NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues

GENES THAT WERE ISOLATED ON THE BASIS OF THEIR DIFFERENTIAL EXPRESSION IN QUIESCENT AND SERUM STIMULATED CELLS

Under starvation conditions (incubation in low serum concentration), confluent cells stop growing and reach the G0 state of the cell cycle. G1 phase re-entry of the starved cells upon treatment with serum triggers the stimulation of immediate early genes, whose expression proceeds shortly after serum stimulation, without the need for de novo protein synthesis (the immediate early terminology was used by comparison with the ordered expression of viral genes upon infection of eukaryotic cells). Characterisation of these immediate genes provided important clues as to the type of genes responsible for the regulation of master switches involved in the control of cell growth.

This experimental approach, which had previously led to the discovery of a variety of signalling proteins and transcription factors, led to the isolation of the murine cyr61 and fisp12 genes.

Fibroblastic cells of different origins have been used to analyse gene expression during serum induced G0/G1 transition. Differential screening of a cDNA library prepared from serum stimulated mouse BALB/c3T3 fibroblasts led to the isolation of cyr61, which encodes a secreted 379 residue polypeptide of apparent molecular mass 42 kDa,⁵ whereas a similar strategy performed with mouse NIH3T3 cells led to the isolation of fisp12, which encodes a secreted 348 residue polypeptide of 37 kDa.⁶ The early expression of these genes in the cell cycle without the need of prior protein synthesis categorised them as immediate early genes.

Early studies established that immediate early genes were expressed transiently and were either required for the G0–G1 transition or for G1 progression to S phase. In the first group, c-fos expression is induced between 20 and 90 minutes and decreases to undetectable levels after two hours, whereas in the second group, c-myc expression is induced between 45 minutes and three hours and is detectable up to six hours after serum stimulation.

Both cyr61 and fisp12 showed the same rapid induction pattern, starting 30 minutes after serum stimulation and peaking between one and two hours after stimulation. In both cases, short half life (30 minutes for cyr61, 10–15 minutes for fisp12) RNA species were produced, and sustained expression was observed over several hours after the addition of serum to starved cells. As reported for other immediate early genes, the decrease in mRNA values requires protein synthesis.

Therefore, it appears that cyr61 and fisp12 represent a new class of genes that are induced in the sequential cellular response to mitogenic signals. The CYR61 and FISP12 proteins are likely to be required for a relatively long period during the progression through the G1 phase.

It is worth pointing out that a second burst of cyr61 RNA synthesis was seen between six and 10 hours after serum stimulation, suggesting that the expression of this protein might also be required at a later stage of cell cycle progression (fig 2 ▶).

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Figure 2

Kinetics of CCN gene expression upon stimulation of cell growth.

GENES THAT WERE IDENTIFIED ON THE BASIS OF THEIR INCREASED EXPRESSION IN CELLS TREATED BY TGFβ1

A considerable number of studies performed with transforming growth factor β (TGFβ) have established the key role of this signalling molecule in the regulation of cell growth and differentiation.

For many years, it has been proposed that the biological effects of TGFβ are mediated by a variety of downstream effectors. To identify genes in which transcription was affected by TGFβ1, a cDNA library was prepared from AKR-2B mouse embryo fibroblasts grown to 90% confluency and treated with cycloheximide before incubation for six hours in the presence of 10 ng/ml TGFβ1. Differential screening of this library with labelled cDNA probes obtained from untreated and TGFβ1 treated cells permitted the cloning of several genes whose expression was increased in TGFβ1 treated cells.⁷ Among them, two structurally related genes were designated βIG-M1 and βIG-M2. Nucleotide sequencing established that of βIG-M1 was identical to the previously described cyr61.

Induction of βIG-M1 in growth arrested cells, as opposed to the induction of CEF10 in cells stimulated to divide, provided the first evidence to suggest that this protein was acting in quite diverse biological processes.⁷

The sequencing of βIG-M2 revealed that it was closely related to βIG-M1 in structure. The βIG-M2 gene was later cloned as ctgf (see below).

GENES THAT WERE IDENTIFIED ON THE BASIS OF THEIR ALTERED EXPRESSION IN TUMOURS

Tumour cells are thought to be altered at the level of the regulatory signals controlling normal cell growth. They have provided an unequalled source of biological material in which to study the molecular events leading to uncontrolled and eventually invasive proliferation.

GENES THAT WERE IDENTIFIED ON THE BASIS OF THEIR DIFFERENTIAL EXPRESSION IN RSV TRANSFORMED FIBROBLASTS

Transformation of CEFs by pp60^v-src of Rous sarcoma virus (RSV) is known to alter, either positively or negatively, the transcription of a variety of regulatory and signalling proteins. Because the expression of pp60^v-src had been reported to block the differentiation of myogenic cells, it was proposed that genes whose expression is decreased by pp60^v-src transformation might act to maintain or induce the differentiated state of target cells.

The strategy used to characterise genes acting in the transduction pathways activated for transformation was usually based on the use of CEFs transformed by a mutant of RSV (tsNY72) expressing a thermosensitive pp60^v-src. Upon shifting to the permissive temperature (35°C), the constitutive tyrosine kinase activity of pp60^v-src induces the cascade of molecular events that lead to oncogenic transformation.

In the first set of experiments, a cDNA library was prepared from tsNY72 infected cells maintained at the permissive temperature for four to six hours in the presence of 75 μM cycloheximide, and screened with a subtracted probe made of labelled cDNA from tsNY72 infected cells grown at 35°C hybridised against mRNA from unstimulated cells. Clones obtained with this strategy corresponded to stabilised mRNA whose expression had been induced by pp60^v-src. Twelve different immediate early genes were induced, including CEF10, the chicken equivalent of cyr61.¹⁷

Upon serum stimulation of starved CEFs, the pattern of CEF10 expression was similar to that of cyr61 in murine cells (fig 2 ▶). However, no late burst of induction was reported for CEF10. Whether this discrepancy results from variations in the protocols and origin of cells used or represents important differences in the biological properties of murine and chicken genes is not yet clear.

The pattern of CEF10 expression in pp60^v-src induced cells is interesting. In pp60^v-src induced cells, CEF10 expression was found to be multiphasic, with two peaks at one and eight hours post shift, a decrease at 12 hours post shift, and a third peak at 24 hours.¹⁷ Under similar conditions, most immediate early genes were persistently induced for 24 hours. When a mitogenic non-transforming myristoylation defective virus NY315 was used, CEF10 was induced in a linear mode for a period of 24 hours.¹⁷ These observations indicated that CEF10 production was increased as a result of the pp60^v-src growth stimulatory effect. The cyclic expression of CEF10, which was not observed with NY315, might be related to the transformation process induced by v-src.

In another set of experiments, the differential display strategy was applied to cDNAs prepared from tsNY72 RSV infected CEFs grown at the non-permissive temperature and either shifted to the permissive temperature or maintained at the non-permissive temperature. Screening of the genes differentially expressed in these cells revealed that the expression of the nov gene was downregulated upon the stimulation of CEF growth induced by pp60^v-src.¹⁸

Nuclear run on assays performed on serum starved uninfected CEFs and wild-type pp60^v-src transformed CEFs established that the differential rate of nov transcription in isolated nuclei did not account for the difference in the steady state of nov transcripts measured in quiescent and transformed CEFs. Therefore, the increase levels detected in uninfected cells might result from an accumulation of the nov transcripts as a result of the long half life (eight hours) of the nov transcripts in quiescent cells.

The use of a mutant strain expressing a thermosensitive pp60^v-src revealed that upon shifting to the non-permissive temperature, there was a decline in the numbers of nov transcripts detected in transformed cells between four and eight hours, with a dramatic reduction after 12 hours, and an absence of transcripts after 24 hours.¹⁸ A similar decline in nov expression was seen when CEFs were stimulated to proliferate either after transformation by various oncogenes or after infection with the non-transforming myristoylation defective NY315 mutant.¹⁸ Therefore, the downregulation of nov expression induced by the stimulation of cell growth and the high concentrations of NOV that were detected in quiescent cells were in striking contrast to the situation observed for other CCN genes, and established that nov was not an immediate early gene (fig 2 ▶). The requirement for protein and RNA synthesis in the decrease of nov transcripts upon stimulation of cellular growth indicated that the reduced expression of nov resulted either from the synthesis of a negative regulator of nov transcription or from an increased turnover of nov transcripts.¹⁸

Our observation that the expression of nov was modulated during the progression of the cell cycle and reached a peak at the G2 phase (S Middendorp and B Perbal, 1995, unpublished results) suggests that the increased synthesis and accumulation of stable nov transcripts at that late stage might be required for proper differentiation.

an amino truncated nov related protein is localised in the nucleus of hela and 143 cells

A 31/32 kDa doublet of NOV protein was identified by western blotting in the nuclear fraction of human epitheloid carcinoma HeLa cells, and human osteosarcoma 143 cells.³⁰ Because the antibodies used in this study were directed against the C-terminus of NOV, the 31/32 kDa NOV isoform was thought to be an amino truncated form of NOV.

The nuclear detection of NOV was reminiscent of a situation reported previously for several other secretory proteins. In the absence of a conventional nuclear localisation signal (NLS) in NOV, its nuclearisation might be governed by the KKGKKCLRTKKS motif, which is similar to the basic motif involved in the nuclear localisation of fibroblast growth factor 3 (FGF3).

Although the biological importance of NOV nuclear targeting remains unclear, our expectation is that the 31/32 kDa NOV isoform might modulate gene expression during normal development. The biochemical identification of this nuclear NOV related protein is under way.

the nuclear 31/32 kda nov related protein colocalises with the transcriptional machinery in the 143 cells

As a first step in establishing the localisation of the nov protein in the nucleus of these cells, we took advantage of the fact that herpes simplex virus 1 (HSV-1) encoded infected cell proteins 4 and 8 (ICP4 and ICP8) can be used as physical markers for the transcription and replication machineries, respectively. The ICP4 protein is a transcriptional transactivator localised in the nucleus of HSV infected cells, where it is responsible for the regulation of HSV gene expression, whereas ICP8 is a single stranded DNA binding protein that is found in the nuclear compartment, where viral DNA synthesis takes place.³¹

To determine whether NOV protein could be detected in the nucleus of actively growing cells, three series of experiments were performed. In the first, uninfected fixed 143 cells were exposed to the anti-nov K19 rabbit antibody and then to biotinylated goat antirabbit immunoglobulin, followed by avidin bound to Texas red. In parallel experiments, cells were treated with the anti-nov K19 antibody and incubated directly with goat antirabbit Texas red conjugated antibodies. Nuclei from uninfected 143 (fig 5A ▶) cells showed a strong positive response to the K19 anti-nov antibody. In uninfected actively growing cells, the nuclear NOV protein appeared to be localised in the nucleoplasm and in nucleoli. Positive staining was also seen at the periphery of the nucleus (probably in the Golgi) and in the cytoplasm of these cells.

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Figure 5

Confocal immunocytolocalisation of NOV in the nucleus of 143 osteosarcoma cells. (A) staining with anti-nov antibodies, (B) double staining with anti-nov and anti-HSV-ICP4 (herpes simplex virus infected cell protein 4) antibodies, (C) double staining with anti-nov and HSV-ICP8 antibodies. The yellow colour indicates colocalisation of ICP4 and NOV.

In the second series of experiments, cells were infected with HSV-1 before incubation with rabbit anti-nov (K19), mouse anti-ICP4, and mouse anti-ICP8 antibodies. It has been reported previously that early after HSV infection, ICP4 is diffusely distributed throughout the infected cell nucleus and that, after the onset of viral DNA replication, staining for ICP4 was associated with discrete dense bodies. In contrast, ICP8 labelling was fairly constant throughout infection, giving rise to a granular pattern that filled the infected nucleus.

Infected 143 cells were first incubated simultaneously with either both the rabbit anti-K19 and the mouse anti-ICP4 antibodies and then with goat antimouse fluorescein isothiocyanate (FITC) conjugated antibodies (reacting with anti-ICP4 and anti-ICP8) or with biotinylated goat antirabbit immunoglobulin before incubation in the presence of Texas red conjugated avidin (allowing indirect visualisation of the K19 antibody).

As shown in fig 5B ▶, ICP4 (green fluorescence) and NOV (red fluorescence) overlapped in the nucleus of infected 143 cells, whereas ICP8 (green fluorescence) and NOV (red fluorescence) did not colocalise (fig 5C ▶). As expected, the nucleoli did not contain dual staining. These experiments suggested that the major fraction of the nuclear NOV isoform was localised in the compartment where transcription occurs and was not associated with the replication machinery (B Perbal, 1997, unpublished data).

THE NOV PROTEIN INTERACTS WITH THE RPB7 SUBUNIT OF RNA POLYMERASE IN A YEAST TWO HYBRID SYSTEM

A yeast two hybrid screening system was used to isolate cDNAs encoding proteins able to interact with the human NOV protein.³⁰ A DNA fragment encoding NOV codons 29 to 357 was amplified by the polymerase chain reaction (PCR) and inserted into pGBT9 (Clontech, Palo Alto, California, USA) in frame with the DNA binding domain of Gal4. The resulting plasmid (pNOV) was used as “bait” to screen three cDNA libraries (an Epstein-Barr virus transformed human peripheral blood lymphocyte library (gift from Aviron Inc.), a HeLa cell library, and a normal human brain library from Clontech, all provided by Dr B Roizman, Kovler Laboratory, University of Chicago, USA), which were fused to the Gal4 transcriptional activation domain in pACT.

Establishing the 3′-proximal sequence of the inserts at the 5′ boundary of the Gal4 DNA domain fusion (B Perbal, 1997, unpublished results; CP Li et al, 2000, unpublished results)³⁰ allowed us to identify several independent clones with sequences that match completely with the published sequence of the rpb7 subunit of human RNA polymerase II. Because it has been established that the rpb7 subunit alone does not give rise to false positives under the conditions used (M Verner, 1998, personal communication), our results strongly suggested that the nuclear amino truncated NOV related protein is involved in the regulation of transcription through its interaction with the RNA polymerase holoenzyme.

A NOV RELATED PROTEIN CAN BE DETECTED AT THE NUCLEAR ENVELOPE

We have shown that the NCI-H295R cells that have been established from an invasive adrenocortical carcinoma secrete increased concentrations of NOV protein (S Kyurkchiev et al, Proceedings of the first international workshop on the CCN family of genes, 17–19 October 2000, Saint-Malo, France). When the immunogold procedure was used to perform an immunocytochemical localisation of the NOV protein in these cells, we found that the cytoplasm, the plasma membrane, and the nuclear envelope stained positive. Unexpectedly, the distribution of the positive grains in the cytoplasm of the NCI-H295R cells and the absence of any recognisable secretory granule or secretory vesicle suggested that shuttling of the NOV protein in these cells did not follow the conventional pathway (G Thomopoulos et al, 2001, in press). The mode of secretion of the protein is currently under investigation.

The detection of an immunoreactive NOV related protein at the nuclear pores of NCI-H295R cells was another piece of evidence suggesting that NOV might be targeted to the cell nucleus.

PROMOTER SEQUENCES AND GENE EXPRESSION

As stated above, the expression of the cyr61 and ctgf genes is induced early upon stimulation of fibroblast proliferation, whereas Elm1 and rCOP-1 exhibit delayed kinetics of induction and the expression of nov is downregulated under similar conditions (fig 2 ▶). This differential expression pattern and the variety of effectors involved in the activation or repression of CCN genes have suggested that distinct regulatory elements govern their expression. Indeed, the comparison of ctgf, cyr61, and nov promoter sequences did not show any obvious conservation.^{3, 37–39}

The induction of cyr61 by serum and PDGF in fibroblasts was reported to be dependent upon a serum responsive element (SRE)-like motif at position −1912/−1933 in the 2 kb 5′-proximal promoter sequences, which are sufficient to confer serum inducibility.³⁷ The expression of ctgf was also found to be stimulated by PDGF and epidermal growth factor (EGF) in human fibroblasts.²³ Similarly, both cyr61 and ctgf were reported to be induced by fibroblast growth factor (FGF).^{5, 23} The expression of cyr61 is also induced by vitamin D3 in human fetal osteoblasts,⁴⁰ by oestrogen and tamoxifen in uterine cells of ovariectomised rats,⁴¹ and hippocampal immortalised cells undergoing neuronal differentiation.⁴²

The rapid induction of cyr61 (peak after 20 minutes, decreased at 60 minutes, and absent at two hours) by 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA)⁵ was in pronounced contrast to the expression of CEF10 (the avian homologue of cyr61), which was reported to be repressed to < 20% of the basal value at 60 minutes, and gradually increased to about 10 times the basal value at 24 hours.¹⁷ Based on the time course for the translocation–activation–deactivation of protein kinase C (PKC), the expression pattern of CEF10 in the presence of 100 ng/ml TPA suggested that the expression of CEF10 is repressed at one hour, when PKC is transiently activated, and derepressed when PKC is degraded.¹⁷ Similarly, stimulation of serum starved cells by 50 ng/ml TPA for six hours resulted in the complete downregulation of nov in CEFs.¹⁸ The downregulation of nov expression induced by serum and by TPA could be abolished by treatment with the H7 inhibitor of PKC, suggesting that PKC might play a role in the decrease in the half life of nov RNA transcripts.¹⁸ The sequences involved in the delayed downregulation of cyr61 and ctgf expression have not been identified as yet.

Although the expression of CEF10/βIG-M1 (the mouse equivalent of cyr61) and βIG-M2 (the mouse equivalent of ctgf) were reported to be inducible by TGFβ in AKR-2B mouse embryo fibroblasts,⁷ the expression of cyr61 was not found to be significantly induced by TGFβ in human skin fibroblasts or NIH3T3 cells.³⁸

These “conflicting” results indicated that the regulation of CCN gene expression may vary in different species (cef10 and cyr61), and depend upon the type of cells used in the same species (AKR-2B versus NIH3T3). These observations stressed once more the fact that one must be extremely cautious in comparing results obtained with different systems.

Given the importance of TGFβ signalling in several fundamental biological processes, much attention has been paid to the relation between TGFβ and ctgf expression. Promoter analysis of ctgf and methylation interference assays have led to the identification of a 13 nucleotide TGFβ putative responsive element (TbRE), which is conserved in the promoter of ctgf throughout species and appears to be specific for TGFβ induced transcriptional activation of ctgf.³⁸ Whether binding of a specific factor to this sequence is responsible for the prolonged induction of ctgf mRNA after short term exposure to TGFβ remains to be established.

The expression of nov was not affected by TGFβ in either NRK-49 F (D Lawrence and B Perbal, 1995, unpublished observations) or NIH3T3 and human skin fibroblasts.³⁸

Ex vivo experiments performed with the human nov promoter indicated that sequences localised between positions −405 and −62 had a negative effect on transcriptional activity when measured by chloramphenicol acetyl transferase (CAT) assays.¹¹ A comparison of the promoter sequences from xenopus, chicken, and human nov did not reveal any significant conservation (fig 7 ▶). A stretch of 19 nucleotides (GCAGGCCCCGGCGCGC CGC) appeared to be conserved in the human and chicken nov promoter. Whether this nov conserved element (NCE) binds a regulatory factor involved in the negative regulation of nov promoter activity is under investigation. The activity of the human nov promoter was downregulated indirectly by the WT1 protein.³⁹ It is worth noting that the ctgf promoter was also recently reported to be a target for WT1 transcriptional regulation.⁴³ Screening of oligonucleotide arrays and northern blotting established that ctgf expression was upregulated in a Wilms's tumour cell line (WiT49A) transfected by a dominant negative mutant of WT1, whereas ctgf promoter activity was repressed by wild-type WT1 protein. As established in the case of nov, the downregulation of ctgf promoter activity was not mediated by canonical WT1 recognition elements.⁴³ These results reinforced the possibility that the alteration of nov expression observed in avian nephroblastomas was indeed related to impaired kidney blastema differentiation and tumour development.

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Figure 7

Schematic structure of the human (A) and chicken (B) nov promoters. The putative binding sites were identified on the basis of their consensus nucleotide sequences.

EXPRESSION PATTERN IN NORMAL EMBRYONIC AND ADULT TISSUES

Early studies indicated that the CCN genes were differentially expressed in normal tissues during development.

Northern blot analysis of chicken embryonic and adult total RNA established that the expression of the nov gene was regulated both temporally and spatially during development,^{9, 12} with nov RNA detected either only at the embryonic stage (heart) and adult stage (lung), or at both stages (brain). Muscle and intestine were also found to express nov at the embryonic stage, whereas adult spleen was positive for nov expression. Embryonic kidneys expressed high amounts of nov, whereas much lower amounts were detected in the kidneys of birds after hatching.⁹ In human tissue samples, abundant amounts of polyA nov RNA were detected by northern blotting in heart, brain, prostate, testes, and pancreas, whereas low amounts were detected in spleen, ovary, small intestine, colon, peripheral blood leucocytes, lung, skeletal muscle, and kidney.³⁶ RNase protection assays performed on total RNA of 8 and 10 week old embryonic human tissues indicated that nov was highly expressed in the adrenal gland and to a lesser extent in kidney, limb bud, and spinal cord, whereas heart, liver, intestine, lung, placenta, and testis were found to be negative for nov expression (S Kocialkowsky et al, 2001, in press).

A cDNA-PCR analysis of WISP RNA expressed in adult and fetal human tissues¹⁶ established that WISP-2 RNAs were to be found in a rather restricted panel of tissues, including adult skeletal muscle, colon, ovary, and fetal lung, whereas WISP-1 RNAs were detected in adult heart, kidney, lung, pancreas, ovary, spleen, and small intestine. In addition to heart, kidney, lung, and spleen, fetal skeletal muscle and liver also contained WISP-1 RNAs. The WISP-3 RNAs were detected only in adult kidney and testis and in fetal kidney. No expression of the WISP genes was detected in brain.¹⁶ In situ hybridisation using whole mount mouse embryos and northern blot analysis of adult rat and mice organs could not detect rCOP1 RNA in brain, heart, lung, kidney, pancreas, spleen, intestine, stomach, and skeletal muscle.¹⁵ The expression of WISP-2 in human skeletal muscle was in contrast to the lack of expression of rCOP-1 in rodents.

Northern blot analysis of ctgf expression in normal adult tissues²⁸ established that ctgf polyA RNAs were found abundantly in spleen, ovary, gastrointestinal tract, prostate, heart, and testis, and at a lower level in thymus, placenta, lung, skeletal muscle, kidney, and pancreas. No expression of ctgf was detected in brain, liver, and peripheral leucocytes.²⁸ The lack of ctgf expression in brain was in contrast to the western blotting identification of a 40 kDa CTGF protein in rat brain homogenates and the immunochemical detection of CTGF in the cerebral cortex, the white matter of spinal cord.⁴⁴ The strongest positive signal for CTGF detection was seen in astrocytes of the cerebral cortex, ependymal cells of the cerebral ventricle, tanycytes along the central canal of the spinal cord, and pyramidal cells in the cortical layers III and V.

RNase protection assays performed on total RNA from BALB/c mouse tissues established that cyr61 expression is highest in lung; moderate in heart, uterus, and skeletal muscle; and low in kidney, adrenal gland, testes, brain, and ovary.⁴⁵

Altogether, these results (see table 2 ▶ for a summary) illustrated the diversity of those tissues in which expression of the CCN genes can be detected at different stages of development, reinforcing the current view that CCN proteins are involved in various biological processes. From table 2 ▶ it can be seen that NOV was the only CCN protein highly expressed in the brain. Indeed, in situ hybridisation and immunocytochemistry performed on developing human, chicken, and rat embryos confirmed that the expression of nov was tightly associated with nervous system differentiation and development (S Kocialkowsky et al, 2001, in press; G Chevalier et al, 2000, unpublished results; W Cai et al, 2000, unpublished results; Y Cherel et al, Proceedings of the first international workshop on the CCN family of genes, 17–19 October 2000, Saint-Malo, France).⁴⁶ The role and functions of NOV in the nervous system are under current investigation.

In situ hybridisation and immunocytochemistry performed on developing chicken embryos allowed us to establish the pattern of nov expression in kidney, muscle, nervous system, and skeletal development (G Chevalier et al, 2000, unpublished results). In general, a good match was seen between nov RNA and NOV protein detection. The expression of nov was detected at embryonic days 3 and 4 (E3 and E4) in the mesonephric mesenchyme, epithelial vesicles, and glomeruli. At later stages (E6 to E12), differentiated tubules were found to be positive for nov. Metanephric epithelial vesicles, glomeruli, and ureteric bud were also positive for nov until E14. From E4 onwards, developing myotome and skeletal muscle stained positive for nov. Nov expression was also detected in smooth muscle cells and cardiomyocytes. The nervous system was found to be strongly positive for nov. At early stages (E3) neuroepithelium was positive at E3, and at later stages (E3–E7) neural tube also showed strong staining for nov. In agreement with our previous detection of high amounts of NOV protein in human neuronal cells and axons,³⁵ high concentrations of NOV protein were detected in chicken neuronal cells, which express low amounts of nov RNA, therefore reinforcing the possibility that in these cells, as well as in podocytes, the NOV protein accumulates or is stabilised.³⁵ Because nov shares identity with the fruit fly axon repellent slit protein, the detection of nov along the chicken and human axons suggests that it might be involved in axonal guidance. Strong expression of nov RNA and proteins was also detected at sites of chondrogenesis, where it is probably involved in the late stages of chondrocyte differentiation and osteogenesis (see below).

In situ hybridisation and immunocytochemistry established that although the distribution of nov RNA and protein was widespread in first trimester human embryos (table 3 ▶), the major sites of nov expression included the central nervous system, the adrenal cortex, and differentiating muscle and kidney (S Kocialkowsky et al, 2001, in press). In all cases, the NOV protein was predominantly cytoplasmic, and weak staining of the extracellular matrix (ECM) was also seen in some instances. In general, there was a fairly good correlation between nov RNA and NOV protein expression, suggesting that nov acts locally rather than distantly. In agreement with results obtained at later developmental stages,⁴⁶ neurones of both the spinal cord and the brain expressed high amounts of nov RNA and the NOV protein was clearly detected in cell bodies and axons. The floor plate of the spinal cord, spinal nerves, and dorsal root ganglia were identified as sites of abundant nov expression.

Muscle was the predominant mesodermal component expressing nov. Myotubes and fusing myoblasts were identified as major sites of nov expression in skeletal muscle. Smooth muscle also expressed nov, but to a lesser extent. At the early stages, cartilage chondrocytes did not express nov, whereas in the older embryo, the NOV protein was demonstrated in the perichondrium and in hypertrophic chondrocytes in the head, ribs, and lower limbs. In the developing urogenital tract, major discrepancies were noted between NOV protein and nov RNA values, suggesting that the NOV protein might accumulate at these sites for a particular function, although we cannot exclude, as yet, that nov RNA species are short lived in these cells. For instance, the concentrations of NOV protein in the podocytes of both mesonephric and metanephric glomeruli are usually extremely high, whereas in the same cells nov RNA species are barely detectable (S Kociazlkowski et al, 2001, in press; G Chevalier et al, 2000, unpublished results).³⁵ Because the NOV protein is fairly stable in the conditioned medium of NOV producing cells (B Perbal, 2000, unpublished results),³⁵ the raised concentrations of NOV detected in podocytes probably results from its accumulation. Whether this accumulation results from its interaction with other proteins at this site or is physiologically important (needed for glomeruli filtration?) remains to be established. The metanephric mesenchyme was strongly positive for nov RNA expression, with increasing staining as differentiation proceeds in the S-shaped bodies, and a dramatic decrease once glomeruli differentiation was achieved. These results were of particular interest because they strongly suggested that nov expression was tightly coupled to the differentiation process in these cells. Examination of immunocytochemical labelling in the heart indicated that at these stages the endocardium was negative for nov, although it is in close contact with the myocardium, an abundant source of NOV protein. This observation provided evidence that the NOV protein acts where it is produced rather than being transported and accumulating at a distant site. Among endodermal derived tissues, the gut mucosa, bronchial epithelium, and pancreatic ducts were positive for nov (table 3 ▶).

The immunocytochemical localisation of CYR61 and FISP12 proteins during mouse embryogenesis showed similarities and differences that were indicative of differential actions in development.⁴⁷ The localisation of CYR61, but not FISP12, in limb bud mesenchyme in vivo and in vitro had suggested that CYR61 might be involved in chondrogenesis.^{47, 48} Purified CYR61 protein was indeed shown to promote the initial cell aggregation responsible for mesenchymal condensation before chondrogenic differentiation and to enhance the synthesis of cartilage specific matrix.⁴⁹ More recently, it has been reported that in rat, cyr61 is expressed in a time dependent manner in mesenchyme cells and osteoblasts from fracture callus,⁵⁰ whereas conventional in situ hybridisation did not detect cyr61 mRNA in normal bone. In the hFOB human osteoblast cell line, cyr61 was identified as a greatly upregulated gene by various factors important for bone metabolism such as 1,25(OH)2-vitamin D3, tumour necrosis factor α (TNF-α), EGF, basic FGF, and interleukin 1β (IL-1β).³⁹

Differential display PCR identified ctgf as being preferentially expressed in HGS-2/8 human chondrosarcoma derived chondrocytic cells.⁵¹ In situ hybridisation established that ctgf was specifically expressed in the hypertrophic chondrocytes of costal cartilage and vertebral column in 17 day old mouse embryos,⁵¹ at a time when cyr61 expression is strongly downregulated,⁴⁸ suggesting that CYR61 and CTGF might have complementary but different functions. Staining of the cost-chondral junctions of newborn mouse ribs indicated that ctgf was expressed in chondrocytes of the hypertrophic zone, but not in the proliferating and resting zone.⁵² Osteoblasts did not express ctgf. Because ctgf mRNA expression was stimulated in HCS2/8 cells treated with TGFβ and bone morphogenic protein 2 (BMP-2), two factors believed to be involved in cartilage and bone formation, it was proposed that CTGF was a mediator of these cytokines in cartilage. Because CTGF promoted the proliferation and differentiation of chondrocytes in culture,⁵³ it was proposed that ctgf expression is involved in endochondral ossification by acting on proliferating and maturating chondrocytes in growth cartilage. Both chondrocytic and osteoblastic cells express a 280 kDa protein that has been described as a receptor for CTGF.^{54, 55}

Because nov expression was also detected in sites of chondrogenesis during chicken development (fig 8 ▶), we postulated that the negative regulatory activity attributed to nov (see above) might be involved at later stages of differentiation to counterbalance the stimulatory effects of CYR61 protein on chondrogenesis, which had been established in micromass cell cultures.⁴⁹ According to this view, CYR61 and NOV protein activities might represent the “yin and the yang” along common regulatory pathways leading to chondrocyte differentiation and osteogenesis.

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Figure 8

Detection of nov RNA species at sites of chondrogenesis in developing chicken. (A) Development stage (St) 3, embryonic day (E) 8.5. (B) St 31, E7. (C) St 35, E8.5. (D) St 38, E12. (E) St 35, E8.5.

To assess the potential role of nov in chondrogenesis and osteogenesis, we first analysed the expression of nov chicken during wing and leg development.⁵⁶ Northern blotting performed on total RNA prepared from limb buds dissected between E4 and E18 established that expression of nov increasing significantly between E8 and E10 and progressively decreased until E14, to reach a basal value at E15.⁵⁶ The expression profile of nov correlated with the time course of limb development, which is completed after 10 days of incubation under normal conditions. Sustained expression indicated that nov was required over an extended period of time during limb development, therefore suggesting that it might be needed for later events. Immunocytochemistry performed on paraffin wax embedded wing bud sections established that nov was mainly detected at E6 in the proliferating chondrocytes and the perichondrium (fig 9A ▶), whereas at E11/E12, staining of nov was essentially detected in the hypertrophic chondrocytes of the central diaphyseal region and to a lower extent in the perichondrium (fig 9B ▶). At E14, nov labelling was greatly reduced and restricted to a few hypertrophic chondrocytes in the developing wing. Flattened chondrocytes were not positive for nov expression at any stage of development (fig 9C ▶). Pretreatment of the sections with hyaluronidase resulted in additional widespread labelling of the cartilaginous matrix, in agreement with nov being associated with the ECM.⁵⁷ Similarly, CYR61 and CTGF were also reported to be ECM associated proteins and suggested to mediate cell–matrix interactions.⁵⁸

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Figure 9

Detection of the NOV protein in developing bone. (A) At stage 29 (embryonic day 6 (E6)), NOV is detected at sites of chondrogenesis in the developing chicken wing. Alcian blue staining identifies cartilage formation. (B) At stages 35–38 (E11–12) strong nov staining is seen in the perichondrium (p) and proliferating chondrocytes (pc). (C) At the same stages, flattened chondrocytes (fc) do not stain positive for nov, whereas hypertrophic chondrocytes (hc) are strongly positive for NOV staining (D).

When mesenchymal cells isolated from the limb buds of 3.5 day old chicken embryos or from 10.5 day old mouse embryos are seeded at high density (10⁷ cells/ml), cellular aggregation proceeds and internodular condensations undergo chondrocytic differentiation, as shown by the formation cartilaginous nodules within three days of culture. When cells are seeded at a lower density (10⁶/ml), chondroblastic differentiation does not proceed and no cartilaginous formation occurs. The use of this system revealed that CYR61 enhances chondrogenic differentiation.⁴⁹ Northern blotting experiments performed on chicken micromass cell cultures established that nov expression, which was not detected at day 0 of culture, increased progressively for two days and decreased at day 3, whereas the expression of collagen II, a marker for determination of progenitor cells to undergo chondroblastic differentiation, increases from day 1 onwards (fig 10 ▶). The expression of nov is dependent upon the productive differentiation of mesenchyme cells, as shown by the lack of nov expression in the absence of cartilaginous nodule formation at low cell density (fig 11 ▶). When cells were seeded at high density, ctgf expression was detected in micromass cultures from day 2 onwards, whereas CEF10 expression was detected from day 0 to day 3.⁴⁹

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Figure 10

Expression of nov RNA species and collagen II (COL II) in chicken micromass cell cultures. Staining of ribosomal RNA species allows relative quantification of the hybridisation signals obtained in two independent experiments.⁵⁶ J0–J3, days of culture; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Figure 11

The expression of nov RNA depends upon chondrogenesis. (A) 10⁶ mesenchymal cells were seeded for micromass cell cultures. (B) 10⁷ mesenchymal cells were seeded for micromass cell cultures. Samples were taken at the day of seeding (J0) and after 24 hours culture (J1). COL II, collagen II; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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, whereas 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 to nephroblastoma, MAV can induce osteopetrosis in chickens, an abnormal proliferation of osteoblasts leading to severe bone diseases.¹⁰ Based on the consideration that nov was a target of MAV in chicken blastemal cells, it is tempting to hypothesise that the induction of osteopetrosis by MAV could also result from an alteration of nov expression in bone precursor cells.

WHERE?

The use of different efficient expression systems (such as baculoviruses) enables the production of recombinant tagged proteins that can be used to identify the sites of CCN protein binding on cells and follow the fate of the secreted proteins once they are presented at the cell surface. The production of specific polyclonal and monoclonal antibodies directed against different portions of the CCN proteins also enables their concentrations to be measured in biological fluids. They can also establish whether quantitative variations of circulating CCN proteins are associated with pathological conditions and can be used as a valuable clinical tool. We are presently using this strategy to measure, under pathological conditions, the variations of NOV released in different biological fluids, such as cerebrospinal fluid, seminal fluid, urine, and serum.

HOW?

This aspect requires the purification of native CCN proteins from a reliable biological source. Although the use of recombinant proteins has proved very fruitful in unravelling the biological properties of several signalling effectors, it must be kept in mind that many biochemical properties of native proteins require post translational modifications, which do not take place (or only partially) in most of the systems used to produce recombinant proteins. Purification of the native protein also permits the isolation of functional complexes whose characterisation has proved to be extremely informative in many instances. Because CCN proteins potentially interact with several other partners, the isolation of complexes in which these proteins are engaged will undoubtely lead to important discoveries concerning their mode of action. The purification of the different CCN proteins can also establish whether they exhibit synergistic or antagonistic properties when tested in combination.

I wish to thank particularly my wife Annick for her help and constant support. Thanks are also due to S Gabaron for reading the manuscript. The research performed in my laboratory was funded by grants from the Ligue Nationale Contre le Cancer (Comités National, du Cher et de l'Indre), Association Française contre les Myopathies (AFM), Matra-Hachettte, Fondation pour la Recherche Médicale (FRM), and Association pour la Recherche contre le Cancer (ARC). I am grateful to Professor B Roizman for his support and for providing the facility to perform experiments with herpes simplex virus and the two hybrid system.