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NOVH increases MMP3 expression and cell migration in glioblastoma cells via a PDGFR--dependent mechanism¹

M. LAURENT^*,2, C. MARTINERIE^*, H. THIBOUT^*, M. P. HOFFMAN, F. VERRECCHIA, Y. LE BOUC^*, A. MAUVIEL and H. K. KLEINMAN

^* INSERM: U 515, "Prolifération, Différenciation et Processus tumoraux," bâtiment Kourilsky, Hôpital Saint-Antoine, Paris, France;
INSERM: U 532, Hôpital Saint-Louis, Paris, France; and
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA

²Correspondence: NSERM U515, Prolifération, Différenciation et Processus tumoraux bâtiment Kourilsky, Hôpital Saint-Antoine, 184 rue du Fbg St-Antoine, 75012 Paris, France. E-mail: laurent{at}st-antoine.inserm.fr.

SPECIFIC AIMS

Nephroblastoma overexpressed (NOV) is a member of the connective tissue-growth factor, cysteine-rich 61, NOV gene (CCN) family, which regulates cellular processes including proliferation, adhesion, apoptosis, and migration. These proteins are involved in implantation, skeletal formation, embryonic development, wound healing, and various disease processes including fibrosis and cancer. However, the function and the mechanisms of action of NOV are not well defined in the central nervous system (CNS), a site of high NOV expression. We have investigated the effects of different levels of human NOV (NOVH) expression on the biology of glial cells and identified and characterized genes regulated by NOV.

PRINCIPAL FINDINGS

1. NOV promotes the adhesion and the migration of human glioblastoma cells
Three transfected glial cell lines N1, N2, and N3, which express different levels of NOVH and one antisense-transfected clone were selected. The expression of NOVH increased cell migration in a dose-dependent manner in the scratch assay and in the Boyden chamber assay. The migration of the NOVH-expressing cells was inhibited in the presence of 2.5 mM EDTA, and the metalloprotease inhibitor GM6001 indicating that the migration of the NOVH-expressing cells is protease-dependent (Fig. 1 ).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of NOVH on cell migration. A) Boyden chamber test. Cell migration from the three NOVH-expressing clones (N1, N2, N3) and from the control clone (N4) was performed overnight through a filter coated with fibronectin. Migrated cells were fixed, stained, and counted by microscopic examination. B) Cell migration from the high NOVH-expressing cells (N1) was also performed in the presence of 2.5 mM EDTA and 100 nM of the metalloprotease inhibitor GM6001 and was compared with untreated cells. The histogram represents the number of cells counted from 10 visual fields in three wells. The data are from a representative experiment repeated three times. The data were analyzed with the Mann-Whitney U test. *, Only P values less than 0.05 were considered to be statistically significant.

2. NOVH expression by glioblastoma cells increases the matrix metalloproteinase-3 (MMP3) and platelet-derived growth factor receptor- (PDGFR-) levels
To identify the molecular mechanisms by which NOVH was regulating cell activity, RNAs from NOVH-expressing and control cells were hybridized to filters containing 1176 known genes. MMP3 and the PDGFR-, which is known to regulate MMP3 gene expression, were up-regulated by NOVH. The differences in MMP3 and PDGFR- mRNA and protein expressions and correlated with the amount of NOVH produced. The high level of PDGFR- resulted in increased proliferation and migration in high NOVH-expressing cells when stimulated by PDGF-AA, which only binds PDGFR-. Parental glioblastoma cells treated for 72 h with recombinant NOVH protein up-regulated MMP3 expression. However, NOVH did not activate the MMP3 promoter in transiently transfected cells treated with recombinant NOVH protein or cotransfected with a cytomegalovirus–NOVH-expressing plasmid. These data indicate that MMP3 is not a direct target of NOV.

3. The endogenous expression of MMP3 is mediated by the increased expression of PDGFR- and potentiated by PDGF-BB
The increased expression of MMP3 was potentiated by serum and cell density, suggesting that regulation of MMP3 expression by NOVH is influenced by cofactors. To identify these factors, NOVH-expressing and control cells were treated with epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2), PDGF-BB, and transforming growth factor-ß1 (TGF-ß1). PDGF-BB was the only growth factor that potentiated the increase of secreted MMP3 by NOVH-expressing cells. In the presence of AG1295 which selectively inhibits PDGFR- autophosphorylation stimulated by PDGF-BB the amount of MMP3 secreted by the NOVH-expressing cells was significantly reduced (Fig. 2 ). MMP3 promoter activity was also significantly increased by PDGF-BB in cells expressing NOVH with no effect on control cells (Fig. 2) . In contrast, FGF2 stimulated MMP3 promoter activity in both cell lines. These data show that PDGF-BB plays a specific role in NOVH-expressing cells by increasing MMP3 promoter activity and protein secretion.

View larger version (49K): [in this window] [in a new window]   Figure 2. Effect of growth factors on the expression of MMP3 and MMP2 proteases. A, B) Cells from control (N4) and two NOVH-expressing clones (N1, N2) were treated for 24 h with (lane b) EGF (40 ng/ml), (lane c) FGF2 (10 ng/ml), (lane d) PDGF-BB (40 ng/ml), and (lane e) TGF-ß1 (4 ng/ml). Untreated cells (lane a) were used as control. Proteins from conditioned media were analyzed by Western blot A) for MMP3 B) for MMP2. C) Cells N4 N1 clones were treated for 24 h with the PDGFR--specific inhibitor tyrphostin AG1295 (10 µM) for 10 min with PDGF-BB (40 ng/ml). Proteins from conditioned media were immunoblotted with the MMP3 monoclonal antibody, and 40 µg of cell lysate was immunoblotted with the PDGFR--specific antibody. D) Cells from the control clone N4 and from the N1 clones were transfected with 0.5 µg MMP3 promoter Luc construct and treated for 24 h with PDGF-BB and FGF2. The mean luciferace activity ± SEM of a representative experiment performed in triplicates is presented.

CONCLUSION AND SIGNIFICANCE

The temporal and spatial expression of NOV suggests it has a role in the development of the CNS. We show that NOVH promotes glioblastoma cell migration and induces the expression of PDGFR- and MMP3, two genes important for brain development and brain tumor progression. The inhibition of the metalloprotease activity by EDTA and by a specific inhibitor blocks NOVH-induced cell migration, indicating that the mechanism of NOVH-induced cell migration is via increased MMP3 expression. The induction of MMP3 in the NOVH-transfected glial cells is partly mediated by overexpression of PDGFR- and is potentiated by the activation of the PDGF signaling pathway. Thus, NOVH triggers a cascade of events including the induction of PDGFR-, whose increased activation induces MMP3, which in turn, activates glial cell migration (Fig. 3 ).

View larger version (30K): [in this window] [in a new window]   Figure 3. NOV induces the expression of PDGFR- and MMP3 and promotes cell migration through the ECM.

The metalloprotease MMP3 degrades noncollagenous matrix components, inactivates serine protease inhibitors, and activates other prometalloproteases that degrade interstitial collagens. Thus, a cascade of extracellular matrix (ECM) degradation is created, which facilitates cell migration. In brain development and cell plasticity, ECM proteolysis is crucial, and MMPs and their inhibitors are involved in the maturation of neural cells and in neurite outgrowth.

During postnatal development of the rat cerebellum, the expression of MMP3 is restricted to the growing dentritic tree of Purkinje cells and to the cell bodies in the adult. Therefore, we can postulate that during brain development, NOV could also affect the MMP3-dependent migration of neuronal cells.

MMPs are invariably up-regulated in cancers and contribute to tumor invasion and metastasis. An induction of PDGFR- is likely an early event in glioma formation. However, NOVH-transfected cells were unable to form tumors, contrary to the parental and control cells and the antisense transfectants did form tumors (data not shown). These data suggest that the induction of MMP-3 and PDGFR-, both important in the formation and the development of brain tumors, is not sufficient for tumor growth. There may be regulation of other unidentified genes by NOVH, which would explain the absence of tumors.

Characterization of MMP3 and PDGFR- as targets of NOV highlights other functions in which NOV may be involved. Expression of PDGFR- is found in glial precursors in various regions of the developing CNS and PDGF signaling appears to play an important role in the differentiation of the O2-A glial progenitors that give rise to oligodendrocytes or type 2 astrocytes. NOV which regulates the expression of the PDGFR- may play a role in the development and functional properties of the brain and spinal cord, two sites of NOV and PDGFR- expression, and in the differentiation of the O2-A cell progenitors. NOV and connective tissue growth factor (CTGF), another CCN family member, are also coexpressed with vascular endothelial growth factor (VEGF) and MMP3 during the remodeling of the CNS and growth plate and in wound healing. The activation of MMP3 expression by NOV could degrade CTGF in the VEGF–CTGF complex and release the angiogenic activity of VEGF. Therefore, the antagonistic activities of NOV and CTGF could be a mechanism to regulate angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1023fje; doi: 10.1096/fj.02-1023fje

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