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EXPRESSION OF NOVH IN NORMAL KIDNEY AND WILMS TUMORS (WT)

Full size copies of these figures are available upon request

novH in heterotypic muscular differentiation

novH in endothelium, neurons, axons

novH in human embryonic kidney

novH in nephroblastomatosis and WT

novH in pseudoglomeruli and WT

For more complete deszcription and discussion see text in:

G. CHEVALIER, H. YEGER, C. MARTINERIE , M. LAURENT, J. ALAMI, S. KOCIALKOWSKI, P.N. SCHOFIELD, et B. PERBAL. (1997). novH: Differential expression in developing kidney and a marker of heterotypic differentiation in Wilms' tumor. Am. J. Pathol. 152, 1563-1575

THE CARBOXY-TERMINAL (CT) DOMAIN OF NOVH IS SUFFICIENT TO PROMOTE INTERACTION WITH FIBULIN 1C.

Bernard Perbal * , Cécile Martinerie, Richard Sainson , Michel Werner , Bin He and Bernard Roizman

Control of cell proliferation and differentiation requires the interplay of many signaling factors among which extracellular matrix proteins play a central role. The existence of a new class of cell growth regulators has been uncovered over the past few years. This emerging family of proteins, often refered as CCN (1), comprise both positive and negative regulators of cell growth (cyr61/cef10 ; ctgf/ fisp12 ; nov ; elm1 and rCop-1) sharing a common multimodular organization. The chicken CEF10 and murine CYR61 genes were first identified as immediate early genes induced by pp60v-src oncogene and serum growth factors respectively (2, 3). The CYR61 protein has been shown to promote cell adhesion, migration and proliferation probably through the potentialisation of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) activities (4). The expression of human cyr61 is associated to normal differentiation of mesenchyme cells into chondrocytes (5) and is differentialy regulated in brain-derived tumor cell lines (6). The human ctgf was also identified as an immediate early gene encoding a connective tissue growth factor (CTGF) (7) showing mitogenic activity for human umbilac vein endothelial cells (HUVECs and fibroblasts in culture (8). On the other hand, the murine Elm1 gene (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells exhibits cell growth inhibitory properties and suppresses the tumorigenic potential of mouse melanoma cells (9). Similarly, the expression of the murine rCop gene is completely abolished following transformation and retroviral-driven expression of rCop-1 had a dramatic cytotoxic effect on transformed cells, but not on untransformed counterparts (10).

The nov gene has been initialy characterized as an integration site for the myeloblastosis associated virus MAV (11) which induces kidney tumors representing a unique model of the Wilms tumor (12). In the human nephroblastomas the levels of WT1 and NOVH are inversely correlated (13) probably as a result of the negative regulation of nov expression by WT1 (14). In other types of human and animal tumors, the expression of the nov gene was found to be altered either positively or negatively (15, 16) Considering that the expression of nov was associated with cell quiescence (17) and that retroviral-driven expression of a full length nov was inhibitory fo cell growth (11) the elevated expression of nov in several types of tumors, including Wilms', appeared paradoxical until it was established that novH encodes a secreed 48 KDa protein whose expression is tightly associated to heterotypic blastemal differentiation in Wilms tumors (18) and to normal differentiation of nervous central system (19), kidney (18), and chondrocytes (Kocialkowsky et al. manuscript in preparation, M. Laurent et al. manuscript in preparation). It therefore appears that under normal circumtances, the expression of NOV is associated or required for some cells to undergo differentiation and its accumulation in differentiated structures such as glomeruli and axons of nervous cells (18) argues for a role of NOV also being important in the maintenance of their differentiated state.

The presence of an insulin-like growth factor (IGF) binding protein (IGFBP)-like motif at the carboxy terminus of NOVH and the extensive homology of NOV, CTGF, CYR61, ELM1, RCOP-1 and IGFBP3 at their 5' terminus raised the posibility that NOV and other related proteins might be acting in the IGF signaling pathway. Along this line, the deletion of its amino-terminal sequences of nov was reported to confer oncogenic properties on the resulting NOV-truncated protein (11). However, recent results indicated that IGFs were not binding to recombinant NOV protein secreted by baculovirus-infected SF9 cells (18) and suggested that NOV and IGFBPs might share common signaling activities through IGF-independent pathways as recently reviewed for IGFBP3 (20, 21). Two motifs sharing identity with Von Willebrand type C domain likely responsible for oligomerisation and with thrombospondin type 1 repeat responsible for interaction with extracellular matrix proteins have been recognized in the NOV protein and other members of the CCN family. Although the biological activity of these motifs remains to be established, their conservation in all members of the CCN family argues for two their functional or strucutral importance. The carboxy-terminus module (CT) of NOV which was proposed to represent a dimerization domain contains a cystine-knot motif present in several growth factors such as nerve growth factor (NGF), transforming growth factor b2 (TGFb2) and platelet derived growht factor BB (PBGFBB). This motif is not represented in the rCop &endash;1 protein (10 ).

The multimodular structure of the NOV and other CCN proteins raises interesting questions as to the participation of each individual module on confering the biological properties to the full length proteins. Either the biochemical functions of the IGFBP, VWC, TSP and CT modules contained in these proteins are indeed conserved and sum up in the full length protein, or the presence of each module confers on the whole protein specific biological function(s) which may substitue or add upon those of individual modules.

As a first step in our study of the NOVH biological function(s) and the putative role of the different modules that it contains, we have used a two hybrid system strtegy to search for factors interacting with NOVH. To avoid selecting preys that would possibly interact only with isolated modules, we have used in the initial screening the full length NOV protein as a bait. We have now report that the secreted NOVH protein physically interacts with fibulin 1C, an extracellular matrix protein interacting with several signaling factors. We also show that a natural amino-truncated isoform of the NOVH protein exhibit a high affinity for fibulin 1C and that the C-terminal domain of NOVH is critical for this interaction.

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