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EXPRESSION AND ROLE OF CCN3(NOV) DURING CHONDROGENESIS

The expression of ccn3(nov) is detected at sites of chondrogenesis during chicken development (Chevalier et al. Manuscript in preparation). Inasmuch as stimulatory effects of CCN1(CYR61) protein on chondrogenesis had been established in micromass cell cultures (Wong M, Kireeva M, Kolesnikova T, Lau L. Cyr61, Product of a Growth Factor-Inducible Immediate-Early Gene, Regulates Chondrogenesis in Mouse Limb Bud Mesenchymal Cells. Developmental Biol 1997; 192:492-508), we have undertaken a study aimed at assessing the potential role of nov in chondrogenesis and osteogenesis.

Expression of ccn3(nov) during wing and leg development in chicken was studied by Northern blotting (Barbot W. DEA Biologie Cellulaire et Moléculaire. Université Paris VI. 1997). The expression of ccn3(nov) was found to increase significantly between day 8 and 10 and progressively decreased until E 14 to reach basal level at E 15. Sustained expression of ccn3(nov) suggested that it might be needed for late events. Immunocytochemistry established that proliferating chondrocytes and the perichondrium stain positive for nov at E 6 (a), whereas hypertrophic chondrocytes of the central diaphyseal region were positive and at at E11-E12 with a labeling of the perichondrium (b), Only a a few hypertrophic chondrocytes in the developing wing were found positive for CCN3(NOV) at E14. Flatten chondrocytes were not positive for nov expression at any stage of development (c).

In micromass cultures ccn3(nov) expression was dependent upon the productive differentiation of mesenchyme cells (Barbot W. DEA Biologie Cellulaire et Moleculaire. Université Paris VI. 1997) . The ccn3(nov) expression was not detected when the cells were seeded, increased progressively for two days and decreased after three days. Under similar condiutions, the expresion of collagen II, a marker for determination of progenitor cells to undergo chondroblastic differentiation, increases from day 1 onwards.

These observations and the results of immunochemical studies performed on sections, suggested that NOV and CTGF were required at late stages of the chondrogenesis/osteogenesis differentiation process while CYR61 was required at early stages. Whether the differential expression of these three CCN genes in the skeletal system is temporally related remains to be established.

It is worth noting that in addition ot nephroblastoma, MAV (myeloblastosis associated virus) is also able to induce osteopetrosis in chicken (Perbal B. Pathogenic potential of myeloblastosis associated viruses. Infectious Agents & Disease, 1995 ; 4 : 212-227) , an abnormal proliferation of osteoblasts leading to severe bone deseases. It is therefore tempting to hypothesize that induction of osteopetrosis by MAV could also result from alteration of ccn3(nov) expression in bone precursor cells.

 

For a discussion of these results and general properties of CCN3(NOV) see the review by Perbal B.

NOV(nephroblastoma overexpressed) and the CCN family of genes : structural and functional issues (2001) Mol. Pathol. 54 57-79.

 

 

EXPRESSION OF NOVH IN 10 WEEK-OLD NORMAL HUMAN EMBRYONIC KIDNEY

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

A DEVELOPMENTAL STUDY OF NOVH GENE EXPRESSION IN HUMAN CENTRAL NERVOUS SYSTEM

Bing yin Su, Wenqin, Cai, Cheng gang Zhang, Hui ci Su , and Bernard Perbal

ABSTRACT

The expression pattern of the human nephroblastoma overexpressed (novH) gene in fetal human central nervous system was examined by in situ hybridization using digoxigenin-labeled novH-specific riboprobes. In spinal cord, the nov-expressing neurons were first detected in the ventral region of spinal cord at G16W and in the dorsal region at G38W. In the medulla, nov-expressing neurons were detected in principal nucleus of inferior olive, hypoglossal nucleus and dorsal motor nucleus of vagus at G16W. Nov-positive neurons were detected at G28W in nucleus of the spinal tract of the trigeminal and cuneate nucleus and at G38W., in abducens nucleus of pons, red nucleus and substantia nigra of midbrain, ventral posterolateral and mediodorsal thalamic nucleus. A strong labeling was also detected in the striatum of cerebrum and cerebral cortex of the parietal lobe. These data established that novH is mainly expresesed in somato motor neurons, at early developmental stages in lower central nervous system and at later stages in higher central nervous system, suggesting that nov may play an important role in neuronal differentiation.

 

DISCUSSION

The results that we have reported indicated that during human CNS development, high levels of novH transcripts were detected along the somato motor axis, first in the lower part of CNS (antrior horns of spinal cord, inferior olive of medulla and dorsal motor nucleus of vagus), and at later developmental stages in higher parts of CNS (pones, midbrain, thalamus and cerebral cortex).

The increasing levels of novH expression observed during the early stages of CNS development, suggested that nov might play an important role in the differenciation and development of the somato-motor neurons in CNS. Inasmuch as the expression of most proto-oncogenes was reported to gradually decrease during CNS development, it is tempting to speculate that the antiproliferative properties of novH are involved in CNS development.

Recent results have established that novH expression is associated with polarized differentiation during chondrogenesis (Laurent et al. manuscript in preparation), and muscular differentiation (Chérel et al. manuscript in preparation, Martinerie et al. manuscript in preparation (10)). Inasmuch as high levels of NOVH protein were found to accumulate along axons in kidney samples, we speculate that NOVH is requested to reach or maintain terminal differentiation.

Primary sequence analysis of the NOV protein (1, 15) revealed that it is composed of several structural modules: i) an IGFBP module, homologous to the to the insulin-like growth factor (IGF)-binding domain present in IGF-binding proteins, ii) a VXC likely to be involved in oligomerisation of von Willebrand factor, iii) a TSP1 module thought to be involved in thrombospondin interaction with extra-cellular matrix proteins, and iv) a carboxy proximal motif proposed to represent a dimerization domain (15). We have previously reported (1) that these motifs are also represented in two groups of positive regulators of cell growth encoded by the immediate early genes ctgf/fisp12 and cyr61/cef10 in chicken, mouse, and human (16-20). Although their functionality remains to be established, we have proposed that the conservation of these domains might be related to the biological function of this emerging family of proteins (3, 10).

The presence of an IGFBP-like motif at the carboxy terminus of NOVH strongly suggests that this protein is acting in the IGF signaling pathway. Along this view, it will be interesting to study the potential interactions of NOVH with IGFs and IGFBPs the role of which in development of CNS has been widely documented (see recent reviews 21,22). The extensive homology of NOVH, CRY61H, CTGF and IGFBP3 at their 3' terminus also suggests that these proteins share common features, among which regulation of CNS neuronal differentiation may be one aspect.

Our recent finding that NOVH is a secreted glycosylated protein (10) which can be associated with the extracellular matrix (Laurent et al. manuscript in preparation) favor the idea that NOVH may play a role in cell-cell communication and be part of the ordered array of events needed to achieve normal NCS differentiation and development. Interestingly, we have previously reported that novH and cyr61H expression was altered in human glioblastomas and that the highest levels of novH were detected in the most differentiated poorly tumorigenic tumor cells (23). Experiments are presently in progress to determine whether the ectopic expression of novH may abrogate the tumorigenicity of human gliobastomas cell lines and interfere with the normal development of CNS in animal models.

 

REFERENCES

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2 - PERBAL, B. (1995). Pathogenic potential of myeloblastosis associated viruses. Infectious Agents & Disease, 4: 212-227

3 - PERBAL, B.1995. Contribution of MAV-1-induced nephroblastoma to the study of genes involved in human Wilms' tumor development. Critical Reviews in Oncogenesis, 5: 589-613.

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21 - SARA V. R. 1992 The role of insulin-like growth factors in the nervous system In: SCHOFIELD P.S. ed. The insulin-like growth factors Oxford Medical Publications, 259-279.

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