Cathleen Conner

Vascular endothelial groth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: Conversion of prothrombin to thrombin results in progelatininase a activation and cell proliferation

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF‐pre‐treated cells with prothrombin and factors X, Va, and VIIa at 37°C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti‐thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP‐1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum‐free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP‐2 abrogated thrombin‐induced progelatinase A activation, it had no significant effect on thrombin‐induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF‐induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane. Int. J. Cancer 75:780–786, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) Binds to the Catalytic Domain of the Cell Surface Receptor, Membrane Type 1-Matrix Metalloproteinase 1 (MT1-MMP)

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2·MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.

Thrombin Induces the Activation of Progelatinase A in Vascular Endothelial Cells PHYSIOLOGIC REGULATION OF ANGIOGENESIS

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombins multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of α-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metalloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Rapid trafficking of membrane type 1-matrix metalloproteinase to the cell surface regulates progelatinase a activation.

Pericellular matrix degradation during cancer invasion and inflammation is dependent on activation of progelatinase A by membrane type 1-matrix metalloproteinase (MT1-MMP); a stoichiometric concentration of tissue inhibitor of metalloproteinase-2 (TIMP-2) is required. Activation of progelatinase A has generally been considered to be a slow process occurring as a result of enhanced expression of MT1-MMP. We herein report that ConA treatment of HT1080 fibrosarcoma cells is followed by MT1-MMP–induced activation of progelatinase A on the cell surface within 1 hour. Cell surface biotinylation, immunohistochemistry, and 125I-labeled TIMP-2 binding to cell surface MT1-MMP were used to characterize the appearance and function of MT1-MMP on the plasma membrane. Treatment of HT1080 cells with ConA resulted in increased specific binding of 125I-labeled TIMP-2 to cell surface receptors within 5 minutes. TIMP-2 binds almost exclusively to activated MT1-MMP on the surface of HT1080 cells. MT1-MMP function at the cell surface was also accelerated by treatment of cells with cytochalasin D, an inhibitor of actin filaments, PMA, a stimulator of protein kinase C, and bafilomycin A1, an inhibitor of lysosome/endosome function. A functional pool of intracellular MT1-MMP available for trafficking to the cell surface was demonstrated by repetitive ConA stimulation. ConA-induced expression of MT1-MMP mRNA (Northern blot analysis) in HT1080 cells was a delayed event (>6 hours). These data suggest that presynthesized MT1-MMP is sorted to a transient storage compartment (trans-Golgi network/endosomes), where it is available for rapid trafficking to the plasma membrane and cell surface proteolytic activity.

Regulation of gelatinases in human airway smooth muscle cells: mechanism of progelatinase A activation.

Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-α and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-α releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-alpha releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.

Comparison of techniques for measurement of gelatinases/type IV collagenases: enzyme-linked immunoassays versus substrate degradation assays

Radiolabeled substrate degradation assays and gelatin zymography are routinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent assays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELISA to standard substrate degradation assays for measurement of MMP-2 and MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as partial purification procedures for MMP-2 and MMP-9. The ELISA data for MMP-2 and MMP-9 are linear on a log:log regression curve over a wide range of MMP concentrations and are specific for the designated gelatinase, with no overlap detected with related metalloproteinases. The minimum detectable concentrations of MMP-2 and MMP-9 were approximately 0.5 ng/ml and 0.2 ng/ml, respectively, in the ELISA as compared to 4 ng/ml and 3 ng/ml, respectively, in gelatin zymography. The [3H]gelatin degradation assay required a combination of >50 ng/ml of MMP-2 and MMP-9 for detection. Although gelatin zymography was less sensitive than ELISA (primarily due to the smaller sample volume employed) and was more difficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.

The 9‐kDa N‐Terminal Propeptide Domain of MT1‐MMP Is Required for the Activation of Progelatinase A

MT1-MMP has a paired basic amino acid cleaving enzyme recognition motif (RRKR) sandwiched between the propeptide and catalytic domains of the latent molecule. 1 Our previous report demonstrated that furin cleaves secreted MT1-MMP lacking the transmembrane domain extracellularly at the RRKR consensus sequence but not intracellularly, and further indicated that furin is not required for the function of MT1-MMP in terms of progelatinase A activation. 2 Based on this finding, we hypothesized that a component of or the entire N-terminal propeptide domain of MT1MMP participates in the process of MT1-MMP–induced progelatinase A activation. In the current study we have examined structure-functional relationships within the propeptide domain of MT1-MMP. Employing recombinant DNA technique, we have constructed an entire N-terminal propeptide domain–deleted MT1-MMP (MT ∆ pro), which theoretically represents an activated form of MT1-MMP. By Western blotting, expressed mutant protein migrated predictably based on the truncated length of the propeptide domain; MT ∆ pro appeared as a protein band of 53 kDa, while MT1-MMP was detected as a 63-kDa protein. In contrast to the activation of progelatinase A induced by MT1-MMP (monitored by gelatin zymogram), transfection of COS-1 cells with the MT ∆ pro failed to result in progelatinase A activation. These data are consistent with our hypothesis that conformational effects induced by the plasma membrane provide functional activity to membrane-bound MT1-MMP without cleavage of the molecule. 2 By transfection of mutant MT1-MMP cDNAs into COS-1 cells, we have demonstrated that deletion of the entire N-terminal propeptide sequence of MT1-MMP resulted in loss of 125 I-TIMP-2 binding activity. This defect was not due to failure of insertion of MT1-MMP protein into COS-1 plasma membranes as demonstrated by surface biotinylation. We then examined whether the prodomain of other secretory MMPs could function in this capacity. A substituted mutation in the prodomain of MT1-MMP was constructed. Since the prodomain of collagenase-3 (which classically is responsible