Mol Pathol 55(4): 250-261

PMC: PMC1187188

PMID: 12147716

doi: 10.1136/mp.55.4.250

A role for CCN3 (NOV) in calcium signalling

^{Laboratoire d’Oncologie Virale et Moléculaire (LOVM), UFR de Biochimie, Université Paris 7-D. Diderot, 2 Place Jussieu, 75005 Paris, France}

^{Department of Microbiology and Immunology, University of Illinois, Chicago, USA}

^{Unité Inserm 339, Hôpital Saint-Antoine, Paris, France}

Correspondence to:
Professor B Perbal, Laboratoire d’Oncologie Virale et Moléculaire (LOVM), UFR de Biochimie, Université Paris 7-D. Diderot, 2 Place Jussieu, 75005 Paris, France;
rf.ueissuj.rcc@labrep

Accepted 2002 May 14.

Abstract

Aims: In animals and humans increased expression of CCN3 (NOV) is detected in tissues where calcium is a key regulator, such as the adrenal gland, central nervous system, bone and cartilage, heart muscle, and kidney. Because the multimodular structure of the CCN proteins strongly suggests that these cell growth regulators are metalloproteins, this study investigated the possible role of CCN3 in ion flux and transport during development, control of cell proliferation, differentiation, and pathobiology.

Methods: The isolation of CCN3 partners was performed by means of the two hybrid system. Yeasts were cotransfected with an HL60 cDNA library fused to the transactivation domain of the GAL4 transcription factor, and with a plasmid expressing CCN3 fused to the DNA binding domain of GAL4. Screening of the recombinant clones selected on the basis of leucine, histidine, and tryptophan prototrophy was performed with a β-galactosidase assay. After the interaction between CCN3 and its putative partners was checked with a GST (glutathione S-transferase) pull down assay, the positive clones were identified by cloning. To establish whether the CCN3 protein affected calcium ion flux, a dynamic imaging microscopy system was used, which allowed the fluorometric measurement of the intracellular calcium concentration. The proteins used in the assays were GST fused with either CCN3 or CCN2 (CTGF) and GST alone as a control.

Results: The two hybrid system identified the S100A4 (mts1) calcium binding protein as a partner of CCN3 and the use of the GST fusion proteins showed that the addition of CCN3 and CCN2 to G59 glioblastoma and SK-N-SH neuroblastoma cells caused a pronounced but transient increase of intracellular calcium, originating from both the entry of extracellular calcium and the mobilisation of intracellular stores.

Conclusions: The interaction of CCN3 with S100A4 may account, in part, for the association of CCN3 with carcinogenesis and its pattern of expression in normal conditions. The increased intracellular calcium concentrations induced by CCN3 and CCN2 both involve different processes, among which voltage independent calcium channels might be of considerable importance in regulating the calcium flux associated with cell growth control, motility, and spreading. These observations assign for the first time a biological function to the CCN3 protein and point out a broader role for the CCN proteins in calcium ion signalling.

Keywords: CCN3 family of genes, NOVH, connective tissue growth factor, S100A4, calcium signalling, calcium channels, store operated channels

Abstract

The CCN3 (NOV) protein is a member of the CCN (CYR61 (cysteine rich), CTGF (connective tissue growth factor), and NOV (nephroblastoma overexpressed)) family of regulatory proteins.^1–⁴ With ELM1, rCOP1, and WISP (wint induced secreted proteins)^5–⁷ the CCN family now consists of six members. The ccn3 gene was originally described as a target for MAV (myeloblastosis associated virus) in avian nephroblastomas,⁸ which represent a unique model of the Wilms’s tumour.⁹ Although its expression was found to be greater in all avian tumours than in normal kidneys, ccn3 was only disrupted in one nephroblastoma and insertional activation of MAV in the vicinity of ccn3 is not a common theme in nephroblastoma (CL Li et al, unpublished data, 2002).

Similar to other members of the CCN family, the CCN3 protein shows some striking features. It is composed of five distinct structural modules encoded by separate exons and contains a total of 38 cysteine residues, the position of which in the four modules has been conserved both throughout evolution and among the different members of the CCN family.¹⁰ The structural conservation of CCN3 in the different species in which it has been identified suggests that this protein fulfills important function(s) that are dictated by a common spatial organisation. It is noteworthy that in the first two modules of CCN3, a total of 11 proline residues are found at the same position in species as far apart as xenopus and humans (fig 1 ▶ ▶). The signal peptide at the N-terminus of the CCN proteins is responsible for the secretion of the full length CCN3 protein, which can be released into the extracellular matrix or remain at the cell membrane.^11,¹² There are increasing numbers of reports of the existence of CCN isoforms lacking one or two of the basic modules. The N-truncation of CCN3 resulting from MAV insertion in target cell DNA conferred on the CCN3 deprived of signal peptide and module 1 (IB; sharing partial identity with insulin-like growth factor binding proteins) a transforming potential,⁸ and high concentrations of an N-truncated CCN3 isoform, deprived of modules 1 and 2 (VWC; sharing identity with the type C repeat of Von Willebrand factor), are detected in the nucleus of cancer cells.¹³ Module 4 (CT; sharing identity with growth factors) is not present in CCN5 (rCOP/WISP2 and CTGFL),^5–^7,¹⁴ and module 2 is absent in WISP1v, a short version of CCN4 (WISP1) expressed in gastric carcinoma.¹⁵ These observations raise interesting questions as to the biological functions of these different isoforms and the mechanisms by which they are generated.

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Figure 1

Part 1: Amino acid sequence alignment in CCN3 proteins. The amino acid sequence of CCN3 proteins from different species has been aligned with the interalign program (University of Sandford, USA). Those residues whose position is conserved throughout evolution are shaded in black. The 38 conserved cysteine residues and conserved prolines are shaded in grey. The position and sequences of the TSP1 (thrombospondin type 1 repeat) and CT (cystin knot) motifs have been indicated in bold above the CCN3 consensus sequences for each domain.

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Figure 1

Part 2: Amino acid sequence alignment in CCN3 proteins. The amino acid sequence of CCN3 proteins from different species has been aligned with the interalign program (University of Sandford, USA). Those residues whose position is conserved throughout evolution are shaded in black. The 38 conserved cysteine residues and conserved prolines are shaded in grey. The position and sequences of the IB (insulin-like growth factor binding related proteins) and VWC (von willebrand type C repeat) motifs have been indicated in bold above the CCN3 consensus sequences for each domain.

The ccn3 gene spans approximately 7.5 kb of DNA (fig 2 ▶) and maps to human chromosome 8q24.¹⁶ Its expression in normal conditions is under tight spatial and temporal regulation. In humans, rodents, and birds the expression of ccn3 during normal development has been associated with the polarised differentiation of different cell types in various systems, including cartilage and bone, central nervous system, kidney, striated muscle, and cardiac muscle.^4,^17–²⁰ The major sites of ccn3 expression in humans, rodents, and chickens were identified by in situ hybridisation and immunocytochemistry as the zona fasciculata of the adrenal, neurones and astrocytes in the nervous system, ganglia, and fusing muscle cells.^4,^17–²⁰ In pathological conditions, the expression of both human ccn3 mRNA species and CCN3 protein is impaired in many different types of tumours (see Perbal⁴ for a review). In neuroblastomas (B Perbal et al, unpublished data, 1997), Wilms’s tumours,¹⁷ osteosarcomas, and chondroblastomas,^21,²² the expression of ccn3 was a marker of differentiation, whereas in renal cell carcinomas,²³ prostate carcinomas,²⁴ and Ewing’s tumours,²¹ the expression of ccn3 was associated with increased cellular proliferation. We have recently reported that CCN3 has antiproliferative activity in glioblastomas and that its expression in Ewing’s sarcoma is associated with a greater risk of developing metastasis.^21,²⁵

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Figure 2

Distribution of sequences encoding the ccn3 gene and multimodular organisation of the CCN3 protein. (A) Schematic representation of the exon distribution in the human ccn3 gene. The sizes are given in base pairs. Boxed sequences are present in the human ccn3 cDNA clone used in previous studies (see Perbal⁴ for review). (B) Schematic representation of the five structural modules of the CCN3 protein. The signal peptide (SP) is encoded by exon 1 (e1) and the domains 1–4 (D1–4) are encoded by exons 2–5 (e2–5). The length of each domain is indicated in amino acid residues. The number of cysteine residues present in each domain is shown in square brackets. The positions of amino acid sequence motifs sharing identity with insulin-like growth factor binding related proteins (IB), von Willebrand type C repeat (VWC), thrombospondin type 1 repeat (TSP1), and cystin knot (CT) are indicated below each module. The consensus sequence for each motif is indicated in fig 1 ▶ ▶.

The variety of expression sites and activities attributed to the CCN proteins has led to the proposal⁴ that the different biological properties of the CCN proteins result from combinatorial events and depend upon their association with multiple partners, the bioavailability of which may vary greatly from one site to the other.

“Our observations raise the possibility that CCN3 may be interacting with a broad range of proteins containing epidermal growth factor-like repeats, the functions of which have been reported to be dependent upon calcium fixation”

We have reported previously that the secreted CCN3 protein interacts with fibulin 1C and that the nuclear CCN3 isoform interacts with the rpb7 subunit of RNA polymerase II.^13,²⁶ More recently, we have also shown that CCN3 interacts with the NOTCH1 protein.^26a We established that the interaction of CCN3 with both fibulin 1C and NOTCH1 occurred at the level of epidermal growth factor (EGF)-like repeats, which are thought to participate in the regulation of matrix and soluble protein interactions. Our observations raise the possibility that CCN3 may be interacting with a broad range of proteins containing EGF-like repeats, the functions of which have been reported to be dependent upon calcium fixation.

Here, we report that CCN3 physically interacts with the calcium binding protein S100A4 and induces a significant transient increase of intracellular calcium in glioblastoma and nervous system cells, suggesting that CCN3 might play a pivotal role in calcium dependent signalling.

Take home messages

Acknowledgments

We are indebted to Professor B Roizman for hosting Professor B Perbal and providing facilities to run the two hybrid screening. Thanks also to Dr R Brandimarti and JM Dupret for stimulating discussion and to A Perbal for her administrative help. The technical assistance of Y Gabadhino is acknowledged. This work was funded by grants to BP from Association pour la Recherche contre le Cancer (ARC), Ligue Nationale contre le Cancer (Comités du Cher et de l’Indre), Ministére de l’Education Nationale, and de la Recherche et de la Technologie (MENRT). CLL and VM were recipients of a fellowship from the Ligue Nationale Contre le Cancer (Comités du Cher et de l’Indre).

Acknowledgments

Abbreviations

CTGF, connective tissue growth factor
DTT, dithiothreitol
EGF, epidermal growth factor
GST, glutathione S-transferase
IP3, inositol 1,4,5-triphosphate
MAV, myeloblastosis associated virus
NOV, nephroblastoma overexpressed
PAGE, polyacrylamide gel electrophoresis
PBS, phosphate buffered saline
PBSc, calcified phosphate buffered saline
PCR, polymerase chain reaction
PMSF, phenylmethylsulfonyl fluoride
SDS, sodium dodecyl sulfate
WISP, wint induced secreted proteins

Abbreviations

Notes

This paper follows the nomenclature proposed by the International CCN Society (http://ccnsociety.jussieu.fr).

Notes

This paper follows the nomenclature proposed by the International CCN Society (http://ccnsociety.jussieu.fr).

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