HIV-1 glycoprotein 120 induces the MMP-9 cytopathogenic factor production that is abolished by inhibition of the p38 mitogen-activated protein kinase signaling pathway.

It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4(-)/CXCR4(+)/CCR5(+) T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 alpha (SDF-1alpha) (CXCL12) and macrophage inflammatory protein 1 beta (MIP-1beta) (CCL4) of their respective coreceptors. Thus, both p38 and SAPK/Jun N-terminal kinase mitogen-activated protein kinases (MAPKs) are activated on stimulation of these cells with either T- or M-tropic gp120, as well as with SDF-1alpha or MIP-1beta. In contrast, extracellular signal-related kinase 1 and 2 MAPKs are only activated by MIP-1beta but not by M-tropic gp120. Importantly, T- and M-tropic gp120 are able to induce the secretion of matrix metalloproteinase 9 (MMP-9), an extracellular metalloproteinase present in cerebrospinal fluid of patients with HIV-1 by T cells or glioma cells. Specific inhibition of MAPK p38 activation resulted in a complete abrogation of the induction of the MMP-9 pathogenic factor expression by gp120 or chemokines in both cell types. Because neurodegenerative features in acquired immune deficiency syndrome dementia may involve demyelinization by MMP-9, the specific targeting of p38 could provide a novel means to control HIV-induced cytopathogenic effects and cell homing to viral replication sites. (Blood. 2001;98:541-547)

In vitro expression of MMP-2 and MMP-9 in glioma cells following exposure to inflammatory mediators.

Progression of glioma is associated with local degenerative processes which are attributed to the activity of gelatinases. As glioma cells are candidate for secretion of these enzymes, we have studied in vitro the potential of cytokines (interleukin-1alpha (IL-1), tumor necrosis factor-alpha (TNFalpha) and transforming growth factor-beta (TGFbeta2)) to regulate the expression of gelatinase A and B (Gels A and B, respectively) in two glioma cells of human (A172) and rat origin (C6). We showed that IL-1 and TNFalpha both induced gene expression and protein secretion of Gel B in both cell lines, as revealed by RT-PCR and gelatin zymography, respectively. In C6 cells, TNFalpha had no effect on Gel A constitutive expression while IL-1 increased its production, but only at high doses. We have also demonstrated that TGFbeta2 inhibited both IL-1- or TNFalpha-induced gene expression and Gel B production in a dose-dependent manner but had no effect on Gel A secretion. The effect of TGFbeta2 on Gel B secretion was reversed by phorbol myristate acetate (PMA). Taken together, these data suggest that IL-1, TNFalpha and TGFbeta2 tightly regulate Gel B secretion in glioma cells, an enzyme which is believed to play an important role in the local invasion of brain tissue by tumor cells.

Multidrug-resistance drug-binding peptides generated by using a phage display library.

A phage display library of random decapeptides was used to generate peptide ligands that can bind multidrug-resistance (MDR) drugs mimicking, in this respect, the drug-binding activity of P-glycoprotein. Seven peptide sequences were identified that specifically bound doxorubicin. Five of these sequences expressed the core consensus motif WXXW. The displacement assay showed that the phages expressing these peptides bound MDR type drugs (vinblastine, doxorubicin, verapamil, and genistein) with the same selectivity as P-glycoprotein and did not interact with non-MDR type drugs, such as arabinosylcytosine (Ara-C) and melphalan. One of the selected peptides that showed a highest capacity for the binding (VCDWWGWGIC) was synthesized and displayed competition with the phage for doxorubicin binding. The structure modeling suggested that all the selected sequences contained a hydrophobic envelope in which MDR drugs could be docked with substantial energy minimization. Western blot analysis showed that monospecific antibody obtained against the phage expressing VCDWWGWGIC peptide could specifically recognize P-glycoprotein in the membrane fraction of MDR phenotype MCF-7ADR cells. The MDR drug-binding sequences generated during this work could provide an important tool for design and screening of new chemotherapeutic agents.

Gelatinase B (MMP-9) production and expression by stromal cells in the normal and adult thymus and experimental thymic lymphoma.

Correlative and functional evidence support a crucial role for metalloproteinase (MMP) activity in tumor progression. Dysregulation of MMP production at local tumor sites is thought to participate in the remodeling of the local stromal tissue necessary for tumor growth. The extent of damages in local tissues is often reflected by the high concentration of MMP released in the bloodstream of cancer patients. The integrity of the thymic architecture plays a crucial role in the development of mature T cells, but it is compromised by extensive remodeling occurring during the development of thymic lymphomas. In the present work, we have used an experimental thymic lymphoma model to investigate the regulation of MMP-9 (gelatinase B) production in animals bearing large thymic lymphomas. We show a 3-fold increase in serum gelatinase B (Gel B) levels in animals bearing thymic lymphoma compared with those found in normal animals and a correlation between these levels and the size of the tumor. Although Gel B was found within the thymic tumor, lymphoma cells did not express it in vivo, indicating that Gel B expression was associated with thymic stromal cells rather than lymphoma cells. This was corroborated by evidence that lymphoma cells have the capacity to stimulate Gel B gene expression in stromal cells. Our results suggest that lymphoma cells can exert a significant control over Gel B expression by local stromal cells, thereby inducing the extensive remodeling necessary for tumor growth.

Flow cytometric analysis of gelatinase B (MMP-9) activity using immobilized fluorescent substrate on microspheres.

Gelatinase B is a member of the metalloproteinase family of enzymes that degrade the extracellular matrix under normal and pathological conditions, including autoimmune diseases and tumor cell dissemination. In the present work, we describe a simple and reliable method that allows qualitative and quantitative measurements of a specific enzymatic reaction (mediated here by gelatinase B) by flow cytometry using fluorochrome-labeled substrate coated on polystyrene microspheres. Using this approach, proteolytic degradation can be monitored by the decrease of the fluorescence emitted by the microspheres following incubation with the enzyme. In most of the experiments, the signal-to-noise ratio between autofluorescent microspheres and those coated with the fluorescein isothiocyanate (FITC)-labeled substrate was near 500. This ratio allows one to measure accurately the enzymatic activity in the presence of chemical or biological inhibitors. FITC labeling and passive adsorption of substrates on the solid support did not cause significant conformational changes or steric hindrance that would interfere with the proteolytic activity of gelatinase B. This method constitutes a powerful tool for the measurement, on a routine basis, of the net activity resulting from the balance between the gelatinase B activity and the presence of natural inhibitors and for the identification of new metalloproteinase inhibitors that could suppress the excessive proteolytic activity that characterizes many disease processes.