Expression and characterization of human tissue inhibitor of metalloproteinases-1 in a baculovirus-insect cell system.

The baculovirus expression system was used to overexpress human tissue inhibitor of metalloproteinases-1 (TIMP-1). Approximately 5 mg of recombinant TIMP-1 was produced per 10(9) Sf9 insect cells infected with the recombinant baculovirus. The optimum time point for the production of biologically active rTIMP-1 was 20 hours postinfection. TIMP-1 activity was demonstrated by a soluble collagenase inhibition assay and reverse zymography. The baculovirus system has the advantage over previously described methods for generating human rTIMP-1 in that it generates large amounts of a fully glycosylated and active protein.

Studies on the effects of laminin, E-8 fragment of laminin and synthetic laminin peptides PA22-2 and YIGSR on matrix metalloproteinases and tissue inhibitor of metalloproteinase expression.

BACKGROUND: Based on a previous observation of laminin-mediated increase in type IV collagenolytic activity of human melanoma (A-2058) and fibrosarcoma (HT-1080) cell lines (Turpeenniemi-Hujanen T, et al. Laminin increases the release of type IV collagenase from malignant cells. J Biol Chem 1986;261:1883-1889), the goal of this study was to identify the proteinases involved. EXPERIMENTAL DESIGN: A soluble type IV collagenase assay and substrate gel electrophoresis were used to assess the effect of laminin and laminin peptides on type IV collagenolytic activity. RESULTS: The laminin-mediated increase in type IV collagenolytic activity was not due to augmented expression or induction of three known type IV collagenolytic matrix metalloproteinases (MMP), i.e., 72-kilodalton gelatinase/type IV collagenase (MMP-2), stromelysin (MMP-3), and 92 kilodalton gelatinase/type IV collagenase (MMP-9). Furthermore, laminin did not modulate the expression of the tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. The E-8 fragment of laminin and the YIGSR laminin peptide had no effect on type IV collagenolytic or MMP/TIMP activities. However, the IKVAV containing PA22-2 laminin peptide selectively stimulated type IV collagenolytic activity of the A-2058 melanoma cell line, although it did not modulate MMP/TIMP activity. Laminin from three different sources of the Engelbreth-Holm-Swarm tumor was found to contain type IV collagenolytic activity. When laminin was added to harvested culture supernatants of the A-2058 and HT-1080 cell lines, the increase in type IV collagenolytic activity was comparable with that observed in supernatants from cells incubated with laminin for 48 hours. Analysis of the laminin preparations revealed five MMP forms ranging in molecular weight from approximately 58 to 105 kilodalton, which may represent latent and active forms of MMP-2 and MMP-9. CONCLUSIONS: These findings suggest that proteinases present in the Engelbreth-Holm-Swarm laminin may account for most, if not all, of the observed laminin-mediated increase in type IV collagenolytic activity. However, the PA22-2-mediated increase in type IV collagenolytic activity of the A-2058 melanoma line remains to be elucidated.

Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase/type IV collagenase in preparations of laminin and Matrigel.

EDTA inhibitable type IV collagenolytic activity copurified with laminin preparations from the Engelbreth-Holm-Swarm (EHS) tumor. Several gelatinolytic and type IV collagenolytic matrix metalloproteinase (MMP) species were visualized in EHS laminin from three different sources by gelatin and type IV collagen substrate gel electrophoresis. Incubation with 4-aminophenylmercuric acetate and trypsin suggested that laminin contained both active and latent MMPs. EHS-derived reconstituted basement membrane, Matrigel, was found to possess an MMP profile identical to that of laminin. The presence of 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinases/type IV collagenases was demonstrated in laminin and Matrigel preparations by Western blot analysis. A rough quantitation of MMP-2 and MMP-9 in 30 micrograms of laminin and 100 micrograms of Matrigel was between 0.3 and 0.6 ng. The presence of these contaminants must be considered in experiments addressing the effects of EHS laminin or Matrigel on cell behavior and, in particular, stimulation of cellular proteolytic activity.

Immunomagnetic separation as a final purification step of liver endothelial cells.

We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.

Immunomagnetic separation as a final purification step of liver endothelial cells.

We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.

Effect of phorbol ester and cytokines on matrix metalloproteinase and tissue inhibitor of metalloproteinase expression in tumor and normal cell lines.

The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) on matrix metalloproteinases (MMP) and metalloproteinase inhibitors was studied in a variety of human cell lines. Expression of the mammalian collagenase (MMP-1), 72-kD gelatinase/type IV collagenase (MMP-2), stromelysin (MMP-3), 92-kD gelatinase/type IV collagenase (MMP-9), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was assessed by zymography and Northern blot analysis. MMP-2 and TIMP-2 activities were refractory to TPA, IL-1 and TNF-alpha treatment in most of the cell lines. In contrast, MMP-3, MMP-9 and TIMP-1 activities were markedly stimulated by TPA in most of the tumor cell lines and human umbilical vein endothelial cells (HUVEC), whereas the fibroblast lines were minimally stimulated or unresponsive to TPA. The MMP-3, MMP-9 and TIMP-1 stimulation in response to IL-1 and TNF-alpha treatment was detected in some of the tumor cell lines and HUVEC. The increase in activity was less marked than in TPA. A breast carcinoma cell line, MDA-MB-231, which did not express MMP-2, had high expression of MMP-3 and MMP-9 which were unaffected by TPA and cytokine treatment. Northern blot analysis of MMP and TIMP mRNA expression reflected the zymogram findings for most of the cell lines. TPA-mediated stimulation of MMP-1 was similar to that of MMP-3 and MMP-9. Exceptions were the fibroblast cell lines which showed either a much more marked mRNA response of MMP-9 to TPA than observed at protein level, or a high constitutive MMP-9 mRNA when MMP-9 activity was not detectable by zymography. TPA-mediated stimulation of MMP-9 and TIMP-1 activity was blocked by staurosporine, an inhibitor of protein kinase C (PKC). A non-PKC-activating phorbol ester, 4 alpha-phorbol-12,13-didecanoate, did not stimulate MMP-9 and TIMP-1 activity. TPA treatment caused the increased expression of c-fos containing AP-1-specific binding activity in selected tumor cell lines. This activity was maximal at 6 h. An association was observed between AP-1 binding activity and increased expression of MMP-1, MMP-3 and MMP-9, which possess TPA-responsive elements (TRE). TPA-sensitive MMPs and TIMP-1 were variably stimulated by biologically relevant cytokines, such as IL-1 and TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)