of MT1-MMP in COS-1 cells (lane 3) or α1PDX-transfected cells (lane 5) was enhanced by the addition of soluble furin to the serum-free culture medium (lanes 4 and 6, respectively).

Figure 5

MT1-MMP processing and activation in CHO-K1 and RPE.40 cells. (A) Processing of soluble and full-length MT1-MMP. CHO-K1 (lanes 1–3) or RPE.40 (lanes 4–6) cells were transfected with a control expression vector (lanes 1 and 4), ΔMT1-MMP (lanes 2 and 5), ΔMT1-MMP and α1PDX (lane 3), or ΔMT1-MMP and furin (lane 6) expression vectors. Cell-free supernatants were subjected to immunoblot analysis with anti-hemopexin domain–specific polyclonal antisera. The arrowheads to the right of lane 6 mark the positions of the pro and mature forms of ΔMT1-MMP, whereas the asterisk marks the position of a minor glycosylated form of proΔMT1-MMP as determined by tunicamycin sensitivity (our unpublished results). In lanes 8–10 and 12–14, Western blotting of Triton X-114 extracts was performed on CHO-K1 or RPE.40 cells expressing full-length MT1-MMP that had been cotransfected with a control expression vector (lanes 8 and 12), a furin expression vector (lanes 9 and 13), or an α1PDX expression vector (lanes 10 and 14). Immunoblots of CHO-K1 or RPE.40 cells transfected with control expression vectors are shown in lanes 7 and 11. In lanes 15–18, RPE.40 cells were transfected with expression vectors for MT1-MMP (lane 15), MT1-MMP/A4 (lane 16), MT1-MMP/AXXR (lane 17), or MT1-MMP/AXXR-A4 (lane 18). (B) MT1-MMP processing in CHO-K1 and RPE.40 cells. Western blotting was performed on CHO-K1 and RPE.40 cell extracts that had been transfected with full-length MT1-MMP (lanes 1 and 2, respectively), cytosolic domain–deleted MT1-MMP (lanes 3 and 4), or a chimeric MT1-MMP variant containing the interleukin 2 receptor transmembrane domain and cytosolic tail (TM swap; lanes 5 and 6). The arrowheads to the right mark the positions of the pro, mature, and truncated forms of MT1-MMP. (C) Processing of progelatinase A by CHO-K1 or RPE.40 cells. CHO-K1 or RPE.40 cells transfected with control expression vectors (lanes 1 and 4), cotransfected with MT1-MMP and control expression vectors (lanes 2 and 5), or cotransfected with MT1-MMP and α1PDX expression vectors (lanes 3 and 6) were incubated with progelatinase A for 16 h, and the supernatants were analyzed by gelatin zymography. The pro, intermediate, and mature forms of gelatinase A are marked by the arrowheads to the right of lane 6.

Figure 6

Maturation of MT1-MMP in CHO-K1 and RPE.40 cells. (A–D) CHO-K1 cells (left panels) or RPE.40 cells (right panels) were transfected with MT1-MMP expression vectors that contained a FLAG insert immediately downstream of 108RRKR. The cells were then fixed, and mature MT1-MMP was immunolocalized with anti-FLAG M1 mAb by confocal laser microscopy (A and B). Nomarski images of the fields are shown for CHO-K1 (C) and RPE.40 (D) cells. (E–H) CHO-K1 or RPE.40 cells were transfected with expression vectors for MT1-MMP/A4 that contained the FLAG insert downstream of 108AAAA. After fixation, anti-FLAG mAb did not stain either CHO-K1 cells (E) or RPE.40 cells (F). Nomarski images of each field are shown in G and H. Cell surface expression of the MT1-MMP/A4-FLAG construct was confirmed with the anti-MT1-MMP 3H7 mAb in CHO-K1 cells (I) and RPE.40 cells (J).

Figure 7

Regulation of MT1-MMP–dependent subjacent proteolytic activity by α1PDX. Control-transfected (A and B), MT1-MMP–transfected (C and D), and MT1-MMP– and α1PDX–cotransfected (E and F) CHO-K1 cells were cultured on surfaces coated with Texas red–labeled gelatin. After 16 h of incubation, cells were visualized by phase-contrast microscopy (A, C, and E), and zones of gelatinolytic activity were identified by confocal laser microscopy (B, D, and F).

Figure 8

Processing of MT1-MMP in HT-1080 cells. (A) Pulse-chase analysis of MT1-MMP processing. HT-1080 cells were pulsed with [35S]methionine as described and chased for 0, 15, 30, 60, and 180 min. Immunoprecipitates of cell lysates were analyzed by SDS-PAGE/autoradiography. The arrowheads mark the positions of pro, mature, and truncated forms of MT1-MMP. (B) Processing of epitope-tagged MT1-MMP in HT-1080 cells. Triton X-114 extracts of HT-1080 cells expressing MT1-MMP/FLAG (lane 2), MT1-MMP/FLAG containing the A4 mutation (lane 3), MT1-MMP/FLAG containing the AXXR mutation (lane 4), MT1-MMP containing both the A4 and AXXR mutations (lane 5), or MT1-MMP/FLAG and α1PDX (lane 6) were analyzed by immunoblotting with anti-FLAG mAb. Glycosylated proMT1-MMP could be detected when processing was blocked by the A4 or AXXR-A4 mutations in lanes 3 and 5. Extracts of HT-1080 cells transfected with a control expression vector did not reveal anti-FLAG immunoreactive products (lane 1).

Figure 9

Prodomain alignments of potential proprotein recognition motifs in soluble and membrane-anchored MMPs. An alignment of MMP prodomains demonstrates that RXKR motifs can be found in stromelysin-3, MT1-, MT2-, MT3-, MT4-, and MT5-MMP, and MMP-23. Either RXXR or KXXR motifs are found in all MMPs except for metalloelastase (MMP-12) and matrilysin (MMP-7) (sequences not shown).

Cited by

An Overview of the Role of Furin in Type 2 Diabetes.
Marafie SK, Al-Mulla F. Marafie SK, et al. Cells. 2023 Oct 5;12(19):2407. doi: 10.3390/cells12192407. Cells. 2023. PMID: 37830621 Free PMC article. Review.
MT1-MMP as a Key Regulator of Metastasis.
Tanaka N, Sakamoto T. Tanaka N, et al. Cells. 2023 Aug 31;12(17):2187. doi: 10.3390/cells12172187. Cells. 2023. PMID: 37681919 Free PMC article. Review.
Allele-Specific Epigenetic Regulation of FURIN Expression at a Coronary Artery Disease Susceptibility Locus.
Yang W, Cao J, McVey DG, Ye S. Yang W, et al. Cells. 2023 Jun 21;12(13):1681. doi: 10.3390/cells12131681. Cells. 2023. PMID: 37443715 Free PMC article.
Cytoplasmic Tail of MT1-MMP: A Hub of MT1-MMP Regulation and Function.
Strouhalova K, Tolde O, Rosel D, Brábek J. Strouhalova K, et al. Int J Mol Sci. 2023 Mar 7;24(6):5068. doi: 10.3390/ijms24065068. Int J Mol Sci. 2023. PMID: 36982142 Free PMC article. Review.
Mint3 as a Potential Target for Cooling Down HIF-1α-Mediated Inflammation and Cancer Aggressiveness.
Tanaka N, Sakamoto T. Tanaka N, et al. Biomedicines. 2023 Feb 14;11(2):549. doi: 10.3390/biomedicines11020549. Biomedicines. 2023. PMID: 36831085 Free PMC article. Review.

References

1. Belien ATJ, Paganetti PA, Schwab ME. Membrane-type 1 matrix metalloproteinase (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter. J Cell Biol. 1999;144:373–384. - PMC - PubMed
1. Benjannet S, Savaria D, Laslop A, Munzer JS, Chretien M, Marcinkiewicz M, Seidah NG. α1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. J Biol Chem. 1997;272:26210–26218. - PubMed
1. Botos I, Scapozza L, Zhang D, Liotta LA, Meyer EF. Batimastat, a potent matrix metalloproteinase inhibitor, exhibits an unexpected mode of binding. Proc Natl Acad Sci USA. 1996;39:2749–2754. - PMC - PubMed
1. Cao J, Drews M, Lee HM, Conner C, Bahou WF, Zucker S. The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A. J Biol Chem. 1998;273:34745–34752. - PubMed
1. Cao J, Rehemtulla A, Bahou W, Zucker S. Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain. J Biol Chem. 1996;271:30174–30180. - PubMed

Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases - PubMed (original) (raw)