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
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.