Ellen E. Rollo

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.

Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages.

Osteopontin (OPN), a secreted acidic phosphoglyeoprotein found in many tissues and body fluids, is produced in increased amounts in response to certain infections and after malignant transformation. In this study we examined the action of OPN on macrophage cytotoxicity and nitric oxide (NO) synthesis. A human OPN cDNA was cloned into the bacteriophage T7‐based vector, pET8C, and the encoded protein purified from an induced culture of Escherichia coli carrying the plasmid. Recombinant OPN inhibited NO production by macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide plus interferon‐γ. OPN also inhibited the cytolytic activity of the activated macrophages toward NO‐sensitive P815 mastocytoma cells, an action that was blocked by the NO synthase inhibitor, NG‐monomethyl‐l‐arginine. Inhibition of NO production correlated with an OPN‐dependent decrease in the abundance of inducible NO synthase mRNA. The shape of the dose‐response curve, with a maximal effect over a narrow range of OPN concentrations, suggested a complex interaction of OPN with cell surface receptors. Our data support the hypothesis that tumor‐cell‐derived OPN functions to protect the tumor cells from macrophage‐mediated destruction. J. Leukoc. Biol. 60: 397–404; 1996.

Osteopontin‐Induced Modifications of Cellular Functionsa

Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the alpha v beta 3 integrin. We have investigated the action on mammalian cells of recombinant OPN made both in E. coli and in human cells. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosphorylation status of several proteins including pp60src and components of focal adhesion complexes. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell peroxide levels, promote the survival of cells exposed to hypoxia, and inhibit the killing of tumor cells by activated macrophages.

Osteopontin (OPN) may facilitate metastasis by protecting cells from macrophage NO-mediated cytotoxicity: evidence from cell lines down-regulated for OPN expression by a targeted ribozyme

Osteopontin (OPN) is a GRGDS-containing phosphoglycoprotein that is capable of facilitating cell adhesion and modulating gene expression via integrin receptors. Three hammerhead ribozymes designed to target three different regions of OPN mRNA were shown to cleave the message catalyticallyin vitro. Plasmid vectors that had been engineered to express the ribozymes in mammalian cells were used to generate stably transfected T24 H-ras-transformed NIH3T3 cells that normally express OPN at high levels. Northern and Western blot analyses showed that OPN mRNA and protein expression were reduced in a subset of these anti-OPN ribozyme-expressing cell lines. Cells whose ability to produce OPN had been impaired exhibited greater sensitivity to the cytotoxic action of activated RAW264.7 macrophage-like cells; they were also less effective at suppressing macrophage NO production. In agreement with previous reports, they were also less tumorigenic and metastatic in an experimental metastasis assay. These results are consistent with the hypothesis that OPN serves as a defense against NO-mediated host cell cytotoxicity and thereby augments the metastatic phenotype.

Differential effects of osteopontin on the cytotoxic activity of macrophages from young and old mice

Osteopontin (OPN) is a secreted phosphoprotein found in body fluids (e.g. plasma, urine, milk) and in mineralized tissues. Its expression is increased in many transformed cells and in normal cells exposed to various cytokines. When stimulated with the inflammatory mediators lipopolysaccharide and interferon‐γ, mouse macrophages secrete nitric oxide (NO) as a cytotoxic agent effective against microbial invaders and tumour cells. This report documents (1) that thioglycollate‐elicited peritoneal macrophages, activated with the inflammatory mediators, produced less NO and exhibited reduced cytotoxicity towards target cells when they were obtained from old animals than when they were obtained from young animals; and (2) that OPN was able to inhibit both the induced NO synthesis and cytotoxicity, but more effectively in macrophages from the young animals than those from the old animals. This may be due to the observed higher level of OPN expression in macrophages from old animals.

The Cytokine Osteopontin Modulates the Severity of Rotavirus Diarrhea

ABSTRACT Osteopontin (OPN) is a sialated phosphoprotein found in tissues and secreted into body fluids. It is an integrin ligand with pleiotropic functions as an extracellular matrix protein in mineralized tissues and a cytokine that is active in cell signaling (A. B. Tuck, C. Hota, S. M. Wilson, and A. F. Chambers, Oncogene 22:1198-1205, 2003). To determine whether OPN may be important in mucosal defense against viral pathogens, we evaluated the OPN response to rotavirus infection and the extent of diarrhea manifested by infected opn null mutant (opn−/−) mice. Reverse transcription-PCR, Northern and Western blots, and immunohistochemical studies of the HT-29 intestinal epithelial cell line and murine intestine were used to evaluate OPN mRNA and product. Intestinal closed loops and diarrheal observations determined disease severity and duration. OPN mRNA levels increased after infection of HT-29 cells, peaking in 4 to 6 h. Infected cultures contained 925 μg of OPN/ml, while for controls the levels were below detection (50 μg/ml). Infection increased OPN mRNA levels in intestinal tissue between 2 and 24 h postinoculation and increased OPN protein in intestinal fluid. The cellular localization of OPN was supranuclear and apical, and responding cells were diffusely distributed on the villus surface. Three days after infection, closed intestinal loops from opn−/− mice contained more fluid than loops from controls, although secretion levels at the onset of illness were similar. Null mutant mice experienced more intense and prolonged diarrhea than controls. Rotavirus infection of intestinal epithelial cells and murine intestine caused marked increases in OPN mRNA levels and secreted OPN protein. OPN-deficient mice suffered prolonged disease.

Attenuation of oxidant-induced lung injury by the synthetic matrix metalloproteinase inhibitor BB-3103.

Acute, diffuse lung injury often complicates sepsis, gastric acid aspiration, extensive trauma, and other conditions. The lung endothelial and epithelial cells are the early targets of this injury leading to increased pulmonary vascular permeability and pulmonary edema. Clinically, this condition is characterized by catastrophic respiratory failure, known as the a dult r espiratory d istress s yndrome (ARDS). Despite advances in our understanding of the pathogenesis of this type of lung injury and in the management of patients with this disorder, the outcome remains grave. There is an urgent need for an effective treatment. Recently matrix metalloproteinases (MMPs), especially gelatinases, have been implicated in the pathogenesis of acute lung injury. Gelatinase A and B and their activated forms are increased in the bronchoalveolar lavage fluid (BAL) of both animal models of acute lung injury and patients with ARDS. 1–4 In this study we sought to investigate the protective effect of an MMP inhibitor in an experimental model of acute lung injury caused by oxygen free radicals. We examined the MMP inhibitor BB-3103, a soluble, low-molecular-weight broad spectrum inhibitor obtained from British Biotechnology Ltd., Oxford, England.

Neutrophil activator of matrix metalloproteinase-2 (NAM).

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.