ABSTRACT
Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades the extracellular matrix, initiates the activation pathway of soluble MMPs and regulates the functionality of cell adhesion signaling receptors, thus playing an important role in many cell functions. Intracellular transport mechanisms, currently incompletely understood, regulate the presentation of MT1-MMP at the cell surface. We have focused our efforts on identifying these mechanisms. To understand the transport of MT1-MMP across the cell, we used substitution and deletion mutants, the trafficking of which was examined using antibody uptake and Chariot delivery experiments. Our experiments have demonstrated that the microtubulin cytoskeleton and the centrosomes (the microtubulin cytoskeleton-organizing centers) are essential for the trafficking and the internalization of MT1-MMP. We determined that after reaching the plasma membrane, MT1-MMP is internalized in the Rab-4-positive recycling endosomes and the Rab-11-positive pericentrosomal recycling endosomes. The microtubular trafficking causes the protease to accumulate in the pericentrosomal region of the cell. We believe that the presence of the transmembrane domain is required for the microtubular vesicular trafficking of MT1-MMP because the soluble mutants are not presented at the cell surface and they are not delivered to the centrosomes. The observed transport mechanisms provide a vehicle for the intracellular targets and, accordingly, for an intracellular cleavage function of MT1-MMP in malignant cells, which routinely overexpress this protease.
Subject(s)
Cell Membrane/enzymology , Metalloendopeptidases/metabolism , Microtubules/enzymology , Animals , Binding Sites, Antibody , CHO Cells , Cell Line , Cell Line, Tumor , Centrosome/enzymology , Centrosome/metabolism , Cricetinae , Cytoskeleton/physiology , Dogs , Humans , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Metalloendopeptidases/immunology , Microtubules/metabolism , Protein Structure, Tertiary/physiology , Protein Transport/genetics , Protein Transport/physiology , Sequence Deletion , Transport Vesicles/enzymology , Transport Vesicles/genetics , Tubulin/physiologyABSTRACT
Understanding the function of invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) is of paramount importance for understanding cancer biology. MT1-MMP is synthesized in cells as a latent zymogen that requires the cleavage of its prodomain to exert the proteolytic activity. The mature alphav integrin subunit is also generated by endoproteolytic cleavage of the alphav subunit precursor (pro-alphav). Cleavage by furin is considered to be a principal event in the activation of both MT1-MMP and pro-alphav. To elucidate the alternative activation pathway of MT1-MMP and pro-alphav, we employed furin-negative LoVo cells, which co-express MT1-MMP with integrin alphavbeta3. In these cells the MT1-MMP proenzyme was rapidly trafficked to the plasma membrane via an unconventional Brefeldin A-resistant pathway and, then, autocatalytically processed on the cell surface. Next, the MT1-MMP activity converted the cell surface-associated pro-alphav into the mature alphav integrin, represented by the disulfide-bonded heavy and light chains, and promoted the formation of the functional integrin alphavbeta3 heterodimer. These events stimulated cell motility in vitro, and malignant invasion and tumor growth in vivo. Our data suggest that in furin-negative colon carcinoma cells MT1-MMP is autocatalytically processed and the active protease then operates as a prointegrin convertase. Our findings argue strongly that the processing by furin is not a prerequisite for the activation of MT1-MMP.
Subject(s)
Colonic Neoplasms/metabolism , Furin/metabolism , Integrin alphaVbeta3/metabolism , Metalloendopeptidases/metabolism , Protein Precursors/metabolism , Protein Subunits/metabolism , Animals , Brefeldin A/metabolism , CHO Cells , Cell Line, Tumor , Cell Membrane/metabolism , Colonic Neoplasms/pathology , Cricetinae , Enzyme Activation , Furin/genetics , Humans , Matrix Metalloproteinase 14 , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , Mice , Mice, Mutant Strains , Neoplasm Invasiveness , Neoplasm Transplantation , Protein Precursors/genetics , Protein Subunits/genetics , Protein Synthesis Inhibitors/metabolism , Protein Transport/physiologyABSTRACT
We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma/metabolism , Cell Membrane/enzymology , Integrins/metabolism , Metalloendopeptidases/metabolism , Cell Adhesion/physiology , Cell Line, Tumor , Cell Movement/physiology , Collagen Type I/metabolism , Enzyme Precursors/metabolism , Extracellular Matrix/metabolism , Female , Humans , Integrin alpha2beta1/metabolism , Integrin alphaVbeta3/metabolism , Matrix Metalloproteinases, Membrane-Associated , Neoplasm Metastasis , Protein Processing, Post-Translational , Protein Subunits/metabolism , Receptor Cross-Talk/physiologyABSTRACT
PURPOSE: To determine in a corneal alkaline burn model of angiogenesis whether the expression of integrins and MMPs is consistent with a VEGF-induced angiogenic response. METHODS: Neovascularization in female Sprague-Dawley rats was induced by alkaline cauterization of the central cornea. RT-PCR for integrins alpha(1), alpha(2), beta(3), and beta(5); the endothelial marker CD31; and metalloproteinases MMP-2 and MT1-MMP was performed on naive corneas and on cauterized corneas 72 and 288 hours after cautery. Analyses of protein and MMP expression were conducted on naive corneas and on cauterized corneas 24, 72, 120, and 168 hours after cautery by immunofluorescence microscopy and gelatin zymography. RESULTS: RT-PCR indicated a correlation between the induced angiogenic response and the expression of alpha(1) and beta(3) integrin subunits and MT1-MMP. Immunohistochemical analysis indicated that alpha(1), alpha(2), alpha(5), and beta(5) integrins and MMP-2 and MT1-MMP were expressed on the newly developing vasculature. The beta(3) integrin was preferentially expressed on platelets. CONCLUSIONS: Integrin expression during neovascularization of rat corneas in response to alkaline injury correlates with an angiogenic response that uses the VEGF/alpha(v)beta(5) pathway. MMP-2 and MT1-MMP, but not MMP-9, are expressed in a pattern consistent with their involvement in the angiogenic response.
Subject(s)
Burns, Chemical/metabolism , Cornea/blood supply , Corneal Neovascularization/metabolism , Eye Burns/chemically induced , Integrins/metabolism , Matrix Metalloproteinase 2/metabolism , Metalloendopeptidases/metabolism , Alkalies , Animals , Burns, Chemical/etiology , Burns, Chemical/pathology , Cornea/pathology , Corneal Injuries , Corneal Neovascularization/etiology , Corneal Neovascularization/pathology , Female , Fluorescent Antibody Technique, Indirect , Integrins/genetics , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers.
