Aims—To establish the expression pattern of ccn3 (nov) in the central nervous system (CNS) of adult rats and to determine whether spatiotemporal variations in the expression of ccn3 (nov) are related to specific developmental stages and/or specific CNS functions.
Methods—The sites of ccn3 (nov) expression have been identified by in situ hybridisation using didoxigenin labelled cRNA and by the reverse transcription-polymerase chain reaction (RT-PCR). The rat CCN3 (NOV) protein was characterised by western blotting performed on brain extracts. The localisation of the CCN3 (NOV) protein in the brain was established by immunocytochemistry.
Results—Increased expression of ccn3 (nov) was detected in the developing brain of rats after birth, as shown by RT-PCR and immunocytochemistry analysis performed on a series of samples taken between day 5 (P5) and day 300 (P300), with a pronounced peak between P15 and P150, suggesting that CCN3 (NOV) might play a role in the maintenance or establishment of specific brain functions. The relatively high amounts of an N-terminal truncated CCN3 (NOV) related protein detected both in the brain tissues and cerebrospinal fluid suggested that post translational processing of CCN3 (NOV) might be particularly prevalent in the brain. Such processing might be of biological importance in the light of the previously reported growth stimulatory effects of N-terminal truncated CCN3 (NOV) isoforms.
Conclusions—The postnatal differential expression of ccn3 (nov) in the brain of developing rats suggests that CCN3 (NOV) might be involved in the acquisition of specific functions. The rat species provides an as yet unequalled system for these studies. Because the CCN3 (NOV) protein is detected in restricted areas of the brain, it will be interesting to establish whether variations of ccn3 (nov) expression are associated with normal cognitive processes and whether ccn3 (nov) expression is affected by aging. In addition, because CCN3 (NOV) is found in the spinal cord and along the axonal processes, it will be of interest to determine the expression of the normal and truncated isoforms of CCN3 (NOV) in various pathological conditions, such as neurodegenerative diseases.
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The ccn3 (nov) gene is a founding member of the expanding CCN family of genes, which encode proteins reported to be involved in a variety of fundamental biological functions (for recent reviews see Lau and Lam,1 Brigstock,2 and Perbal3). The ccn3 (nov) gene was identified originally as an integration site of myeloblastosis associated virus type 1-N (MAV1 N)4 in avian nephroblastomas, which provide a unique model of the Wilms's tumour.5 Although raised concentrations of CCN3 (NOV) are detected in all avian tumours, integration of MAV in the vicinity of ccn3 (nov) is a rare event (CL Li et al, 2001, unpublished data) and overexpression of ccn3 (nov) in the avian tumours might be either a consequence of tumorigenesis or a necessary step for nephroblastoma development. Indeed, alterations of the ccn3 (nov) locus are not significantly associated with Wilms's tumours (V Huff, et al, 1997, unpublished data) and ccn3 (nov) expression can be altered either positively or negatively in Wilms's tumours.6
These observations suggested that ccn3 (nov) might encode a protein whose function was associated with abnormal cell growth and that alterations of ccn3 (nov) expression in the avian and human tumours were manifestations of uncontrolled proliferative activity of the tumour cells.
Because long terminal repeat driven expression of a full length ccn3 (nov) gene interfered with chicken embryo fibroblast (CEF) growth,4 and expression of ccn3 (nov) is associated with quiescence of CEF cells,7 we proposed that this gene might encode a negative regulator of cell growth, the alteration of which would result in uncontrolled cell proliferation.3, 5, 8, 9
We have previously established that expression of the CCN3 (NOV) protein was developmentally regulated in humans and chickens.4 Major sites of expression included the adrenal gland, the nervous system, cartilage, muscle, and the kidney.3, 4, 10–13 In the chicken, differential regulation of ccn3 (nov) RNA expression has been reported in embryos and adults.4 Studies performed with a large panel of Wilms's tumours, including WAGR (Wilms, aniridia, growth retardation) and DDS (Denis Drash syndrome), have shown that raised expression of ccn3 (nov) is a marker of the heterotypic differentiation of blastemal cells.11 These studies also found increased concentrations of the CCN3 (NOV) protein along the axonal processes and in the glomerular podocytes, where low amounts of RNA were detected, which suggests that the CCN3 (NOV) protein might play a role in the function of these structures.11 A survey of ccn3 (nov) expression in tumour samples or cell lines of different origins and nature established that increased expression of ccn3 (nov) was associated with: (1) a good prognosis in neuroblastomas (H Yeger et al, 2000, unpublished data), glioblastomas,14 and chondrosarcomas (C Yu et al, Proceedings of the first international workshop on the CCN family of genes, 17–19 October, 2000, Saint-Malo, France) or (2) increased tumorigenic potential and metastasis in renal cell carcinomas (L Glukhova et al, Cancer Genet Cytogenet, in press), prostate carcinoma (M Maillard, 2001, J Clin Pathol: Mol Pathol, in press) and Ewing's tumours (K Scotlandi et al, AACR, 92nd annual meeting, March 24–28, 2001, New Orleans, USA). In adrenocortical tumours, variation in ccn3 (nov) expression alone was not a useful prognostic or diagnostic marker.3
Analysis of the ccn3 (nov) expression pattern in the human CNS during normal development indicated that ccn3 (nov) RNA species were present mainly in somato motor neurones in the lower CNS, both at early and later developmental stages.15 As a first step to identifying its biological function in the CNS, we have studied the pattern of expression of ccn3 (nov) RNA and protein in the brain of adult rats. The results obtained in our study support the idea that CCN3 (NOV) is required for specific functions in the CNS. In addition, the overlapping pattern of ccn3 (nov) RNA and protein expression generally observed suggests that, in most cases, the CCN3 (NOV) protein probably functions at its site of production.
Material and methods
Adult Wistar rats weighing between 200 and 230 g were used for immunocytochemistry and in situ hybridisation experiments. Care and treatment of the animals were in accordance with the Animal Scientific Procedures Act and were submitted for the approval of the Third Military Medical University animal use committee.
Brain tissue taken from rats at different developmental stages (embryonic day 18 (E18), postnatal day 5 (P5), P15, P30, P60, P150, and P300) was used as the source of RNA. Total RNA was purified after treatment with 4 M guanidium isothiocyanate, followed by phenol/chloroform extraction and isopropanol precipitation, as described previously.16
REVERSE TRANSCRIPTION (RT)
RT was performed in 30 μl final volume samples containing 50 mM Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.125 mM each dNTP, 500 ng oligo-dT, 600 units of Moloney murine leukaemia virus reverse transcriptase (Life Technologies, Rockville, Maryland, USA), 40 units of RNAsin (Promega, Madison, Wisconsin, USA), and 2 μg of RNA. Incubation was performed at 37°C for 90 minutes before denaturation at 100°C for five minutes.
PCR amplifications were carried out in 12.5 μl final volume samples containing 100 ng of genomic DNA template, 20 ng of ccn3 (nov) specific primers, 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 0.1 mg/ml gelatin, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.5 units of Taq polymerase. The amplifications were performed in a Perkin Elmer Cetus thermal cycler for 30 cycles at 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds.
The K19M rabbit polyclonal anti-human CCN3 (hCCN3; NOVH) antibody11 was used at a 1/250 dilution. The streptavidin–biotin peroxidase complex kit (Zymed; Beijing Zhong Shan Biological Technology Co, Beijing, China) was used at a 1/200 dilution as recommended by the supplier. Diaminobenzidine (DAB) was purchased from Sigma (St Louis, Missouri, USA). For immunohistochemistry, the rats were anaesthetised with sodium penbarbital and perfusion fixed through the left ventricle of the heart, first with physiological saline and then with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains were immediately removed and cut into small pieces, which were immersed in the same fixative for two hours at 4°C and transferred to sodium phosphate buffer containing 30% sucrose for at least 18 hours at 4°C.
Cryostat sections (20 μm) were cut and free floated in 0.01 M sodium citrate (pH 6.0). Antigen retrieval was performed in a microwave oven.
PREPARATION OF RAT PROTEIN SAMPLES AND WESTERN BLOTTING
Adrenals and brains were obtained from 6 week old rats. After decapitation, organs were removed and 0.1 g of each tissue was crushed on ice, washed twice with cold phosphate buffered saline (PBS), and centrifuged at 1000 ×g for seven minutes. Pellets were resuspended in 1% deoxycholate (DOC; pH 11.3), boiled for five to 10 minutes, and cooled on ice before centrifugation for 30 minutes at 15 000 ×g. Samples (40 μl) were boiled with 10 μl of 5× concentrated Laemmli denaturing buffer,16 and electrophoresed in a 12% sodium dodecyl sulphate (SDS) polyacrylamide gel under reducing conditions.16 The proteins were then electrotransferred on to a polyvinylene difluoride (PVDF) membrane (0.45 μm pore size; Amersham, Les Ulis, France) at 250 mA for two hours. The membranes were washed twice with PBS then treated for one hour at room temperature with PBS containing 5% non-fat dry milk to saturate non-specific binding sites. The membranes were then incubated for one hour at room temperature in the same buffer containing the K19M anti-hCCN3 (NOVH) polyclonal antibody (final dilution, 1/1000). After washing in PBS, the PVDF membranes were incubated for one hour at room temperature with a secondary antibody (antirabbit IgG antibody coupled to horseradish peroxidase, final dilution 1/20 000). After further washing in PBS, the immunoreactive proteins were revealed by the Amersham chemiluminescence detection system, according to the manufacturer's instructions.
IN SITU HYBRIDISATION OF CCN3 (NOV) RNA EXPRESSED IN ADULT RAT BRAIN
In situ hybridisation of frozen sections with digoxigenin labelled ccn3 (nov) riboprobes enabled us to establish the distribution of ccn3 (nov) RNA in adult brain rat tissues (figs 1 and 2). In the telencephalon, positive neurones were widely distributed in the cerebral cortex, especially in the temporal cortex (auditary area) and piriform cortex. The striated cortex showed several weakly labelled neurones. Many cells showing strong positive labelling were detected in the CA1 to CA3 regions of the hypocampus, whereas the CA4 region had moderate labelling. The strongest labelling was seen in the amygdala, particularly in the basomedial amygdaloid nucleus.
In the diencephalon, we found high to moderate labelling of thalamic neurones. A strong hybridisation signal was seen in the ventromedial, ventral posterolateral, and mediodorsal nuclei. The cell bodies of positive cells in the zona incerta displayed a moderate to dense accumulation of grains. The hypothalamus showed moderate to high neurone labelling .
The brain stem, spinal cord, and cerebellum showed a low degree of expression of ccn3 (nov) RNA in a variable number of positive cells. The ambiguous and facial nuclei contained a high proportion of moderately labelled neurones. Large cells scattered throughout the pontine reticular formation only showed weak labelling. In the ventral horn of the spinal cord, large motor neurones were positive for ccn3 (nov) expression. In the cerebellum, ccn3 (nov) RNA expressing cells were found mainly in the Purkinje layer.
EXPRESSION OF CCN3 (NOV) IN BRAIN TISSUES IS DEPENDENT UPON DEVELOPMENTAL STAGE IN ADULT RATS
Expression of ccn3 (nov) RNA in the rat brain was measured at different developmental stages, both by in situ hybridisation and RT-PCR. The RT-PCR results showed that ccn3 (nov) expression increased significantly between P5 and P30, reached a plateau between P30 and P60, and decreased from P60 to a basal low level at P150 (fig 3).
In situ hybridisation performed in parallel allowed us to establish that the distribution of ccn3 (nov) positive cells increased with brain differentiation. From P0 to P4, positive neurones were weakly labelled and localised mainly in the facial nucleus, trigemal nucleus, and the ventral and dorsal horn of the spinal cord. At P5, a small number of positive neurones was detected in the thalamic and pyramidal layers of the hippocampus. At P8, the number of positive neurones increased. Labelled cells were widely distributed in the limbic system, including the hippocampus, amygdaloid nucleus, globus pallidus, and lateral septal nucleus. At this stage, the hybridisation signal was clearly seen in the ventral posteromedial thalamic nucleus and ventral posterolateral thalamic nucleus (fig 4), whereas weak labelling was seen in the cerebral cortex and hypothalamus.
At P15, the number of ccn3 (nov) RNA expressing neurones increased in the mediodorsal thalamic, posterior thalamic, dorsal hypothalamic, ventromedial hypothalamic, and paraventricular hypothalamic nuclei, and in the cerebral cortex. At P30, the strongest positive signal was found in the facial nucleus, vestibular nucleus, and the dorsal and ventral cochlear nuclei. The hippocampus, thalamus, and hypothalamus showed an intermediate hybridisation signal. In the positive areas, staining was mostly associated with large and middle sized cells (figs 4 and 5). At P60, an increased positive signal was seen in the cerebral cortex (fig 6). High amounts of ccn3 (nov) RNA were still detected in the pons and medulla, and the hybridisation signal was increased in the giganocellular reticular, suprofacial, and dorsal cochlear nuclei. The labelling of the Purkinje cells was particularly obvious. ccn3 (nov) labelling in the cerebral cortex, the thalamus, and the hypothalamus was identical to that observed at P30.
At P300, the number of ccn3 (nov) positive neurones was greatly reduced. In the cerebral cortex, hippocampus, amygdaloid nucleus, and globus pallidus the positive neurones expressed significantly lower amounts of ccn3 (nov) RNA (data not shown).
DETECTION OF THE CCN3 (NOV) PROTEIN IN ADULT RAT BRAIN
Because the C-terminal peptide of the hCCN3 (NOVH) protein (KNNEAFLQELELKTTRGKM) is extremely well conserved (QNNEAFLQELELKTTRGKM) in the rat protein (rCCN3),14 we used the polyclonal K19M antibody raised against the hCCN3 (NOVH) protein11 to characterise the rCCN3 (NOVR) protein and to establish its distribution in brain tissue.
Immunohistochemistry performed on rat brain sections with the K19M antibody enabled the detection of the CCN3 (NOV) protein in most of the areas that were positive for ccn3 (nov) RNA expression (table 1; fig 7). The endopiriform nucleus, temporal cortex (auditary area), temporal lobus, piriform cortex, and striate cortex stained strongly for CCN3 (NOV). In general, the immunoreactive cells were middle to large sized, and were round, spindle, or pyramidal in shape (fig 8). Distinct CCN3 (NOV) positive cells were also seen in the hippocampus (CA1 to CA4 fields), the thalamus, the hypothalamus, the pons, and the cerebellum. The ventral horn of the spinal cord showed only a limited number of CCN3 (NOV) reactive cells. The CCN3 (NOV) protein was not detected in the ventroposterior thalamic nucleus (medial and lateral parts), which were positive for ccn3 (nov) RNA expression. On the contrary, high amounts of the CCN3 (NOV) protein were detected in the endopiriform nucleus, which was negative for ccn3 (nov) RNA (fig 8).
Western blotting analysis performed on adrenal and brain extracts allowed us to detect different CCN3 (NOV) related proteins. In both extracts, two proteins with apparent molecular masses of 46 and 38 kDa (fig 9) were detected by the K19M antibody. Another fast migrating protein with an apparent molecular mass of 18 kDa was also detected in both extracts, although very faintly in the adrenal. Because hccn3 (nov) and rccn3 (novR) RNA species encode an extremely well conserved protein,14 the 48 kDa protein probably corresponded to the previously described full length hCCN3 (NOVH) protein.11 Although the exact origin of the 38 kDa and 18 kDa proteins remains to be established, they are probably processed isoforms similar to those that have already been reported for CCN2 (CTGF) and CCN3 (NOV).2, 3
The CCN3 (NOV) protein (as with all other members of the CCN family except one) is composed of the following: (1) a signal peptide involved in protein secretion, (2) a module sharing identity with insulin-like growth factor (IGF) binding proteins, (3) a module similar to the C repeat of the von Willebrand factor, (4) a module similar to the thrombospondin TSP1 motif involved in interaction with extracellular matrix proteins, and (5) a C-terminal (CT) motif containing a cystin knot proposed to represent a dimerisation domain.
In human samples, the 48 kDa CCN3 (NOV) isoform usually predominates, and only a small proportion is in the form of the N-terminal truncated isoform (apparent molecular mass, 32–35 kDa).3, 11 Truncated CCN2 (CTGF) and CCN3 (NOV) that are biologically active have been described previously.2, 3 The post translational processes that generate these truncated isoforms remain to be established. However, several lines of evidence suggest that truncation of the CCN proteins might represent a key element in the regulation of their biological activities.3
Because the K19M antibody was raised against a C-terminal peptide of CCN3 (NOV), the 38/40 kDa and 18 kDa proteins detected in the brain and adrenal extracts from adult rats are likely to be N-terminal truncated polypeptides.
The detection of large amounts of N-terminal truncated CCN3 (NOV) protein in the brain tissues is quite unusual and raises several interesting questions as to the origin of this protein. It is tempting to propose that the increased quantities of truncated protein reflects active post translational processing of CCN3 (NOV) taking place in brain tissues, and that the truncated CCN3 (NOV) protein is needed either for terminal differentiation or neuronal function. The two equally abundant proteins giving rise to the 38/40 kDa band doublet might either result from post translational modifications, such as proteolysis17 or glycosylation11 of the CCN3 (NOV) protein, or from alternative splicing of the ccn3 (nov) gene in brain tissues. The identification and biochemical characterisation of the CCN3 (NOV) related proteins detected in rat brain extracts is currently being undertaken.
The distribution of ccn3 (nov) RNA and CCN3 (NOV) protein in tissues of the CNS of adult rats overlapped with the expression pattern established previously in the human developing embryo.15 In most cases, a good correlation was seen both for RNA and protein localisation. However, in some cases, large quantities of CCN3 (NOV) protein were detected in areas (such as the endopiriform nucleus) where very low amounts of RNA were expressed. This situation, which is reminiscent of that described previously in human kidney glomeruli,11 suggests that the CCN3 (NOV) protein either accumulates or is stabilised at these sites. Whether the same biological function of CCN3 (NOV) is required at both sites remains to be established and is under current investigation.
Because large amounts of CCN (NOV) are detected in sensitive areas, preliminary experiments were performed to investigate whether the expression of ccn3 (nov) was affected by different olfactory states (B-Y Su and W-Q Cai, 2000, personal communication). In the first model, volatilised ammonia water was used to stimulate the olfactory system of the rats. Increased expression of the ccn3 (nov) gene was seen in the anterior olfactory nucleus, frontal cortex, and polymorphic layer three hours after contact. ccn3 (nov) expression remained particularly high for two days and decreased at day 3. In the second model, exposure of rats to formaldehyde gas for three hours resulted in a sharp decrease of ccn3 (nov) expression for 12 hours, after which normal expression was regained. In the third model, the rats were exposed to relatively high concentrations of methyl bromide, which induces the destruction of neurones and sustentacular cells in over 95% of the olfactory epithelium, hence inducing anosmia at a very early stage. Surprisingly, the expression of ccn3 (nov) was strongly induced in the olfactory nucleus, frontal cortex, and polymorphic layer, with a significant change in cell morphology. In the fourth model, anosmia was induced by intranasal perfusion of zinc sulphate. Again, the expression of ccn3 (nov) was strongly induced for three hours and gradually decreased. These results strongly suggested that the CCN3 (NOV) protein might play a role in the olfactory system of the rat and in the response to stress stimulation.
The association of the CCN3 (NOV) protein with the limbic system also raised the possibility that it plays a role in the acquisition of cognitive processes. Preliminary experiments indicated that increased expression of ccn3 (nov) parallelled the increased memory and learning ability of developing rats, as shown by the acquisition and retention of active avoidance and passive avoidance (B-Y Su and W-Q Cai, 2000, personal communication).
Current investigations should enable these preliminary observations to be validated and shed new light on the biological role of CCN3 (NOV) and other members of this new family of proteins.
This work was supported by grant 399 00078 from National Science Founding of China to B-Y Su and grants from ARC (Association pour la Recherche contre le Cancer), Ligue Nationale Contre le Cancer (Comités du Cher et de l'Indre), and PRA (Programme de Recherche Avancée Franco-Chinois) to Professor B Perbal. Thanks to S Gabaron for reading the manuscript and to A Perbal for help and support.
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