Kaisa Lehti

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

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 MT1ΔC with more robust pro-MMP-2 activation and cell surface expression than wild-type MT1-MMP in transfected cells. MT1ΔC 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 MT1ΔC 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.

Expression of collagenases-1 and -3 and their inhibitors TIMP-1 and -3 correlates with the level of invasion in malignant melanomas.

SummarySince proteolysis of the dermal collagenous matrix and basement membranes is required for local invasive growth and early metastasis formation of cutaneous melanomas, we have analysed the activities/expression levels of certain metalloproteinases in melanomas and cultured melanoma cells by in situ hybridization and Northern analysis. In addition to collagenases-1 and -3 that have been implicated in invasive growth behaviour of various malignant tumours, we analysed the levels of 72-kDa gelatinase and its activators MT1-MMP and TIMP-2 in cultured melanoma cells. The lesions examined included three cases of lentigo maligna and 28 cases of Clark grade I–V melanomas. The premalignant as well as the grade I tumours were consistently negative for collagenase-1 and -3 and TIMP-1 and -3. The collagenases were predominantly expressed in the cancer cells of Clark grade III and IV tumours. TIMP-1 and -3 were abundantly expressed in the cancer and/or stromal cells of grade III and IV melanomas, while TIMP-2 protein was detected also in melanomas representing lower invasive potential. Northern analysis of seven melanoma cell lines showed that the expression of collagenase-1 and TIMPs-1 and -3 was associated with 72-kDa gelatinase positivity. All melanoma cell lines were positive for MTI-MMP and TIMP-2 mRNAs. Our results suggest that overexpression of collagenases-1 and -3 and TIMPs -1 and -3 is induced during melanoma progression. Expression of TIMPs may reflect host response to tumour invasion in an effort to control MMP activity and preserve extracellular matrix integrity.

Plasmin activates pro-matrix metalloproteinase-2 with a membrane-type 1 matrix metalloproteinase-dependent mechanism

Membrane‐type 1 matrix metalloproteinase (MT1‐MMP) has been implicated as a physiological activator of progelatinase A (MMP‐2). We previously reported that plasmin treatment of cells results in proMMP‐2 activation and increased type IV collagen degradation. Here, we analyzed the role of MT1‐MMP in plasmin activation of MMP‐2 using HT‐1080 cells transfected with MT1‐MMP sense or antisense cDNA. Control, vector‐transfected cells that expressed endogenous MT1‐MMP, and antisense cDNA transfectants with very low levels of MT1‐MMP did not activate proMMP‐2. Conversely, cells transfected with sense MT1‐MMP cDNA expressed high MT1‐MMP levels and processed proMMP‐2 to 68/66‐kDa intermediate activation products. Control cells and MT1‐MMP transfectants had much higher levels of cell‐associated MMP‐2 than antisense cDNA transfectants. Addition of plasmin(ogen) to control or MT1‐MMP‐transfected cells generated active, 62‐kDa MMP‐2, but was ineffective with antisense cDNA transfectants. The effect of plasmin(ogen) was prevented by inhibitors of plasmin, but not by metalloproteinase inhibitors, implicating plasmin as a mechanism for proMMP‐2 activation independent of the activity of MT1‐MMP or other MMPs. Plasmin‐mediated activation of proMMP‐2 did not result from processing of proMT1‐MMP and did not correlate with αvβ3 integrin or TIMP‐2 levels. Thus, plasmin can activate proMMP‐2 only in the presence of MT1‐MMP; however, this process does not require the catalytic activity of MT1‐MMP.

Epilysin (MMP-28) induces TGF-β mediated epithelial to mesenchymal transition in lung carcinoma cells

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase (MMP) family. Although it is expressed in a number of tissues, no biological substrates or functions for this enzyme have been identified yet. We have expressed recombinant epilysin in A549 lung adenocarcinoma cells and found that this resulted in stable and irreversible epithelial to mesenchymal transition (EMT) accompanied by loss of cell surface E-cadherin, proteolytic processing of latent TGF-β-complexes and increased levels of active TGF-β. The cascade of events leading to the onset of EMT is prevented by the MMP inhibitor GM6001 or antibodies neutralizing the activity of TGF-β. Once EMT had occurred the cell phenotype could, however, not be reversed by the MMP-inhibitor. Importantly, the expression of epilysin also resulted in upregulation of MT1-MMP and gelatinase-B (MMP-9) and in the collagen invasive activity of A549 cells. Further, we found that epilysin and the recombinant hemopexin domain were targeted to the surface of epithelial cells. This cell surface interaction was sensitive to the proteolytic activity of MT1-MMP, and was lost after EMT. Current results indicate that epilysin can induce EMT and cell invasion through a TGF-β-dependent mechanism suggesting novel biological roles for this enzyme in the regulation of epithelial cell function and in the induction of carcinogenesis.

Secretion of active membrane type 1 matrix metalloproteinase (MMP-14) into extracellular space in microvesicular exosomes.

Membrane type 1 matrix metalloproteinase (MT1‐MMP, MMP14) is an efficient extracellular matrix (ECM) degrading enzyme that plays important roles in tissue homeostasis and cell invasion. Like a number of type I membrane proteins, MT1‐MMP can be internalized from the cell surface through early and recycling endosomes to late endosomes, and recycled to the plasma membrane. Late endosomes participate in the biogenesis of small (30–100 nm) vesicles, exosomes, which redirect plasma membrane proteins for extracellular secretion. We hypothesized that some of the endosomal MT1‐MMP could be directed to exosomes for extracellular release. Using cultured human fibrosarcoma (HT‐1080) and melanoma (G361) cells we provide evidence that both the full‐length 60 kDa and the proteolytically processed 43 kDa forms of MT1‐MMP are secreted in exosomes. The isolated exosomes were identified by their vesicular structure in electron microscopy and by exosomal marker proteins CD9 and tumor susceptibility gene (TSG101). Furthermore, exosomes contained β1‐integrin (CD29). The exosomes were able to activate pro‐MMP‐2 and degrade type 1 collagen and gelatin, suggesting that the exosomal MT1‐MMP was functionally active. The targeting of MT1‐MMP in exosomes represents a novel mechanism for cancer cells to secrete membrane type metalloproteolytic activity into the extracellular space. J. Cell. Biochem. 105: 1211–1218, 2008.

MT1-MMP releases latent TGF-β1 from endothelial cell extracellular matrix via proteolytic processing of LTBP-1

Targeting of transforming growth factor beta (TGF-beta) to the extracellular matrix (ECM) by latent TGF-beta binding proteins (LTBPs) regulates the availability of TGF-beta for interactions with endothelial cells during their quiescence and activation. However, the mechanisms which release TGF-beta complexes from the ECM need elucidation. We find here that morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) resulted in membrane-type 1 matrix metalloproteinase (MT1-MMP) -mediated solubilization of latent TGF-beta complexes from the ECM by proteolytic processing of LTBP-1. These processes required the activities of PKC and ERK1/2 signaling pathways and were coupled with markedly increased MT1-MMP expression. The functional role of MT1-MMP in LTBP-1 release was demonstrated by gene silencing using lentiviral short-hairpin RNA as well as by the inhibition with tissue inhibitors of metalloproteinases, TIMP-2 and TIMP-3. Negligible effects of TIMP-1 and uPA/plasmin system inhibitors indicated that secreted MMPs or uPA/plasmin system did not contribute to the release of LTBP-1. Current results identify MT1-MMP-mediated proteolytic processing of ECM-bound LTBP-1 as a mechanism to release latent TGF-beta from the subendothelial matrix.

Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor

Notch signaling plays a central role in cell-fate determination, and its role in lateral inhibition in angiogenic sprouting is well established. However, the role of Notch signaling in lymphangiogenesis, the growth of lymphatic vessels, is poorly understood. Here we demonstrate Notch pathway activity in lymphatic endothelial cells (LECs), as well as induction of delta-like ligand 4 (Dll4) and Notch target genes on stimulation with VEGF or VEGF-C. Suppression of Notch signaling by a soluble form of Dll4 (Dll4-Fc) synergized with VEGF in inducing LEC sprouting in 3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears promoted lymphangiogenesis, which was augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal VEGFR-2 Ab as well as soluble VEGF and VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the tip cell position over nontransduced cells in 3D sprouting assays, suggesting an analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These results indicate that the Notch pathway controls lymphatic endothelial quiescence, and explain why LECs are poorly responsive to VEGF compared with VEGF-C. Understanding the role of the Notch pathway in lymphangiogenesis provides further insight for the therapeutic manipulation of the lymphatic vessels.

KSHV-Initiated Notch Activation Leads to Membrane-Type-1 Matrix Metalloproteinase-Dependent Lymphatic Endothelial-to-Mesenchymal Transition

Kaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread.

EphA2 cleavage by MT1-MMP triggers single cancer cell invasion via homotypic cell repulsion

Metalloproteinase-mediated cleavage of EphA2 induces breast tumor cells to shift from collective invasion to single-cell invasion.

Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFβ1 in diabetic eyes undergoing vitrectomy.

Purpose:  Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia‐induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase‐2 and matrix metalloproteinase‐9 (MMP‐2 and MMP‐9), angiopoietin‐1 and angiopoietin‐2 (Ang‐1 and Ang‐2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor‐β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker.