And the CCN Family of Genes: Structural and Functional Issues

J Clin Pathol: Mol Pathol 2001;54:57–79 57

Review Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from

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

B Perbal

Abstract terminus is expected to confer on the The CCN family of genes presently con- truncated polypeptide constitutive posi- sists of six distinct members encoding tive or negative activities. proteins that participate in fundamental (J Clin Pathol: Mol Pathol 2001;54:57–79) biological processes such as cell proliferation, attachment, migration, diVerentia- Keywords: cancer; diVerentiation; signalling; develop- tion, wound healing, angiogenesis, and ment; angiogenesis; fibrosis; ctgf; cyr61; wisp; CCN several pathologies including fibrosis and tumorigenesis. Whereas CYR61 and CTGF were reported to act as positive The regulation of cellular growth, diVerentia- regulators of cell growth, NOV (nephrob- tion, and death is central to a myriad of lastoma overexpressed) provided the first biological processes that govern the appear- example of a CCN protein with negative ance, maintenance, and termination of life. regulatory properties and the first exam- Although many genes whose products are ple of aberrant expression being associ- reported to play a key role in cell growth con- ated with tumour development. The trol have been described, several basic regula- subsequent discovery of the ELM1, tory circuits involve proteins yet to be identi- rCOP1, and WISP proteins has broadened fied. http://mp.bmj.com/ The discovery of three structurally related the variety of functions attributed to the genes whose expression was modulated by CCN proteins and has extended previous growth factors and altered in cancer cells has observations to other biological systems. led to the recognition of a new family of cell This review discusses fundamental ques- growth regulators with unique biological prop- tions regarding the regulation of CCN erties. gene expression in normal and pathologi- Bork1 first proposed that these genes should on September 29, 2021 by guest. Protected copyright. cal conditions, and the structural basis for be designated the CCN family of genes, which their specific biological activity. After dis- stands for Cyr61 (cystein rich protein), Ctgf cussing the role of nov and other CCN (connective tissue growth factor), and Nov proteins in the development of a variety of (nephroblastoma overexpressed gene). The diVerent tissues such as kidney, nervous existence of this family of genes has recently system, muscle, cartilage, and bone, the been reinforced by the discovery of three new altered expression of the CCN proteins in members (see below). various pathologies is discussed, with an For many years, the CCN proteins seemed emphasis on the altered expression of nov not to share much more than their strikingly in many diVerent tumour types such as conserved primary structure and no physi- Wilms’s tumour, renal cell carcinomas, ological or biological properties had been prostate carcinomas, osteosarcomas, clearly assigned to them. chondrosarcomas, adrenocortical carci- The bulk of the recent results presented at nomas, and neuroblastomas. The possible the first international workshop on the CCN Laboratoire d’ use of nov as a tool for molecular medicine family of genes and the increasing pace at Oncologie Virale et is also discussed. The variety of biological Moléculaire, UFR de which papers are being published in this field Biochimie, Université functions attributed to the CCN proteins point to the increased attention being focused Paris 7-D. Diderot, 2 has led to the proposal of a model in which on these proteins. Their roles are being clearly Place Jussieu, 75 005 physical interactions between the amino established in fundamental biological proc- Paris, France and carboxy portions of the CCN proteins esses such as cell proliferation, attachment, B Perbal modulate their biological activity and migration, and diVerentiation, wound healing, Correspondence to: ensure a proper balance of positive and angiogenesis, and several pathologies including Professor Perbal negative signals through interactions with fibrosis and tumorigenesis. Furthermore, it [email protected] other partners. In this model, disruption appears that in spite of their highly conserved Accepted for publication of the secondary structure of the CCN structure, these proteins display non- 9 January 2001 proteins induced by deletions of either redundant functions, which are apparently

www.molpath.com 58 Perbal

cyr61 Cystein rich 61 CEF10 (chicken embryo fibroblasts) (Ck) nuclear run on transcription assays. This (1p22.3) βIG1 (TGFβ induced gene) cyr61 (Mu) aspect must be approached with extreme cau-

tion because it has a pronounced impact on Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from ctgf Connective tissue Ctgf (Ck) ctgf (Sw) ctgf (Xn) our way of thinking when analysing the (6q23.1) growth factor bIG2 (TGFβ induced gene) (Mu) biological properties of potential regulatory Fisp 12 (fibroblast inducible secreted protein) (Mu) molecules, such as CCN proteins, whose nov Nephroblastoma nov (Ck) novM (Mu) nov (Sw) nov (Xn) (8q24.1) overexpressed expression is developmentally regulated in a spatiotemporal way. WISP-1 Wnt-1 induced Elm1 (expressed in low metastatic type 1 cells) (Mu) (8q24.1–24.3) secreted protein Wisp 1 (Mu) GENES THAT WERE ISOLATED ON THE BASIS OF THEIR DIFFERENTIAL EXPRESSION IN QUIESCENT WISP-2 Wnt-1 induced rCop-1 (expression lost after transformation) (Mu) AND SERUM STIMULATED CELLS (20q12–13) secreted protein HICP (heparin induced CCN-like protein) Under starvation conditions (incubation in low Wisp 2 (Mu) serum concentration), confluent cells stop WISP-3 Wnt-1 induced growing and reach the G0 state of the cell (6q23.1) secreted protein cycle. G1 phase re-entry of the starved cells Figure 1 The CCN family of genes. The names and chromosomal localisations of the upon treatment with serum triggers the stimu- human CCN genes are indicated in the left column. lation of immediate early genes, whose expres- required at diVerent locations and at diVerent sion proceeds shortly after serum stimulation, times along the same biological pathways. without the need for de novo protein synthesis Understanding the structural and functional (the immediate early terminology was used by bases for the molecular diversity of the CCN comparison with the ordered expression of family is an important challenge. It will set the viral genes upon infection of eukaryotic cells). stage for understanding the role of these genes Characterisation of these immediate genes in the control of cellular proliferation, diVeren- provided important clues as to the type of tiation, and death, thereby allowing the use of genes responsible for the regulation of master these genes and proteins in modern biomedi- switches involved in the control of cell growth. cal technologies for early diagnosis and This experimental approach, which had pretreatment. viously led to the discovery of a variety of Because recent publications have dealt in signalling proteins and transcription factors, detail with the description of the CCN genes led to the isolation of the murine cyr61 and and corresponding proteins,2–4 this review will fisp12 genes. deal with some fundamental questions regard- Fibroblastic cells of diVerent origins have ing the expression of CCN proteins in normal been used to analyse gene expression during and pathological conditions, and the structural serum induced G0/G1 transition. DiVerential basis for their specific biological activity, with screening of a cDNA library prepared from

an emphasis on studies performed on the nov serum stimulated mouse BALB/c3T3 fibro- http://mp.bmj.com/ gene in my laboratory. blasts led to the isolation of cyr61, which encodes a secreted 379 residue polypeptide of Isolation of the CCN genes apparent molecular mass 42 kDa,5 whereas a The CCN family of genes presently consists of similar strategy performed with mouse six distinct members in the human (fig 1), the NIH3T3 cells led to the isolation of fisp12, orthologues of which have been described in which encodes a secreted 348 residue polypep- several diVerent species ranging from xenopus tide of 37 kDa.6 The early expression of these on September 29, 2021 by guest. Protected copyright. to rodents. genes in the cell cycle without the need of prior The way in which the CCN genes were iso- protein synthesis categorised them as immedi- lated might provide valuable information for ate early genes. understanding their diVerential expression and Early studies established that immediate biological properties. The diVerent strategies early genes were expressed transiently and were that have led to the isolation of CCN genes either required for the G0–G1 transition or for include: subtractive hybridisation and diVeren- G1 progression to S phase. In the first group, tial display of RNAs whose expression is c-fos expression is induced between 20 and 90 aVected in normal cells stimulated to grow by minutes and decreases to undetectable levels serum, growth factors, and viruses, or in after two hours, whereas in the second group, tumour cells as compared with their normal c-myc expression is induced between 45 counterparts. minutes and three hours and is detectable up to The diVerential screening strategies are six hours after serum stimulation. based on the fact that RNA species are under Both cyr61 and fisp12 showed the same represented or lacking in one of the two situa- rapid induction pattern, starting 30 minutes tions analysed. Although most authors at- after serum stimulation and peaking between tribute increased RNA values to stimulation of one and two hours after stimulation. In both transcription, it is important to keep in mind cases, short half life (30 minutes for cyr61, that RNA metabolism can be aVected dra- 10–15 minutes for fisp12) RNA species were matically by the events that lead to transfor- produced, and sustained expression was ob- mation or induction of proliferation in re- served over several hours after the addition of sponse to viral infection and growth factors. serum to starved cells. As reported for other Only in a few instances was it possible to check immediate early genes, the decrease in mRNA whether increased or decreased gene expres- values requires protein synthesis. sion was indeed responsible for the diVerential Therefore, it appears that cyr61 and fisp12 levels of transcripts detected, by means of represent a new class of genes that are induced

www.molpath.com NOV and the CCN family of genes 59

fos myc cyr61/ctgf cyr61 elm1 nov cop1 Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from

0 20 min 30 min 1 h 2 h 4 h 6 h 8 h 24 h Figure 2 Kinetics of CCN gene expression upon stimulation of cell growth.

in the sequential cellular response to mitogenic The âIG-M2 gene was later cloned as ctgf (see signals. The CYR61 and FISP12 proteins are below). 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 GENES THAT WERE IDENTIFIED ON THE BASIS OF cyr61 RNA synthesis was seen between six and THEIR ALTERED EXPRESSION IN TUMOURS 10 hours after serum stimulation, suggesting Tumour cells are thought to be altered at the that the expression of this protein might also be level of the regulatory signals controlling required at a later stage of cell cycle progres- normal cell growth. They have provided an sion (fig 2). unequalled source of biological material in which to study the molecular events leading to uncontrolled and eventually invasive prolifera- http://mp.bmj.com/ GENES THAT WERE IDENTIFIED ON THE BASIS OF tion. THEIR INCREASED EXPRESSION IN CELLS TREATED BY TGFâ1 A considerable number of studies performed Isolation of nov with transforming growth factor â (TGFâ) Early studies performed in our laboratory had have established the key role of this signalling established that the chicken myeloblastosis molecule in the regulation of cell growth and associated virus (MAV) induced nephroblas- on September 29, 2021 by guest. Protected copyright. diVerentiation. toma constitutes a unique model of Wilms’s For many years, it has been proposed that tumour, a paediatric tumour aVecting approxi- the biological eVects of TGFâ are mediated by mately one in 10 000 children.8 Histologically, a variety of downstream eVectors. To identify Wilms’s tumours and the avian nephroblasto- genes in which transcription was aVected by mas are very much alike, with varying amounts TGFâ1, a cDNA library was prepared from of heterotypic diVerentiation occurring, along AKR-2B mouse embryo fibroblasts grown to with the development of kidney tumours.9 For 90% confluency and treated with cyclohex- this reason, Wilms’s tumour is often considered imide before incubation for six hours in the as a “diVerentiated” tumour.8 presence of 10 ng/ml TGFâ1. DiVerential MAV belongs to a group of tumorigenic rep- screening of this library with labelled cDNA lication competent retroviruses that do not probes obtained from untreated and TGFâ1 carry oncogenic sequences of cellular origin treated cells permitted the cloning of several and usually induce tumours by integrating in genes whose expression was increased in the vicinity of growth regulatory genes in the TGFâ1 treated cells.7 Among them, two struc- host genome.10 Cloning of the proviral integra- turally related genes were designated âIG-M1 tion sites of avian and murine retroviruses led and âIG-M2. Nucleotide sequencing estab- to the identification of most of the oncogenes lished that of âIG-M1 was identical to the pre- and tumour suppressors that turned out to be viously described cyr61. major players in the control of cellular growth. Induction of âIG-M1 in growth arrested This strategy is particularly useful because it cells, as opposed to the induction of CEF10 in does not rely on any particular type of function cells stimulated to divide, provided the first for the target gene. evidence to suggest that this protein was acting Because the genetic basis of Wilms’s tumour in quite diverse biological processes.7 development is complex and has not been The sequencing of âIG-M2 revealed that it completely elucidated,11 the avian tumours was closely related to âIG-M1 in structure. provided a unique system in which to identify

www.molpath.com 60 Perbal

the genes underlying the aberrant diVerentia- alterations as a consequence of rearrangements tion process and the molecular events involved in this chromosome region might also involve

in the initiation and maintenance of the aberrant expression of nov. Although our Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from tumour state. Our analysis of MAV integration search for such cases has not proved successful sites enabled us to establish that in one tumour as yet, this point certainly deserves more atten- the MAV proviral genome was integrated into tion and should be the matter of a thorough nov,12 a gene sharing structural identity with analysis. The recent mapping of WISP-1 to the cyr61, cef10, and ctgf. Based on the observa- same region (see below) reinforces the possi- tion that the amounts of nov transcripts were bility that alterations of CCN genes might play significantly increased in avian nephroblasto- a key role in some pathologies involving mas, we assumed that the alteration of nov was chromosome 8q24.1, previously attributed to related to tumour development. Whether it was alterations of c-myc. a cause or a consequence still remains to be Only recently, the aberrant expression of established. CCN genes in tumours and transformed cells The discovery of nov had three immediate has provided the basis for the identification of implications: three new members of the family. Again, it is (1) The structural relation that had been not surprising that the various strategies that reported between cyr61 and ctgf was were used led to the isolation of genes involved extended to another gene, thereby leading at diVerent levels of tumorigenesis and growth to the concept of a new family of genes1 (a control. family is usually defined as a group of at least three subjects sharing common char- Isolation of Elm1 acteristics). In that case, diVerential display methodology (2) nov provided the first example of aberrant was applied to isolate genes expressed at diVer- expression of a CCN gene being associated ent levels in low (C23) and high (M2) with tumour development, therefore indi- metastatic K-1735 murine metastatic cells.14 It cating that the biological properties of this is important to recall that tumour cell popula- new class of growth regulators extended tions are heterogenous with regard to their beyond the normal response to growth ability to disseminate and give rise to metas- stimulation. tases. Low and high metastatic tumour cells (3) The growth inhibitory eVect of nov on that are both part of the tumour cell population chicken embryonic fibroblasts (CEFs) also are believed to exhibit diVerent biological provided the first evidence that the CCN properties, resulting from the diVerential ex- family of genes contains negative regula- pression of genes governing fundamental proc- tors and suggested that nov might counter- esses such as adhesiveness, spreading, motility, balance the stimulatory eVects of the and proliferation.

immediate early CCN proteins. DiVerential screening of RNA species ex- http://mp.bmj.com/ For many years, these last two features did pressed in the low and high metastatic K-1735 not receive the attention that they deserved. variants led to the isolation of Elm1, a gene One possible explanation is that it seemed encoding a 367 amino acid protein, the expres- somewhat paradoxical that a negative regulator sion of which was detected in low metastatic of growth should be overexpressed in tumour but not high metastatic cells.14 The acquisition cells prone to active division and proliferation. of a metastatic potential is also accompanied by In fact, the very nature of the nephroblastoma profound modiﬁcations of the post transcrip- on September 29, 2021 by guest. Protected copyright. provides a satisfactory explanation for this tional events that can result in an increased apparent contradiction. As stated above, neph- stabilisation or degradation of RNA tran- roblastomas contain an increased number of scripts. To assess the biological importance of developed structures and heterotypic tissues, the expression of Elm1 being associated with a the diVerentiation of which also requires nega- low metastatic potential, the eVects of ectopic tive regulatory factors. It is therefore not so expression of Elm1 on the tumorigenic poten- heretical that a gene involved in normal diVer- tial of high metastatic K-1735 M2 cells were entiation processes (see below) is aberrantly studied.14 For this purpose, an Elm1 expression expressed in highly diVerentiated tumours. vector was used to transfect and select G418 The inhibition of growth resulting from resistant K-1735 M2 derived stable transform- retroviral driven expression of nov in secondary ants expressing diVerent amounts of Elm1 CEFs was not observed upon infection of RNA, from no expression up to six times the BALB/c 3T3 mouse cells with retroviral amount of Elm1 expressed in the C23 low constructs expressing the murine orthologue of metastatic variant. nov (P Jolicoeur, 1994, personal communica- The results obtained indicated that the tion; EL Suchman et al, 1995, unpublished expression of Elm1 had pronounced eVects on results), therefore suggesting that the growth the in vivo properties of transfected cells, inhibitory properties of nov might be eVective whereas little eVect was seen on the ex vivo only in early passage cells, and not in cell lines. growth properties of the transfectants, which Expression of nov was neither detected in qui- only showed a slight increase of doubling time escent 3T3 mouse cells, nor in human and slight decreased saturation density.14 ﬁbroblastic cells (S Kyurkchiev and B Perbal, The in vivo eVects were much more 1999, unpublished results). informative because the expression of Elm1 The location of nov on human chromosome appeared to aVect several parameters.14 First, 8q24.1, distal to c-myc,13 raised the possibility the absolute number of tumours obtained for a that some pathologies attributed to c-myc given period of time was dramatically reduced

www.molpath.com NOV and the CCN family of genes 61

by the expression of Elm1. Second, as a result CCN gene expression obtained in embryonic of the pronounced inhibitory eVect of Elm1 on ﬁbroblasts and established cell lines.

the growth properties of transfected cells in Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from vivo, the time needed for tumours to reach a Isolation of WISP genes given size was considerably increased. Third, The suppressive subtractive hybridisation the transfectants that expressed high amounts (SSH) strategy was used to identify targets of of Elm1 gave rise to a much smaller number of the Wnt-1 protein by screening RNAs diVeren- lung metastases than those expressing low lev- tially expressed in C57MG mouse mammary els of Elm1. epithelial cells and their transformed counter- It therefore appeared that Elm1 was another part obtained after infection with a wnt-1 example of a CCN protein with a negative type expressing retrovirus.16 The WNT-1 protein of activity on cell growth. The induction of was originally isolated as an insertion site for Elm1 expression seen three hours after serum mouse mammary tumour virus in murine stimulation of BALB/c 3T3 cells (fig 2), at a mammary tumour virus (MMTV) induced much later time than cyr61 and ctgf, would mammary adenocarcinomas. It is a member of argue for a role for Elm1 at a relatively late an expanding family of signalling proteins stage of the G1 phase.14 involved in the control of normal cell proliferation and in tumorigenesis. Isolation of rCOP1 Two genes (WISP1 and WISP2),16 the The identification of genes responsible for expression of which was upregulated in wnt-1 oncogenic transformation of normal cells has transformed cells, were shown to be identical to been the subject of many studies over the past the previously described Elm1 and rCOP1 20 years. Despite the considerable amount of genes, respectively. Their expression was not information that has emerged from these stud- increased by overexpression of wnt-4, which ies, most of the molecular events that govern does not induce morphological transformation progression from the normal to immortalised of the C57MG cells, therefore suggesting that and transformed stages remain to be uncov- WISP expression might play a role in the trans- ered. Thus, the functional bases for the formation process. The WISP-1 gene has been cooperation between the p53 tumour suppres- mapped to chromosome 8q24.1–8q24.3 in the sor and activated H-ras oncogene in inducing vicinity of nov, whereas the WISP-2 gene has oncogenic transformation are still obscure. been mapped to chromosome 20q12–20q13.1, The diVerential display strategy that was a region frequently amplified in node negative used to screen mRNAs expressed in normal rat breast cancers with poor prognosis and in embryo fibroblasts and their counterparts many colon cancers. transformed by p53 mutants in cooperation The expression patterns and biological with activated H-ras has allowed the identifica- properties of WISP1 and WISP2 have both

tion of rCOP1, a new gene of the CCN family, reinforced the idea that the biological proper- http://mp.bmj.com/ encoding a 250 amino acid protein, the expres- ties of CCN proteins might be dependent upon sion of which was downregulated in trans- the cellular context and have pointed out the formed cells.15 complexity of the regulatory circuitry in which The expression of rCOP1 was detected in these proteins function. mouse BALB/c 3T3 cells but was repressed in For example, the upregulation of rCOP1/ several transformed derivatives, and retroviral WISP-2 in wnt-1 transformed C57MG epithe- driven ectopic expression of rCOP1 estab- lial cells16 was in apparent contradiction to the on September 29, 2021 by guest. Protected copyright. lished that rCOP1 expression had a strong downregulation of this gene upon transforma- negative eVect on the growth of rat trans- tion of BALB/c 3T3 fibroblastic cells.15 This formed cells, leading to a cell population show- suggests that the CCN proteins may have ing enrichment in sub-G1 DNA content. Fur- opposing functions in diVerent cell types. Fur- thermore, rCOP1 overexpression in stably thermore, in primary human colon adenocarci- transfected rat cells transformed by p53 and nomas, the expression of WISP-2 was signifi- H-ras significantly reduced their tumorigenic- cantly decreased, and it was not detected in the ity when tested by subcutaneous injection in epithelial tumour cells of mammary carcinoma athymic mice. obtained from wnt-1 transgenic mice.16 In the These results15 identified rCOP1 as the third WISP positive stroma, spindle shaped cells member of the CCN family to exhibit proper- adjacent to capillary vessels stained positive for ties of a negative regulator of growth. WISP-2 expression in these tumours. Interestingly, rCOP1 expression was de- Similarly, the expression of Elm1/WISP-1, tected only after repeated passages of rat and which had been reported to suppress the meta- mouse embryo fibroblasts and not in primary static potential of murine melanoma cells,14 fibroblasts.15 In addition, adult rat tissues did was significantly increased in most human not appear to contain any rCOP1 RNA colon carcinomas.16 Increased expression of species, indicating that the expression of WISP-1 was seen in the stroma of mouse rCOP1 was not induced as the result of growth mammary carcinomas produced in wnt-1 arrest in non-proliferating, fully diVerentiated transgenic mice (which stained strongly posi- tissues. tive for WISP-1 expression), whereas tumour This is another example of the expression of epithelial cells expressed low amounts of some CCN genes being susceptible to events WISP-1.16 leading to the “establishment” of cell lines (see In apparent contradiction to the tumour above for nov) and stresses the fact that care suppressor type of activity assigned to Elm1,14 should be taken when comparing patterns of overexpression of WISP1 induced accelerated

www.molpath.com 62 Perbal

growth and morphological transformation of cyclic expression of CEF10, which was not NRK-49F ﬁbroblastic cells.16 Furthermore, observed with NY315, might be related to the

expression of WISP-1 was found to increase transformation process induced by v-src. Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from the tumorigenicity of two NRK-49F cell lines In another set of experiments, the diVerential established after infection by retrovirus con- display strategy was applied to cDNAs pre- structs expressing WISP-1. Because these cells pared from tsNY72 RSV infected CEFs grown would not grow without anchorage, the overex- at the non-permissive temperature and either pression of WISP-1 was not suYcient to allevi- shifted to the permissive temperature or main- ate the need for proper interactions with the tained at the non-permissive temperature. extracellular matrix and attachment.16 Screening of the genes diVerentially expressed in these cells revealed that the expression of the GENES THAT WERE IDENTIFIED ON THE BASIS OF nov gene was downregulated upon the stimula- v-src 18 THEIR DIFFERENTIAL EXPRESSION IN RSV tion of CEF growth induced by pp60 . TRANSFORMED FIBROBLASTS Nuclear run on assays performed on serum Transformation of CEFs by pp60v-src of Rous starved uninfected CEFs and wild-type sarcoma virus (RSV) is known to alter, either pp60v-src transformed CEFs established that the positively or negatively, the transcription of a diVerential rate of nov transcription in isolated variety of regulatory and signalling proteins. nuclei did not account for the diVerence in the Because the expression of pp60v-src had been steady state of nov transcripts measured in qui- reported to block the diVerentiation of myo- escent and transformed CEFs. Therefore, the genic cells, it was proposed that genes whose increase levels detected in uninfected cells expression is decreased by pp60v-src transforma- might result from an accumulation of the nov tion might act to maintain or induce the diVer- transcripts as a result of the long half life (eight entiated state of target cells. hours) of the nov transcripts in quiescent cells. The strategy used to characterise genes The use of a mutant strain expressing a ther- acting in the transduction pathways activated mosensitive pp60v-src revealed that upon shifting for transformation was usually based on the to the non-permissive temperature, there was a use of CEFs transformed by a mutant of decline in the numbers of nov transcripts RSV (tsNY72) expressing a thermosensitive detected in transformed cells between four and pp60v-src. Upon shifting to the permissive eight hours, with a dramatic reduction after 12 temperature (35°C), the constitutive tyrosine hours, and an absence of transcripts after 24 kinase activity of pp60v-src induces the cascade hours.18 A similar decline in nov expression was of molecular events that lead to oncogenic seen when CEFs were stimulated to proliferate transformation. either after transformation by various onco- In the first set of experiments, a cDNA genes or after infection with the non- library was prepared from tsNY72 infected transforming myristoylation defective NY315 18 cells maintained at the permissive temperature mutant. Therefore, the downregulation of http://mp.bmj.com/ for four to six hours in the presence of 75 µM nov expression induced by the stimulation of cycloheximide, and screened with a subtracted cell growth and the high concentrations of probe made of labelled cDNA from tsNY72 NOV that were detected in quiescent cells were infected cells grown at 35°C hybridised against in striking contrast to the situation observed for mRNA from unstimulated cells. Clones ob- other CCN genes, and established that nov was tained with this strategy corresponded to stabi- not an immediate early gene (fig 2). The lised mRNA whose expression had been requirement for protein and RNA synthesis in on September 29, 2021 by guest. Protected copyright. induced by pp60v-src. Twelve diVerent immedi- the decrease of nov transcripts upon stimula- ate early genes were induced, including tion of cellular growth indicated that the CEF10, the chicken equivalent of cyr61.17 reduced expression of nov resulted either from Upon serum stimulation of starved CEFs, the synthesis of a negative regulator of nov the pattern of CEF10 expression was similar to transcription or from an increased turnover of that of cyr61 in murine cells (fig 2). However, nov transcripts.18 no late burst of induction was reported for Our observation that the expression of nov CEF10. Whether this discrepancy results from was modulated during the progression of the variations in the protocols and origin of cells cell cycle and reached a peak at the G2 phase used or represents important diVerences in the (S Middendorp and B Perbal, 1995, unpub- biological properties of murine and chicken lished results) suggests that the increased genes is not yet clear. synthesis and accumulation of stable nov tran- The pattern of CEF10 expression in pp60v-src scripts at that late stage might be required for induced cells is interesting. In pp60v-src induced proper diVerentiation. cells, CEF10 expression was found to be mul- tiphasic, with two peaks at one and eight hours GENES THAT WERE ISOLATED ON THE BASIS OF AN post shift, a decrease at 12 hours post shift, and IMMUNOLOGICAL CROSSREACTIVITY OR A 17 a third peak at 24 hours. Under similar STRUCTURAL IDENTITY WITH CCN GENES conditions, most immediate early genes were Polyclonal antibodies raised against platelet persistently induced for 24 hours. When a derived growth factor (PDGF) were used to mitogenic non-transforming myristoylation de- characterise and isolate the protein(s) responsi- fective virus NY315 was used, CEF10 was ble for the PDGF related mioattractant activity induced in a linear mode for a period of 24 that was detected in conditioned medium from hours.17 These observations indicated that human umbilical vein endothelial cells (HU- CEF10 production was increased as a result of VEC).19 This approach has led to the identifi- the pp60v-src growth stimulatory eVect. The cation of an anti-PDGF crossreacting protein

www.molpath.com NOV and the CCN family of genes 63

with a reported apparent molecular mass of which have been conserved throughout the 38 kDa. This protein, which was shown to pos- family members and during evolution, sug-

sess the major chemotactic and mitogenic gested that they might share some kind of Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from activity previously attributed to PDGF, was functionality. designated CTGF (connective tissue growth Until now, most of the studies performed factor).19 Cloning of CTGF revealed that it was with the various CCN genes and proteins have the human orthologue of fisp12 and âIG-M2 been descriptive and have been performed on (see above). As expected from its structural diVerent biological systems. Unfortunately, identity with fisp12, the ctgf gene was shown to only in a very few instances, were studies be rapidly induced by serum in human skin performed in parallel to compare the expres- fibroblasts (fig 2), independently of protein sion or the activity of diVerent members of the synthesis, therefore confirming that it was family. This type of approach might turn out to indeed an immediate early gene.20 be very fruitful in the case of CCN proteins for In agreement with previous studies that led which no biological assay is available as yet. to the discovery of âIG-M2, the ctgf gene, Obtaining this type of information through which is expressed in unstimulated fibro- collaborative eVorts would undoubtedly lead to blasts,21 22 was reported to be induced by TGFâ an integrated view of CCN gene expression in the absence of protein synthesis.72023 From and functions at a very reduced cost. these observations, a potential role for CTGF The use of diVerent systems has increased in fibrotic diseases has progressively emerged, the number of results that appear to be and it is now well established that ctgf mRNA “conflicting” if one does not take into account and protein are overexpressed in fibrotic the variety of physiological conditions consid- lesions of several major organs in humans, ered. There is no doubt that if the CCN including liver, kidney, gingiva, cardiovascular proteins play, as one can guess, key roles in so system, pancreas, bowel, and eye (reviewed by many diVerent fundamental regulatory proc- Brigstock3 and Essam and colleagues4). How- esses, the major issue is understanding the ever, the relation between TGFâ and ctgf is not structural basis for their biological functions. obligatory because several studies have shown This goal will be reached by the combination of that the activation of ctgf expression can diVerent techniques, including purification of proceed independently of TGFâ (reviewed by biologically active CCN proteins, identification Essam and colleagues4). of their targets and cofactors, directed muta- Because of its identification as a downstream genesis of crucial amino acids, the construction target of TGFâ, ctgf has been studied exten- of chimaeras, and the manipulation of genes in sively over the past few years and our growing the context of living animals. knowledge indicates that, as reported for other Before this goal can be achieved, there are CCN genes, its biological properties and regu- issues that remain unresolved and need to be

lation of expression are dependent upon the considered for each member in the context of http://mp.bmj.com/ cellular context and the nature of the cells in the CCN family as a whole. which it is produced and/or acting. Initially Based on the results that we have obtained reported to induce cell proliferation, survival, with nov I will consider below: (1) the relation and extracellular matrix production, CTGF between the structure and subcellular localisa- has also been shown to be apoptotic in human tion of CCN proteins, (2) the mechanisms aortic smooth muscle cells and human breast governing the temporal and spatial regulation cancer MCF-7 cells.24–26 of CCN gene expression, (3) the implication of on September 29, 2021 by guest. Protected copyright. Another experimental approach, based on the CCN proteins in pathological conditions, the screening of expressed sequence tag (EST) and (4) the identification of proteins and databases, with the WISP-1 protein sequence,16 ligands that interact with CCN proteins. allowed the identification of WISP-3, a gene encoding a 354 amino acid new member of the Structure and subcellular localisation of CCN family with slightly diVerent structural CCN proteins features to the other members: it is the only Figure 3 shows the modular structure of the member thus far that does not show the CCN proteins. Although they have a very con- characteristic conservation of cysteines. served multimodular organisation, with four Until recently, very little information was modules sharing identity with insulin-like available about the biological properties of growth factor binding proteins (IGFBPs), Von WISP-3. The WISP-3 gene has been reported Willebrand factor, thrombospondin, and a to be significantly overexpressed in human cystein knot containing family of growth regu- colon carcinoma,16 and putative loss of func- lators, the CCN proteins have distinctive tion mutations in the WISP-3 gene have been biological properties and are diVerentially associated with progressive pseudorheumatoid regulated. dysplasia in humans, an autosomal recessive Because of their partial identity with form of spondyloepiphyseal dysplasia.27 IGFBPs and the low aYnity binding of IGF-I On the one hand, the diVerent systems in and IGF-II to CTGF,28 a few groups in the IGF which the CCN genes have been identified field have attempted to rename the CCN pro- made it obvious that their expression ought to teins using an IGFBP related system. Although be involved in a wide variety of biological proc- it is quite possible that both IGFBPs and CCN esses in normal and pathological conditions. proteins participate in common regulatory However, on the other hand, the striking pathways, there is as yet no functional evidence conservation of their modular organisation to support such a renaming.29 From discus- with 38 cysteins, the position and number of sions at the first international workshop on the

www.molpath.com 64 Perbal

Table 1 Proposed nomenclature for CCN proteins identiﬁed at the N-terminus of NOV and all other CCN proteins. Although increased ex- CCN nomenclature Current names Alternative designations

pression of nov was detected in all nephroblas- Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from CCN 1 CYR61/CCE10 IGFBP-rP 4 tomas tested (including those induced by the CCN 2 CTGF/FISP12 IGFBP-rP 2, Hcs 24 MAV2-O strain, which also induces osteopet- CCN 3 NOV IGFBP-rP 3 CCN 4 WISP-1/ELM 1 rosis), none of them was found to contain a CCN 5 WISP-2/rCOP-1 physically altered nov gene (CL Li et al, 2000, CCN 6 WISP-3 unpublished results).12 CCN names were given according to their chronological identification. It is suggested that a lower The construction of retroviral competent case letter should be used to indicate species when needed (for example, hCCN1 for human ovian recombinants, expressing either the full cyr61). length nov RNA or the truncated version of CCN family of genes, a unifying nomenclature nov expressed in tumour 725, allowed us to is now proposed for the CCN genes and establish that the ectopic overexpression of the proteins (table 1). It is also suggested that the full length nov in actively growing CEFs nomenclature committee of the CCN society (which did not express endogenous nov) inhib- 12 ([email protected]) should be con- ited their growth. Recombinant viruses repli- tacted for the attribution of names for new cated in these cells (as shown by RNA expres- CCN members. sion) and gave rise to viral stocks; the growth The multimodular organisation of the CCN inhibitory eVect of nov did not induce cell proteins raises interesting questions as to the death but rather blocked the cells in the cell participation of each module in the function of cycle, probably at the G2–M transition. On the the full length protein. Either the activities of contrary, the expression of the truncated nov each module add up or they confer on the RNA species did not interfere with the growth whole protein specific functions that might of the CEFs and induced their morphological 12 substitute or add to the function of the transformation. From these results it was individual modules. Such a modular structure concluded that nov was a protooncogene with also suggests that the diVerent modules corres- antiproliferative activity. Therefore, it could be pond to functional domains that can interact concluded that the development of nephroblas- either sequentially or simultaneously with tomas in chickens was associated with an other partners, and that the final biological increased expression of either a full length or a properties of the CCN proteins might be truncated NOV protein. dependent upon diVerent combinatorial ef- Because the fates of truncated and full length fects. NOV proteins are expected to be quite Studies performed with NOV, CTGF, and diVerent (fig 4), these results suggested that the WISP-2 have provided valuable information biological properties of the NOV proteins about the structural basis for some biological might depend upon their structure and their properties of the CCN proteins. subcellular localisation. http://mp.bmj.com/ The nov gene was discovered as an integra- The immunofluorescence detection of a tion site for the MAV retrovirus in avian neph- NOV related protein in the nucleus of several roblastomas (see above).12 This gene was diVerent cell lines and frozen tissue prepara- designated nov because it was highly expressed tions raised the intriguing possibility that it in all nephroblastomas compared with adult might be involved more directly in the kidney, where its expression was low but still regulation of gene expression. Because the 9 amount of nuclear NOV protein was found to detectable. In one sample (tumour 725), the on September 29, 2021 by guest. Protected copyright. integration of MAV into the second intron of vary greatly (from zero to high concentrations) nov resulted in the U5 long terminal repeat among various cell lines (B Perbal, 1997, (LTR) driven expression of a chimaeric nov unpublished observations and PN Schofield, mRNA containing 91 3'-proximal viral 1998, personal communication), the nuclear nucleotides and 2.0 kb of cellular sequences. localisation of NOV might be related to their As a consequence, the first putative initiation proliferation and cell cycle status. codon corresponded to an internal methionine Several lines of evidence confirmed the codon in the full length NOV protein, and the existence of a nuclear NOV related protein and resulting chimaeric protein, composed of 253 amino acids, was expected not to be secreted because it was deprived of the signal peptide

%ID/HO IGFBP VWC TSP1 CT RSV 5' truncated nov CTGF 100/100 Variable

CYR61 44/60 Variable

NOV 48/64 Variable

ELM1/WISP1 41/57 Variable RSV Full length nov COPI/WISP2 32/41 Variable Figure 4 Fate of the full length and truncated proteins. WISP3 36/52 Variable Rous sarcoma virus (RSV) driven expression of the truncated protein in chicken embryonic ﬁbroblasts (CEFs) Figure 3 Multimodular structure of the CCN proteins. CT,cystein knot containing family leads to the cytoplasmic accumulation (and further of growth regulators-like domain; IGFBP,insulin-like growth factor binding protein-like processing?) of the truncated NOV protein, whereas the domain; TSP1, thrombospondin-like domain; and VWC, Von Willebrand factor-like presence of the signal peptide in full length NOV governs its domain. secretion.

www.molpath.com NOV and the CCN family of genes 65

suggested that it was indeed involved in the cells, three series of experiments were per- regulation of transcription. formed. In the ﬁrst, uninfected ﬁxed 143 cells

were exposed to the anti-nov K19 rabbit Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from antibody and then to biotinylated goat antirab- AN AMINO TRUNCATED NOV RELATED PROTEIN IS bit immunoglobulin, followed by avidin bound LOCALISED IN THE NUCLEUS OF HeLa AND 143 CELLS to Texas red. In parallel experiments, cells were A 31/32 kDa doublet of NOV protein was treated with the anti-nov K19 antibody and identified by western blotting in the nuclear incubated directly with goat antirabbit Texas fraction of human epitheloid carcinoma HeLa red conjugated antibodies. Nuclei from unin- cells, and human osteosarcoma 143 cells.30 fected 143 (fig 5A) cells showed a strong posi- Because the antibodies used in this study were tive response to the K19 anti-nov antibody. In directed against the C-terminus of NOV, the uninfected actively growing cells, the nuclear 31/32 kDa NOV isoform was thought to be an NOV protein appeared to be localised in the amino truncated form of NOV. nucleoplasm and in nucleoli. Positive staining The nuclear detection of NOV was reminis- was also seen at the periphery of the nucleus cent of a situation reported previously for sev- (probably in the Golgi) and in the cytoplasm of eral other secretory proteins. In the absence of these cells. a conventional nuclear localisation signal In the second series of experiments, cells (NLS) in NOV, its nuclearisation might be were infected with HSV-1 before incubation governed by the KKGKKCLRTKKS motif, with rabbit anti-nov (K19), mouse anti-ICP4, which is similar to the basic motif involved in and mouse anti-ICP8 antibodies. It has been the nuclear localisation of fibroblast growth reported previously that early after HSV infec- factor 3 (FGF3). tion, ICP4 is diVusely distributed throughout Although the biological importance of NOV the infected cell nucleus and that, after the nuclear targeting remains unclear, our expecta- onset of viral DNA replication, staining for tion is that the 31/32 kDa NOV isoform might ICP4 was associated with discrete dense modulate gene expression during normal bodies. In contrast, ICP8 labelling was fairly development. The biochemical identification constant throughout infection, giving rise to a of this nuclear NOV related protein is under granular pattern that filled the infected nu- way. cleus. Infected 143 cells were first incubated simultaneously with either both the rabbit THE NUCLEAR 31/32 kDa NOV RELATED PROTEIN COLOCALISES WITH THE TRANSCRIPTIONAL anti-K19 and the mouse anti-ICP4 antibodies MACHINERY IN THE 143 CELLS and then with goat antimouse fluorescein As a first step in establishing the localisation of isothiocyanate (FITC) conjugated antibodies

the nov protein in the nucleus of these cells, we (reacting with anti-ICP4 and anti-ICP8) or http://mp.bmj.com/ took advantage of the fact that herpes simplex with biotinylated goat antirabbit immuno- virus 1 (HSV-1) encoded infected cell proteins globulin before incubation in the presence of 4 and 8 (ICP4 and ICP8) can be used as Texas red conjugated avidin (allowing indirect physical markers for the transcription and rep- visualisation of the K19 antibody). lication machineries, respectively. The ICP4 As shown in fig 5B, ICP4 (green fluores- protein is a transcriptional transactivator local- cence) and NOV (red fluorescence) overlapped ised in the nucleus of HSV infected cells, where in the nucleus of infected 143 cells, whereas it is responsible for the regulation of HSV gene ICP8 (green fluorescence) and NOV (red fluo- on September 29, 2021 by guest. Protected copyright. expression, whereas ICP8 is a single stranded rescence) did not colocalise (fig 5C). As DNA binding protein that is found in the expected, the nucleoli did not contain dual nuclear compartment, where viral DNA syn- staining. These experiments suggested that the thesis takes place.31 major fraction of the nuclear NOV isoform was To determine whether NOV protein could localised in the compartment where transcrip- be detected in the nucleus of actively growing tion occurs and was not associated with the

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.

www.molpath.com 66 Perbal

replication machinery (B Perbal, 1997, unpub- A NOV RELATED PROTEIN BINDS THE PROMOTER lished data). OF HUMAN PLASMINOGEN ACTIVATOR INHIBITOR

TYPE 2 Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from THE NOV PROTEIN INTERACTS WITH THE RPB7 In recent studies performed to identify the fac- SUBUNIT OF RNA POLYMERASE IN A YEAST TWO tors responsible for the transcriptional regula- HYBRID SYSTEM tion of the human plasminogen activator A yeast two hybrid screening system was used inhibitor type 2 (PAI-2) gene, competition to isolate cDNAs encoding proteins able to electrophoretic mobility shift assays identified interact with the human NOV protein.30 A two cDNAs from a HeLa cell library encoding DNA fragment encoding NOV codons 29 to partial peptides that specifically bound to the 357 was amplified by the polymerase chain transcriptional regulatory motif (TRM), previ- reaction (PCR) and inserted into pGBT9 ously found to be essential for constitutive (Clontech, Palo Alto, California, USA) in PAI-2 transcription. Sequencing of the cDNAs frame with the DNA binding domain of Gal4. established that one of these peptides was The resulting plasmid (pNOV) was used as identical to the fifth domain of NOV,32 “bait” to screen three cDNA libraries (an therefore reinforcing the possibility that a NOV Epstein-Barr virus transformed human pe- related protein might indeed be involved in ripheral blood lymphocyte library (gift from transcriptional regulation. Aviron Inc.), a HeLa cell library, and a normal Because there is no evidence as yet for alter- human brain library from Clontech, all native splicing of the nov RNA, the detection of provided by Dr B Roizman, Kovler Labora- a NOV related protein in the nucleus and in the tory, University of Chicago, USA), which were cytoplasm of the cells raises fundamental ques- fused to the Gal4 transcriptional activation tions as to the biochemical constitution of these domain in pACT. proteins and their association. To investigate Establishing the 3'-proximal sequence of the these questions, we have now constructed inserts at the 5' boundary of the Gal4 DNA diVerent types of vectors expressing tagged full domain fusion (B Perbal, 1997, unpublished length and truncated NOV proteins. Studies results; CP Li et al, 2000, unpublished aimed at characterising processes governing results)30 allowed us to identify several inde- the turnover and subcellular localisation of the pendent clones with sequences that match diVerent NOV related proteins and their func- completely with the published sequence of the tional relation have been undertaken. rpb7 subunit of human RNA polymerase II. In the absence of compelling evidence for the Because it has been established that the rpb7 expression of new members of the CCN family subunit alone does not give rise to false exhibiting a nuclear localisation, it is tempting positives under the conditions used (M to relate these various observations to the Verner, 1998, personal communication), our detection of an amino truncated NOV isoform

results strongly suggested that the nuclear in the conditioned medium of nov expressing http://mp.bmj.com/ amino truncated NOV related protein is cells. involved in the regulation of transcription The analysis of conditioned medium from through its interaction with the RNA polymer- SF9 insect cells infected with a nov expressing ase holoenzyme. recombinant baculovirus identified a 25 kDa NOV protein as an amino truncated isoform. A NOV RELATED PROTEIN CAN BE DETECTED AT N-terminal sequencing performed on the puri- THE NUCLEAR ENVELOPE fied secreted protein revealed that the trun- on September 29, 2021 by guest. Protected copyright. We have shown that the NCI-H295R cells cated protein was made of the two C-proximal that have been established from an invasive domains of NOV, with an N-terminal sequence adrenocortical carcinoma secrete increased (AYRPE), suggesting that it was the result of a concentrations of NOV protein (S Kyurkchiev proteolytic cleavage of the full length secreted et al, Proceedings of the first international NOV protein.33 The existence of a truncated workshop on the CCN family of genes, 17–19 form of CTGF with a similar N-terminal October 2000, Saint-Malo, France). When the sequence34 suggests that specific proteolysis of immunogold procedure was used to perform these CCN proteins might be a key element in an immunocytochemical localisation of the the regulation of their biological activity. NOV protein in these cells, we found that the A truncated NOV protein with an apparent cytoplasm, the plasma membrane, and the molecular mass of 30–32 kDa can be detected nuclear envelope stained positive. Unexpect- in the conditioned medium of naturally ex- edly, the distribution of the positive grains in pressing cells such as NCI-H295R (G the cytoplasm of the NCI-H295R cells and the Thomopoulos et al, 2001, in press); transfected absence of any recognisable secretory granule Madin Darby canine kidney (MDCK) cells35; or secretory vesicle suggested that shuttling transfected human glioblastomas (B Perbal, of the NOV protein in these cells did 2000, unpublished results); in five day old not follow the conventional pathway (G human myoblasts, but not in myotubes (S Thomopoulos et al, 2001, in press). The mode Kyurkchiev and B Perbal, 1999, unpublished of secretion of the protein is currently under results); and in diVerent human biological flu- investigation. ids, such as urine, amniotic fluid,36 and The detection of an immunoreactive NOV cerebrospinal fluid.36 Our working hypothesis is related protein at the nuclear pores of NCI- based on the assumption that the elimination H295R cells was another piece of evidence of the N-proximal modules confer on the trun- suggesting that NOV might be targeted to the cated NOV protein growth stimulatory proper- cell nucleus. ties, as opposed to the inhibitory properties of

www.molpath.com NOV and the CCN family of genes 67

protein, the C-terminus of NOV was not C-ter N-ter accessible to the antibodies, either because of

conformational constraints or because it was Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from combined with other partners. The possibility that physical interactions C-ter C-ter between the C-proximal and N-proximal N-ter domains of NOV can regulate the activity of the native protein is under current investiga- N-ter tion. C-ter C-ter Thus, the balanced production of truncated and full length NOV isoforms might be tightly R regulated and be dependent upon physiological C-ter C-ter conditions. This aspect might be particularly N-ter T important in the case of cancer cells, which overproduce many diVerent kinds of proteases. T The disregulation of proteolysis would increase the relative ratio of truncated over full length C-ter isoforms, therefore leading to abnormal signal- Cytoplasm C-ter N-ter ling. However, the situation is probably not that Nucleus simple. In the course of the studies that we have initiated to identify the molecular events Figure 6 Schematic hypothetical pathways leading to the potential internalisation of the amino truncated NOV protein. In all cases, the secretion of a full length NOV protein is leading to the secretion of NOV isoforms, we thought to be required for further processing. The full length NOV protein might be secreted have established that the concentration of and processed at the membrane site, the C-proximal moiety of the protein being internalised truncated NOV protein in the conditioned directly or indirectly through specific interactions with either a receptor or a transporter. Alternatively, the NOV protein could be released into the medium or the extracellular medium of eukaryotic cells is not related matrix where it would be submitted to proteolysis. The amino part of the protein might itself directly to the amount of the released full play an important role in signalling, either directly or indirectly. C-ter, amino truncated length 48 kDa protein (V Martinez and B Per- protein; N-ter, N-proximal moiety of the protein; R, receptor; T,transporter. bal, 2000, unpublished results), therefore sug- the full length NOV protein (see above). The gesting that the production of the amino trun- oncogenic properties of the truncated NOV cated NOV isoform is not the sole result of protein expressed in avian nephroblastoma (see NOV post translational processing. above), and the growth inhibitory properties of The possibility that the detection of a rCOP1,15 might therefore result from the nuclear NOV protein did not result from the disruption of the regulatory balance existing in intracellular addressing of NOV, but was the full length protein, between a negative related to the secretion of a full length NOV, N-proximal domain and a positive C-terminal was suggested by experiments in which inhibi- domain. In the case of an amino truncated tion of the secretion of the full length NOV did http://mp.bmj.com/ NOV protein, constitutive positive properties not increase the concentration of nuclear NOV would result from the lack of negative regula- in the treated cells (B Perbal, 1997, unpub- tion conferred by the first module, whereas in lished observations). It is tempting to speculate that the truncated NOV protein is targeted to the case of rCOP1, the lack of positive eVects the nucleus after post translational proteolysis would result from the lost of the C-terminal of the secreted NOV protein, either at the sur- module. face of the cell or in the matrix by one of the Indirect evidence suggesting that the on September 29, 2021 by guest. Protected copyright. mechanisms depicted in fig 6. C-terminus of NOV is not freely accessible in The isolation of several biologically active the native protein came from studies aimed at truncated forms of CTGF in biological flu- setting up an enzyme linked immunosorbent ids334 is another example that highlights the assay (ELISA) to measure the concentrations potential importance of post translational of NOV in biological fluids (S Kyurkchiev et al, modifications of CCN proteins, and stresses Proceedings of the first international workshop the need for tight control to ensure balanced on the CCN family of genes, 17–19 October production of the various isoforms if process- 2000, Saint-Malo, France). By performing ing and biological activity are related. In that ELISA with phosphate buVered saline (PBS) case, the levels of low mass stable CTGF or Triton treated NCI-H295R cells we estab- isoforms were proposed to be qualitatively and lished that the NOV protein was present at the quantitatively dependent upon several factors, cell membrane and that a considerable amount such as tissue of origin, species, or stage of the of NOV could be found in the cytoplasm of cell cycle.3 Again, the amino truncated 10 kDa these cells. This conclusion was confirmed by CTGF, which contains only the C-terminal indirect immunofluorescence assays, which module of CCN proteins, exhibited mitogenic indicated that the NOV protein was widely dis- properties, confirming that the N-proximal tributed in the cytoplasm of the NCI-H295R modules are not required for the positive regu- cells, sometimes associated with perinuclear lation of growth.3 However, the complexity of granules, suggesting labelling of the Golgi. the structure–activity relation is exemplified by Unexpectedly, the NOV protein was not the fact that the 10 kDa CTGF molecule is detected when absorption ELISA was per- mitogenic for BALB/c 3T3 cells, vascular formed with conditioned medium from NCI- smooth muscle cells, and endometrial stromal H295R cells in which the presence of secreted cells but not endothelial cells.3 NOV had been checked for by western The study of the mechanisms governing the blotting. This suggests that in the native NOV production of the truncated CCN proteins

www.molpath.com 68 Perbal

should result in a better insight into the the decrease in the half life of nov RNA biochemical properties of the four constitutive transcripts.18 The sequences involved in the

modules and their functional interactions. delayed downregulation of cyr61 and ctgf Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from expression have not been identified as yet. Regulation of CCN RNA and protein Although the expression of CEF10/âIG-M1 expression (the mouse equivalent of cyr61) and âIG-M2 Another fundamental question regarding the (the mouse equivalent of ctgf) were reported to biological properties of the CCN proteins con- be inducible by TGFâ in AKR-2B mouse cerns the way their expression is regulated both embryo fibroblasts,7 the expression of cyr61 in space and time. Because a large quantity of was not found to be significantly induced by information concerning the patterns of cyr61 TGFâ in human skin fibroblasts or NIH3T3 and ctgf expression has been published in cells.38 2–4 recent reviews, I will focus on results These “conflicting” results indicated that the obtained with nov and will attempt to highlight regulation of CCN gene expression may vary in the specificities of each system. diVerent species (cef10 and cyr61), and depend upon the type of cells used in the same PROMOTER SEQUENCES AND GENE EXPRESSION species (AKR-2B versus NIH3T3). These As stated above, the expression of the cyr61 observations stressed once more the fact that and ctgf genes is induced early upon stimula- one must be extremely cautious in comparing tion of fibroblast proliferation, whereas Elm1 results obtained with diVerent systems. and rCOP-1 exhibit delayed kinetics of induc- Given the importance of TGFâ signalling in tion and the expression of nov is downregu- several fundamental biological processes, lated under similar conditions (fig 2). This dif- much attention has been paid to the relation ferential expression pattern and the variety of between TGFâ and ctgf expression. Promoter e ectors involved in the activation or repres- V analysis of ctgf and methylation interference sion of CCN genes have suggested that distinct assays have led to the identification of a 13 regulatory elements govern their expression. nucleotide TGFâ putative responsive element Indeed, the comparison of ctgf, cyr61, and nov (TbRE), which is conserved in the promoter of promoter sequences did not show any obvious ctgf throughout species and appears to be spe- conservation.3 37–39 cific for TGFâ induced transcriptional activa- The induction of cyr61 by serum and PDGF 38 in fibroblasts was reported to be dependent tion of ctgf. Whether binding of a specific upon a serum responsive element (SRE)-like factor to this sequence is responsible for the motif at position −1912/−1933 in the 2 kb prolonged induction of ctgf mRNA after short 5'-proximal promoter sequences, which are term exposure to TGFâ remains to be suYcient to confer serum inducibility.37 The established.

expression of ctgf was also found to be The expression of nov was not aVected by http://mp.bmj.com/ stimulated by PDGF and epidermal growth TGFâ in either NRK-49 F (D Lawrence and B factor (EGF) in human fibroblasts.23 Similarly, Perbal, 1995, unpublished observations) or 38 both cyr61 and ctgf were reported to be NIH3T3 and human skin fibroblasts. induced by fibroblast growth factor (FGF).523 Ex vivo experiments performed with the The expression of cyr61 is also induced by human nov promoter indicated that sequences vitamin D3 in human fetal osteoblasts,40 by localised between positions −405 and −62 had oestrogen and tamoxifen in uterine cells of a negative eVect on transcriptional activity on September 29, 2021 by guest. Protected copyright. ovariectomised rats,41 and hippocampal im- when measured by chloramphenicol acetyl 11 mortalised cells undergoing neuronal diVeren- transferase (CAT) assays. A comparison of tiation.42 the promoter sequences from xenopus, The rapid induction of cyr61 (peak after 20 chicken, and human nov did not reveal any minutes, decreased at 60 minutes, and absent significant conservation (fig 7). A stretch of 19 at two hours) by 100 ng/ml 12-O- nucleotides (GCAGGCCCCGGCGCGC tetradecanoylphorbol-13-acetate (TPA)5 was CGC) appeared to be conserved in the human in pronounced contrast to the expression of and chicken nov promoter. Whether this nov CEF10 (the avian homologue of cyr61), which conserved element (NCE) binds a regulatory was reported to be repressed to < 20% of the factor involved in the negative regulation of nov basal value at 60 minutes, and gradually promoter activity is under investigation. The increased to about 10 times the basal value at activity of the human nov promoter was down- 24 hours.17 Based on the time course for the regulated indirectly by the WT1 protein.39 It is translocation–activation–deactivation of pro- worth noting that the ctgf promoter was also tein kinase C (PKC), the expression pattern of recently reported to be a target for WT1 tran- CEF10 in the presence of 100 ng/ml TPA sug- scriptional regulation.43 Screening of oligonu- gested that the expression of CEF10 is cleotide arrays and northern blotting estab- repressed at one hour, when PKC is transiently lished that ctgf expression was upregulated in a activated, and derepressed when PKC is Wilms’s tumour cell line (WiT49A) transfected degraded.17 Similarly, stimulation of serum by a dominant negative mutant of WT1, starved cells by 50 ng/ml TPA for six hours whereas ctgf promoter activity was repressed resulted in the complete downregulation of nov by wild-type WT1 protein. As established in in CEFs.18 The downregulation of nov expres- the case of nov, the downregulation of ctgf pro- sion induced by serum and by TPA could be moter activity was not mediated by canonical abolished by treatment with the H7 inhibitor of WT1 recognition elements.43 These results PKC, suggesting that PKC might play a role in reinforced the possibility that the alteration of

www.molpath.com NOV and the CCN family of genes 69

A –700 –581 –455 –434 –283 –222 –179 –139 –39 –29 –15 +30 +87 +211 +298 Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from

ATG

TG (20) SP1 AP1 NF1/CTPS1 AP2 NCE NF1/CTF AP2TATA SP1 NF1/CTF E1 SP1 E2

B –235 –190 –171 –89–114–70 –48 –40 +6 +28 +117+136 +185

ATG

AP2 NCE NFKE2 SP1SP1SP1 AP2 TATA SP1 E1 SP1 SP1 E2

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. nov expression observed in avian nephroblasto- expression of the WISP genes was detected in mas was indeed related to impaired kidney brain.16 In situ hybridisation using whole blastema diVerentiation and tumour develop- mount mouse embryos and northern blot ment. analysis of adult rat and mice organs could not detect rCOP1 RNA in brain, heart, lung, EXPRESSION PATTERN IN NORMAL EMBRYONIC kidney, pancreas, spleen, intestine, stomach, AND ADULT TISSUES and skeletal muscle.15 The expression of Early studies indicated that the CCN genes WISP-2 in human skeletal muscle was in con- were diVerentially expressed in normal tissues trast to the lack of expression of rCOP-1 in during development. rodents. Northern blot analysis of chicken embryonic Northern blot analysis of ctgf expression in and adult total RNA established that the normal adult tissues28 established that ctgf expression of the nov gene was regulated both polyA RNAs were found abundantly in spleen, temporally and spatially during develop- 912 ovary, gastrointestinal tract, prostate, heart, ment, with nov RNA detected either only at and testis, and at a lower level in thymus, the embryonic stage (heart) and adult stage placenta, lung, skeletal muscle, kidney, and http://mp.bmj.com/ (lung), or at both stages (brain). Muscle and pancreas. No expression of ctgf was detected in intestine were also found to express nov at the brain, liver, and peripheral leucocytes.28 The embryonic stage, whereas adult spleen was lack of ctgf expression in brain was in contrast positive for nov expression. Embryonic kidneys to the western blotting identification of a expressed high amounts of nov, whereas much 40 kDa CTGF protein in rat brain homoge- lower amounts were detected in the kidneys of nates and the immunochemical detection of birds after hatching.9 In human tissue samples, CTGF in the cerebral cortex, the white matter on September 29, 2021 by guest. Protected copyright. abundant amounts of polyA nov RNA were of spinal cord.44 The strongest positive signal detected by northern blotting in heart, brain, prostate, testes, and pancreas, whereas low for CTGF detection was seen in astrocytes of amounts were detected in spleen, ovary, small the cerebral cortex, ependymal cells of the cer- intestine, colon, peripheral blood leucocytes, ebral ventricle, tanycytes along the central lung, skeletal muscle, and kidney.36 RNase pro- canal of the spinal cord, and pyramidal cells in tection assays performed on total RNA of 8 the cortical layers III and V. and 10 week old embryonic human tissues RNase protection assays performed on total indicated that nov was highly expressed in the RNA from BALB/c mouse tissues established adrenal gland and to a lesser extent in kidney, that cyr61 expression is highest in lung; limb bud, and spinal cord, whereas heart, liver, moderate in heart, uterus, and skeletal muscle; intestine, lung, placenta, and testis were found and low in kidney, adrenal gland, testes, brain, 45 to be negative for nov expression (S Kocial- and ovary. kowsky et al, 2001, in press). Altogether, these results (see table 2 for a A cDNA-PCR analysis of WISP RNA summary) illustrated the diversity of those tis- expressed in adult and fetal human tissues16 sues in which expression of the CCN genes can established that WISP-2 RNAs were to be be detected at diVerent stages of development, found in a rather restricted panel of tissues, reinforcing the current view that CCN proteins including adult skeletal muscle, colon, ovary, are involved in various biological processes. and fetal lung, whereas WISP-1 RNAs were From table 2 it can be seen that NOV was the detected in adult heart, kidney, lung, pancreas, only CCN protein highly expressed in the ovary, spleen, and small intestine. In addition brain. Indeed, in situ hybridisation and immu- to heart, kidney, lung, and spleen, fetal skeletal nocytochemistry performed on developing muscle and liver also contained WISP-1 RNAs. human, chicken, and rat embryos confirmed The WISP-3 RNAs were detected only in adult that the expression of nov was tightly associ- kidney and testis and in fetal kidney. No ated with nervous system diVerentiation and

www.molpath.com 70 Perbal

Table 2 Expression of CCN RNA in diVerent species

H BR PL LU LI S/M K PA SP TH PR T OV CO S/I LEU AD Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from c-novE++++NT−− + + NTNTNTNTNTNTNT+NTNT c-nov A − ++ NT ++ − − NT NT + NT NT NT NT NT NT NT NT h-novE−+−−−NT+NTNTNTNTNTNTNT−NT++ h-novA++++−+−+++++−++++++++ NT ctgf ++ −++−+ ++++++++++++++−NT cyr61+++/−NT+/−−+++−−NTNT+/−+/−NT−NT+ WISP-1E + − NT + +/− +/− ++ NT NT − NT NT NT NT NT NT NT WISP-1A +/− − +/− +/− − − +/− +/− +/− − − − ++ − ++ − NT WISP-2E − − NT + − − − NT NT − NT NT NT NT NT NT NT WISP-2A−−−−−+−−−−−−++−− NT WISP-3E − − NT − − − ++ NT NT − NT NT NT NT NT NT NT WISP-3A−−−−−−++−−−−+−−−− NT rCOP-1E − − NT − NT − −−−NTNTNTNTNT−NTNT

Origin of tissues: H, heart; BR: brain; PL, placenta; LU, lung; LI, liver; S/M, skeletal muscle; K, kidney; PA, pancreas; SP, spleen; TH, thymus; PR, prostate; T, testis; OV, ovary; CO, colon; S/I, small intestine; LEU, leukocytes; AD, adrenal. c-nov, chicken nov RNA; h-nov human nov RNA; E, embryonic; A, adult; NT, not tested.

Table 3 Distribution of nov transcripts in 10 week human embryo chicken and human axons suggests that it might be involved in axonal guidance. Strong Ectoderm Endoderm Mesoderm expression of nov RNA and proteins was also Brain ++ Gut endothelium ++ Adrenal cortex +++++ detected at sites of chondrogenesis, where it is Spinal cord (mt,fp) +++ Bronchial epithelium + Kidney tubules +++ probably involved in the late stages of chondro- Cranial ganglia +++ Pancreatic ducts + Fusing skeletal muscle +++ Auditory apparatus +++ Gut smooth muscle ++ cyte diVerentiation and osteogenesis (see Dorsal root ganglia +++ IndiVerent gonad ++ below). Heart + Mesonephros + In situ hybridisation and immunocytochem- Perichondria + istry established that although the distribution of nov RNA and protein was widespread in ﬁrst Data are from S Kocialkowski (2001, in press). et al trimester human embryos (table 3), the major development (S Kocialkowsky et al, 2001, in sites of nov expression included the central press; G Chevalier et al, 2000, unpublished nervous system, the adrenal cortex, and diVer- results; W Cai et al, 2000, unpublished results; entiating muscle and kidney (S Kocialkowsky Y Cherel et al, Proceedings of the ﬁrst et al, 2001, in press). In all cases, the NOV pro- international workshop on the CCN family of tein was predominantly cytoplasmic, and weak genes, 17–19 October 2000, Saint-Malo, staining of the extracellular matrix (ECM) was France).46 The role and functions of NOV in also seen in some instances. In general, there the nervous system are under current investiga- was a fairly good correlation between nov RNA

tion. and NOV protein expression, suggesting that http://mp.bmj.com/ In situ hybridisation and immunocytochem- nov acts locally rather than distantly. In agree- istry performed on developing chicken em- ment with results obtained at later develop- bryos allowed us to establish the pattern of nov mental stages,46 neurones of both the spinal expression in kidney, muscle, nervous system, cord and the brain expressed high amounts of and skeletal development (G Chevalier et al, nov RNA and the NOV protein was clearly 2000, unpublished results). In general, a good detected in cell bodies and axons. The floor match was seen between nov RNA and NOV plate of the spinal cord, spinal nerves, and dor- protein detection. The expression of nov was sal root ganglia were identified as sites of abun- on September 29, 2021 by guest. Protected copyright. detected at embryonic days 3 and 4 (E3 and dant nov expression. E4) in the mesonephric mesenchyme, epithe- Muscle was the predominant mesodermal lial vesicles, and glomeruli. At later stages (E6 component expressing nov. Myotubes and fus- to E12), diVerentiated tubules were found to ing myoblasts were identified as major sites of be positive for nov. Metanephric epithelial nov expression in skeletal muscle. Smooth vesicles, glomeruli, and ureteric bud were also muscle also expressed nov, but to a lesser positive for nov until E14. From E4 onwards, extent. At the early stages, cartilage chondro- developing myotome and skeletal muscle cytes did not express nov, whereas in the older stained positive for nov. Nov expression was embryo, the NOV protein was demonstrated in also detected in smooth muscle cells and the perichondrium and in hypertrophic cardiomyocytes. The nervous system was chondrocytes in the head, ribs, and lower found to be strongly positive for nov. At early limbs. In the developing urogenital tract, major stages (E3) neuroepithelium was positive at discrepancies were noted between NOV pro- E3, and at later stages (E3–E7) neural tube also tein and nov RNA values, suggesting that the showed strong staining for nov. In agreement NOV protein might accumulate at these sites with our previous detection of high amounts of for a particular function, although we cannot NOV protein in human neuronal cells and exclude, as yet, that nov RNA species are short axons,35 high concentrations of NOV protein lived in these cells. For instance, the concentra- were detected in chicken neuronal cells, which tions of NOV protein in the podocytes of both express low amounts of nov RNA, therefore mesonephric and metanephric glomeruli are reinforcing the possibility that in these cells, as usually extremely high, whereas in the same well as in podocytes, the NOV protein cells nov RNA species are barely detectable (S accumulates or is stabilised.35 Because nov Kociazlkowski et al, 2001, in press; G Chevalier shares identity with the fruit fly axon repellent et al, 2000, unpublished results).35 Because the slit protein, the detection of nov along the NOV protein is fairly stable in the conditioned

www.molpath.com NOV and the CCN family of genes 71

medium of NOV producing cells (B Perbal, with TGFâ and bone morphogenic protein 2 2000, unpublished results),35 the raised con- (BMP-2), two factors believed to be involved in

centrations of NOV detected in podocytes cartilage and bone formation, it was proposed Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from probably results from its accumulation. that CTGF was a mediator of these cytokines Whether this accumulation results from its in cartilage. Because CTGF promoted the interaction with other proteins at this site or is proliferation and diVerentiation of chondro- physiologically important (needed for cytes in culture,53 it was proposed that ctgf glomeruli filtration?) remains to be established. expression is involved in endochondral ossifica- The metanephric mesenchyme was strongly tion by acting on proliferating and maturating positive for nov RNA expression, with increas- chondrocytes in growth cartilage. Both ing staining as diVerentiation proceeds in the chondrocytic and osteoblastic cells express a S-shaped bodies, and a dramatic decrease once 280 kDa protein that has been described as a glomeruli diVerentiation was achieved. These receptor for CTGF.54 55 results were of particular interest because they Because nov expression was also detected in strongly suggested that nov expression was sites of chondrogenesis during chicken devel- tightly coupled to the diVerentiation process in opment (fig 8), we postulated that the negative these cells. Examination of immunocytochemi- regulatory activity attributed to nov (see above) cal labelling in the heart indicated that at these might be involved at later stages of diVerentia- stages the endocardium was negative for nov, tion to counterbalance the stimulatory eVects although it is in close contact with the myocar- of CYR61 protein on chondrogenesis, which dium, an abundant source of NOV protein. had been established in micromass cell cul- This observation provided evidence that the tures.49 According to this view, CYR61 and NOV protein acts where it is produced rather NOV protein activities might represent the than being transported and accumulating at a “yin and the yang” along common regulatory distant site. Among endodermal derived tis- pathways leading to chondrocyte diVerentia- sues, the gut mucosa, bronchial epithelium, tion and osteogenesis. and pancreatic ducts were positive for nov To assess the potential role of nov in (table 3). chondrogenesis and osteogenesis, we first ana- The immunocytochemical localisation of lysed the expression of nov chicken during CYR61 and FISP12 proteins during mouse wing and leg development.56 Northern blotting embryogenesis showed similarities and diVer- performed on total RNA prepared from limb ences that were indicative of diVerential actions buds dissected between E4 and E18 estab- in development.47 The localisation of CYR61, lished that expression of nov increasing signifi- but not FISP12, in limb bud mesenchyme in cantly between E8 and E10 and progressively vivo and in vitro had suggested that CYR61 decreased until E14, to reach a basal value at might be involved in chondrogenesis.47 48 Puri- E15.56 The expression profile of nov correlated

fied CYR61 protein was indeed shown to pro- with the time course of limb development, http://mp.bmj.com/ mote the initial cell aggregation responsible for which is completed after 10 days of incubation mesenchymal condensation before chondro- under normal conditions. Sustained expression genic diVerentiation and to enhance the indicated that nov was required over an synthesis of cartilage specific matrix.49 More extended period of time during limb develop- recently, it has been reported that in rat, cyr61 ment, therefore suggesting that it might be is expressed in a time dependent manner in needed for later events. Immunocytochemistry mesenchyme cells and osteoblasts from frac- performed on paraYn wax embedded wing on September 29, 2021 by guest. Protected copyright. ture callus,50 whereas conventional in situ bud sections established that nov was mainly hybridisation did not detect cyr61 mRNA in detected at E6 in the proliferating chondro- normal bone. In the hFOB human osteoblast cytes and the perichondrium (fig 9A), whereas cell line, cyr61 was identified as a greatly at E11/E12, staining of nov was essentially upregulated gene by various factors important detected in the hypertrophic chondrocytes of for bone metabolism such as 1,25(OH)2- the central diaphyseal region and to a lower vitamin D3, tumour necrosis factor á (TNF- extent in the perichondrium (fig 9B). At E14, á), EGF, basic FGF, and interleukin 1â nov labelling was greatly reduced and restricted (IL-1â).39 to a few hypertrophic chondrocytes in the DiVerential display PCR identified ctgf as developing wing. Flattened chondrocytes were being preferentially expressed in HGS-2/8 not positive for nov expression at any stage of human chondrosarcoma derived chondrocytic development (fig 9C). Pretreatment of the sec- cells.51 In situ hybridisation established that tions with hyaluronidase resulted in additional ctgf was specifically expressed in the hyper- widespread labelling of the cartilaginous ma- trophic chondrocytes of costal cartilage and trix, in agreement with nov being associated vertebral column in 17 day old mouse with the ECM.57 Similarly, CYR61 and CTGF embryos,51 at a time when cyr61 expression is were also reported to be ECM associated pro- strongly downregulated,48 suggesting that teins and suggested to mediate cell–matrix CYR61 and CTGF might have complemen- interactions.58 tary but diVerent functions. Staining of the When mesenchymal cells isolated from the cost-chondral junctions of newborn mouse ribs limb buds of 3.5 day old chicken embryos or indicated that ctgf was expressed in chondro- from 10.5 day old mouse embryos are seeded cytes of the hypertrophic zone, but not in the at high density (107 cells/ml), cellular aggrega- proliferating and resting zone.52 Osteoblasts did tion proceeds and internodular condensations not express ctgf. Because ctgf mRNA expres- undergo chondrocytic diVerentiation, as shown sion was stimulated in HCS2/8 cells treated by the formation cartilaginous nodules within

www.molpath.com 72 Perbal Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from http://mp.bmj.com/ on September 29, 2021 by guest. Protected copyright.

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. three days of culture. When cells are seeded at cultures established that nov expression, which a lower density (106/ml), chondroblastic diVer- was not detected at day 0 of culture, increased entiation does not proceed and no cartilagi- progressively for two days and decreased at day nous formation occurs. The use of this system 3, whereas the expression of collagen II, a revealed that CYR61 enhances chondrogenic marker for determination of progenitor cells to diVerentiation.49 Northern blotting experi- undergo chondroblastic diVerentiation, in- ments performed on chicken micromass cell creases from day 1 onwards (ﬁg 10). The

www.molpath.com NOV and the CCN family of genes 73 Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from

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).

expression of nov is dependent upon the These observations and the results of immu- productive diVerentiation of mesenchyme nochemical studies performed on sections sug- cells, as shown by the lack of nov expression in gested that NOV and CTGF were required at

the absence of cartilaginous nodule formation late stages of the chondrogenesis/osteogenesis http://mp.bmj.com/ at low cell density (ﬁg 11). When cells were diVerentiation process, whereas CYR61 was seeded at high density, ctgf expression was required at early stages. Whether the diVeren- detected in micromass cultures from day 2 tial expression of these three CCN genes in the onwards, whereas CEF10 expression was skeletal system is temporally related remains to detected from day 0 to day 3.49 be established. It is worth noting that in addition to nephroblastoma, MAV can induce osteopetro- on September 29, 2021 by guest. Protected copyright. C F J0 J1 J3J2 J0 J1J2 J3 sis in chickens, an abnormal proliferation of osteoblasts leading to severe bone diseases.10 Based on the consideration that nov was a tar- Nov 2.2 kb get 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. 4.2 kb COL II

EXPRESSION PATTERN IN PATHOLOGICAL CONDITIONS

GAPDH 1.3 kb Evidence indicating that CCN proteins participate in various diseases has been accumulating at an increasing pace over the past years. The relation that was established between ctgf expression and TGFâ has resulted in con- 28S siderable interest in the potential role of CTGF Et. Br. in fibrosis and its possible medical applications. This has led to the widely accepted conclusion 18S that the profibrogenic properties of TGFâ result from ctgf induction, stimulation of fibroblast proliferation, and overproduction of Figure 10 Expression of nov RNA species and collagen II (COL II) in chicken extracellular matrix. As discussed in depth by micromass cell cultures. Staining of ribosomal RNA species allows relative quantification of 4 the hybridisation signals obtained in two independent experiments.56 J0–J3, days of culture; Essam et al, the relation between ctgf and GAPDH, glyceraldehyde-3-phosphate dehydrogenase. TGFâ expression is not absolute and there are

www.molpath.com 74 Perbal

in agreement with the ex vivo downregulation abof nov promoter activity by WT1.39 However,

studies performed with a larger panel of Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from samples35 representative of sporadic, Wilms, aniridia, growth retardation (WAGR) and Denis Drash syndrome (DDS) histological COL II types of tumours did not confirm that inverse relation and raised the possibility that the variations of nov expression resulted from diVerent relative amounts of WT1 isoforms contained in these tumours.35 These studies also established that nov expression was a marker of heterotypic diVer- entiation.35 In striated muscle tumours, the NOV expression of nov was shown to occur at an earlier stage than desmin during the heterotypic diVerentiation of the blastemal cells that takes place in these tumours,35 and confocal microscopy established that the NOV protein was colocalised with desmin in heterotypic muscle fibres (G Chevalier, 1998, unpublished observation). GAPDH Studies that we have performed with several other types of tumours have established a strong association between nov expression and tumour diVerentiation in rhabdomyosarcomas, J0 J1 J0 J1 neuroblastomas, and cartilage tumours (H Yeger et al,andCYuet al, Proceedings of the first international workshop on the CCN family of genes, 17–19 October 2000, Saint-Malo, Figure 11 The expression of nov RNA depends upon France). In the case of neuroblastomas with chondrogenesis. (A) 106 mesenchymal cells were seeded for poor prognostic features, nov staining was low/ 7 micromass cell cultures. (B) 10 mesenchymal cells were moderate within the small tumour cells, seeded for micromass cell cultures. Samples were taken at the day of seeding (J0) and after 24 hours culture (J1). whereas in tumours with a favourable progno- COL II, collagen II; GAPDH, glyceraldehyde-3-phosphate sis and no N-myc amplification, nov staining dehydrogenase. was strong in the cytoplasm of diVerentiated

several studies indicating that ctgf expression ganglion-type cells (H Yeger et al, 1998, http://mp.bmj.com/ may be independent of TGFâ in fibroblasts. unpublished results). These results indicated Nonetheless, ctgf expression is involved in that nov expression can be used as a valuable many types of diseases, including fibrotic molecular marker for neuroblastoma typing diseases, atherosclerosis, inflammatory dis- and for tumour prognosis. eases, and scleroderma. The expression of ctgf Because the adrenal was identified as a major also increases in wound healing. Because these site of nov expression in the human embryo aspects of CTGF biology have been reviewed (see above), and because there is no molecular on September 29, 2021 by guest. Protected copyright. extensively,2–4 I will focus on studies that iden- prognosis factor for adrenocortical carcinoma, tify CCN proteins as key players in tumour we examined whether alterations in nov development. expression occurred in these highly lethal can- For many years, the only evidence suggesting cers with a mortality rate over 50%. In the nor- that CCN genes might be involved in cancer mal adult adrenal cortex, immunocytochemi- development came from studies performed cal studies showed a distinct positive labelling with nov. for nov in the zona fasciculata, the middle layer Because nov was originally shown to be of the adrenal cortex. The labelling was fairly highly expressed in all avian nephroblastomas, uniform and confined to the cytoplasm (fig we initiated studies aimed at establishing 12A). In the adrenocortical adenomas there whether nov expression was also altered in was clear staining of nov in cells that probably human Wilms’s tumours and whether an mimic the zona fasciculata (fig 12B). However, alteration of nov expression was associated staining in the adenomas was not homogenous, with other types of tumours. These studies therefore suggesting that the amount of nov were conducted with two hopes, namely: (1) detected in the cells of these benign tumours that measurement of nov in biological fluids or was dependent upon their metabolic state or in tumours might be of clinical value for early was related to hormone production. Adreno- diagnosis, typing, and prognosis; and (2) that cortical carcinomas stained either negative (fig the antiproliferative activity of nov could be 12C) or positive for nov. No clearcut relation used in cancer treatment. could be established between the degree of nov The studies that we have performed with expression and tumour stage. Wilms’s tumours established that they con- In pronounced contrast and apparent contained abnormal amounts of nov RNA, which tradiction to these reports, our studies also were in some cases inversely related to that of established that the expression of nov corre- the WT1 RNAs detected in the same sam- lated with the metastatic potential of tumour ples,59 an observation that, at first glance, was cells in the case of Ewing’s sarcomas (MC

www.molpath.com NOV and the CCN family of genes 75

200 250 Nov

200 Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from 150

150 Nov Passage 100

Days 100

50 50 Latency

0 0 RCC RCC RCC RCC RCC RCC 41 49 1 47 44 7 Figure 13 Expression of nov in renal cell carcinomas (RCCs). Increased amounts of nov were correlated with increased tumorigenicity. Nov, levels of nov RNA estimated from northern blots; Latency, time in days required to reach a tumour of a given size.

of secreted NOV protein was detected by western blotting in the conditioned medium of fast growing tumours. These results indicated that there was an inverse relation between the amount of NOV secreted by the tumour cells and the time that they required to establish tumours in vivo and for the tumour to reach a given size (ﬁg 13). Finally, results that were obtained in our study of glioblastoma cell lines suggested that nov expression was correlated with reduced tumorigencity and reduced metastatic potential in these brain tumour cells.61 Whether we can use the negative growth regulatory properties attributed to nov as a tool for treatment of these highly aggressive tumours is currently the matter of intense investigation. Therefore, it appeared that the expression of

nov was altered in many types of tumours and http://mp.bmj.com/ Figure 12 Detection of the NOV protein in normal (A) that high concentrations of NOV could be and tumoral adrenal tissues (B and C). The normal area was isolated from a patient with adenoma. Note the uneven associated with either diVerentiation or in- nov staining in the adenoma (B). An example of NOV creased proliferation and metastasis. This negative adrenal carcinoma is shown (C). Photographs complex and apparently conﬂicting situation is were kindly provided by H Yeger. not unique to nov among the CCN proteins. As reported above: (1) the WISP genes whose Manara , 2000, unpublished results), pros- expression is upregulated in Wnt transformed

et al on September 29, 2021 by guest. Protected copyright. tate cancer (B Cadot et al, Proceedings of the cells are either upregulated or downregulated first international workshop on the CCN fam- in colon cancers, and (2) the expression of ily of genes, 17–19 October 2000, submitted WISP-1 increased the tumorogenicity of transfor publication) and renal cell carcinoma (L fected cells whereas overexpression of Elm1 Glukhova et al, 2001, submitted for publi- had opposite eVects. cation; L Glukhova et al, Proceedings of the It is widely accepted that the establishment first international workshop on the CCN fam- and development of tumours rely on the ability ily of genes, 17–19 October 2000, submitted of the tumour cells to overcome hypoxia and for publication). In addition to the labelling of establish a network of blood vessels that will NOV in the cytoplasm of acinar epithelial cells, ultimately permit the tumour to expand. Neo- prostate hyperplasia showed intense luminal vascularisation is therefore controlled by the labelling suggestive of NOV secretion in semi- tumour cells that secrete angiogenic factors to nal fluid. Whether measurement of NOV in attract endothelial cells, which in turn are seminal fluid can be of value as a marker of believed to produce growth factors stimulating prostate disease is currently being investigated. tumour growth. Several positive and negative In prostate tumour cell lines, the expression of regulators of angiogenesis have been described. nov was detected in the cytoplasm of three cell The angiogenic properties of CYR61 and lines derived from metastasis to bone (PC3), CTGF26263 suggest that these proteins might brain (DU145), and lymph node (LNCap). No be involved in tumour growth. Indeed, gastric expression was detected in the SV40-T antigen adenocarcinoma RF-1 cells transfected with immortalised PNT1B cells, an observation in cyr61 gave rise to larger and more vascularised contrast to a recent study indicating that tumours than parental cells when injected into SV40-T immortalised P69 cells were positive nude mice.62 Many tumour cell lines were for nov expression.60 In renal cell carcinomas reported to express cyr61 at various levels. (RCCs), which represent 85–90% of all kidney Although it was reported that cyr61 expressing tumours, a significantly higher concentration tumour cells were more tumorigenic than those

www.molpath.com 76 Perbal

that did not express this gene,2 no clearcut with this, I would like to propose that the bio- association of this kind was drawn from studies logical activity of the CCN proteins is depend-

that we performed with glioblastoma cell ent upon diVerent combinatorial eVects. Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from lines.64 Downregulation of cyr61 expression Therefore, a search for proteins and ligands was reported in rhabdomyosarcomas,65 and in interacting with nov has been undertaken. about 50% of prostate tumours samples,66 whereas increase expression of cyr61 has been Identification of CCN protein partners detected in pancreatic cancers (C Wenger and As a first step in identifying the proteins that TM Gress, Proceedings of the first inter- might interact with NOV and other CCN pro- 30 33 national workshop on the CCN family of teins we have used the two hybrid system genes, 17–19 October 2000, Saint-Malo, strategy, which has been used successfully over France) and in about 30% of invasive breast the past few years to uncover many protein carcinomas.67 interactions involved in cell growth signalling. Increased expression of ctgf has also been To avoid selecting targets that might only detected by northern blotting in about 70% of interact with isolated modules, we used the full 11 human invasive mammary ductal carcino- length NOV protein fused to the DNA binding domain of Gal4 DNA as bait. The interactions mas,68 in 100% of nine dermatofibromas that were detected with this system were examined, in pyogenic granuloma, in endothe- usually validated by pull down assays per- lial cells of angiolipomas and angioleiomyo- formed with a panel of glutathione mas,69 and in 15 of 19 pancreatic tumours.70 S-transferase (GST) fusion proteins containing However, five of seven cases of dermatofibro- diVerent portions of nov. The use of these sarcoma protuberans and two cases of malig- recombinant proteins also allowed us to nant fibrous histiocytoma were negative for ctgf 69 identify the domains of NOV that were expression. A more recent immunocyto- involved in the interactions. chemical study performed with 18 chondro- In addition to RNA polymerase II subunit sarcomas representative of various histological 7,30 we identified fibulin 1C as a protein inter- grades established that ctgf expression closely acting with NOV.33 The potential interaction of 71 correlated with increasing malignancy. NOV and fibulin 1C provided the first evidence Although the number of reports concerning for a role of NOV in cell adhesion signalling. the expression of cyr61 and ctgf in tumours is Fibulin 1C is a 100 kDa protein of the ECM still limited, the general picture is that neither a that has been reported to bind fibronectin, straightforward nor a general correlation can laminin, fibrinogen, and basement membrane be drawn between the expression of cyr61 or protein nidogen.33 The expression of fibulin 1 ctgf and tumour development. in the perichondrium and calcifying regions of A clue to understanding the discrepancies developing bones, in the gut subepithelium,

observed for the expression of the CCN genes and in peripheral nerves of human embryos of http://mp.bmj.com/ in cancer cells might come from the multi- gestational weeks 4–10 correlated with sites of modular structure of the CCN proteins and nov expression at the same developmental their potential ability to interact sequentially or stages, and provided another indication that simultaneously with diVerent partners, the NOV and ﬁbulin might indeed interact in vivo. bioavailability of which might be a key element Furthermore, the levels of nov and ﬁbulin 1C in the manifestation of the positive or negative RNAs correlated inversely in several cell lines,33 and ectopic expression of nov is accompanied

eVects attributed to the CCN proteins. In line on September 29, 2021 by guest. Protected copyright. by a decrease of fibulin 1C concentrations, Negative factors therefore suggesting that a downregulation of ? fibulin 1C transcription might take place in Ct cells expressing high amounts of nov. These studies also established that the C-terminal module of NOV was suYcient to TSP1 promote the interaction of NOV with fibulin IGFBP 1C. The association of NOV and fibulin Ct Hinge through the C-terminal portion of fibulin 1C, which contains five of nine EGF repeats present in fibulin 1C, raised the possibility that VWC TSP1 the binding of NOV might mediate the poten- IGFBP tial role of fibulin 1C in the assembly of the ECM and cell adhesion.33 It is worth noting Hinge that the C-terminal domain of NOV was also VWC shown to mediate the interaction of NOV with CTGF in the two hybrid system, therefore TSP1 suggesting that heterodimerisation of CCN Hinge proteins might take place in vivo and that cross ? talk between CCN proteins is involved in the Positive factors modulation of their biological activities. CCN 72 Figure 14 Model for the structural organisation of the CCN proteins. Potential proteins such as rCOP1 and CTGF-L, which interactions with positive and negative regulators are thought to be disrupted upon are deprived of the C-terminal domain, might truncation of the N-proximal and C-proximal modules of CCN proteins. Ct, cystein knot play an important role in these processes and containing family of growth regulators-like domain; IGFBP,insulin-like growth factor binding protein-like domain; TSP1, thrombospondin-like domain; and VWC, Von exhibit a whole range of dominant negative Willebrand factor-like domain. type activities.

www.molpath.com NOV and the CCN family of genes 77

Upregulated, suppressor responsible for the regulation of NOV biologi- nov cal activity. Establishing whether other CCN

Unregulated, inducder proteins also interact with the NOV targets Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from wisp1 remains a major goal. Downregulated, suppressor Full length More generally, there is a dramatic need for Elm1 comparative studies to be performed with diVerent members of the CCN family. The availability of the reagents and the develop- Upregulated ment of biological assays for several members oncogenic of the CCN family should greatly ease such nov studies, the potential value of which is obvious NH truncated 2 in the light of the diversity of the systems used thus far. Upregulated To answer the question “what is nov doing?” wisp2 I have embarked my laboratory on a path Downregulated, suppressor aimed at examining several complementary COOH truncated Wisp2/rCOP-1 aspects of nov biology. Similar approaches per- Figure 15 CCN proteins and cancer. This schematic drawing summarises the diversity of formed with other CCN genes would certainly situations encountered for CCN proteins in tumours. Whether the subcellular localisation of the CCN proteins other than NOV can result in opposing biological properties remains to be prove to be extremely fruitful. Thus, I think it is established. essential to establish where, how, and with whom the CCN proteins act to answer later the Our working hypothesis is based on the questions regarding the regulation of their assumption that under native conditions the activity and the consequences of altered CCN proteins adopt a compact structure (ﬁg expression. 14), where two halves interact with each other as a result of the hinge region that is found WHERE? between modules 3 and 4 (sharing identity with The use of diVerent eYcient expression von Willebrand factor type C repeat and systems (such as baculoviruses) enables the thrombospondin, respectively). This interac- production of recombinant tagged proteins tion would permit the N-proximal half to exert that can be used to identify the sites of CCN a negative regulatory control on the stimula- protein binding on cells and follow the fate of tory properties of the C-proximal half. The the secreted proteins once they are presented at folded structure would also allow a certain the cell surface. The production of speciﬁc number of interactions with positive and nega- polyclonal and monoclonal antibodies directed tive eVectors. The bioavailability of these eVec- against diVerent portions of the CCN proteins tors, coupled with the temporal and spatial also enables their concentrations to be

expression of the CCN proteins, would be cru- measured in biological fluids. They can also http://mp.bmj.com/ cial factors that dictate the constitution of the establish whether quantitative variations of cir- complexes that can be formed, and subse- culating CCN proteins are associated with quently the nature of the signals transmitted by pathological conditions and can be used as a these complexes. Truncations of the CCN pro- valuable clinical tool. We are presently using teins at either their N-terminus or C-terminus this strategy to measure, under pathological would disrupt these regulatory interactions, conditions, the variations of NOV released in alter the balance of positive and negative regu- diVerent biological fluids, such as cerebrospi- on September 29, 2021 by guest. Protected copyright. latory signals (fig 15), and result in conferring nal fluid, seminal fluid, urine, and serum. to these proteins a constitutive repressive or stimulatory eVect on cell growth, as exempli- HOW? fied by the case of WISP-2/rCOP1, and by the This aspect requires the purification of native production of an oncogenic truncated NOV CCN proteins from a reliable biological protein in MAV induced nephroblastoma (see source. Although the use of recombinant above). The implications of this working model proteins has proved very fruitful in unravelling are presently being tested. the biological properties of several signalling eVectors, it must be kept in mind that many Future directions and prospects biochemical properties of native proteins re- In the light of the recent results reported in this quire post translational modifications, which review, it seems that CCN proteins might act as do not take place (or only partially) in most of multipotent matchmakers, allowing the scaf- the systems used to produce recombinant pro- folding of a complex network of regulatory teins. Purification of the native protein also proteins responsible for the regulation of permits the isolation of functional complexes several fundamental functions during normal whose characterisation has proved to be life, development, and probably death. extremely informative in many instances. The identification of other proteins poten- Because CCN proteins potentially interact tially interacting with NOV has established that with several other partners, the isolation of each of the four constitutive domains of the complexes in which these proteins are engaged native protein is required alone or in combina- will undoubtely lead to important discoveries tion with others to promote the interactions. In concerning their mode of action. The purifica- some cases, only the full length NOV protein tion of the diVerent CCN proteins can also was found to interact with potential targets. establish whether they exhibit synergistic or Deciphering this complex situation will cer- antagonistic properties when tested in combi- tainly lead to the identification of major players nation.

www.molpath.com 78 Perbal

WITH WHOM? 10 Perbal B. Pathogenic potential of myeloblastosis associated viruses. Infect Agents Dis 1995;4:212–27. Immunoprecipitation of complexes, covalent 11 Chevalier G, Perbal B. Genetic alterations associated with

crosslinking, and solid phase assays are among pathologic diVerentiation of Wilms’ tumors. Bull Cancer Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from the biochemical strategies being used currently (Paris) 1997;84:289–303. 12 Joliot V, Martinerie C, Dambrine G, et al. Proviral to identify ligands interacting with the CCN rearrangements and overexpression of a new cellular gene proteins. The use of biological systems such as (nov) in myeloblastosis-associated virus type 1-induced nephroblastomas. Mol Cell Biol 1992;12:10–21. phage display, the two hybrid system, etc 13 Martinerie C, Viegas-Péquignot E, Guénard I, et al. Physical should also permit the identification of proteins mapping of human loci homologous to the chicken nov interacting with some, or many members, of protooncogene. Oncogene 1992;7:2529–34. 14 Hashimoto BY, Shindo-Okada N, Tani M, et al. Expression the CCN family. Establishing whether cross of the ELM1 gene, a novel gene of the CCN (connective interactions exist among the diVerent CCN tissue growth factor, cyr61/cef10, and neuroblastoma overexpressed gene) family, suppresses in vivo tumor growth proteins will be of great interest. and metastasis of K-1735 murine melanoma cells. JExp Questions regarding the biological activity of Med 1998;187:289–96. 15 Zhang R, Averboukh L, Zhu W, et al. Identification of the CCN proteins in vitro or ex vivo are also rCop-1, a new member of the CCN protein family, as a worth considering. There are several systems in negative regulator for cell transformation. Mol Cell Biol 1998;18:6131–41. which to study the eVects of CCN proteins on 16 Pennica D, Swanson TA, Welsh JW, et al. WISP genes are proliferation, diVerentiation, and cell death. members of the connective tissue growth factor family that Again, comparative studies performed with are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors. Proc Natl Acad Sci diVerent members of the CCN family in the USA1998;95:14717–22. same system should greatly improve our 17 Simmons D, Levy D, Yannoni Y, et al. Identification of a phorbol ester-repressible v-w-c- inducible gene. Proc Natl understanding of the biology of CCN proteins. Acad Sci USA 1989;86:1178–82. Post transcriptional and post translational 18 Scholz G, Martinerie C, Perbal B, et al. Transcriptional down regulation of the nov proto-oncogene in fibroblasts processes are thought to be involved in the transformed by p60v-src. Mol Cell Biol 1996;16:481–6. regulation of the bioavailability and functions 19 Bradham DM, Igarashi A, Potter RL, et al. Connective tissue growth factor: a cysteine-rich mitogen secreted by of CCN proteins. PKC is involved in the regu- human vascular endothelial cells is related to the SRC- lation of CCN RNA post translational turno- induced immediate early gene product CEF-10. J Cell Biol ver. Activation of PKC has recently been 1991;114:1285–94. 73 20 Ryseck RP, Macdonald-Bravo H, Mattei MG, et al. reported to inhibit the expression of ctgf. It Structure, mapping, and expression of fisp-12, a growth would be worth examining more precisely the factor-inducible gene encoding a secreted cysteine-rich protein. Cell Growth DiVer 1991;2:225–33. relation that might exist between PKC directed 21 Lin J, Liliensiek B, Kanitz M, et al. Molecular cloning of phosphorylation and CCN gene expression. genes diVerentially regulated by TNF-á in bovine aortic endothelial cells, fibroblasts and smooth muscle cells. Car- Last, but not least, the bulk of studies aimed diovasc Res 1998;38:802–3. at establishing whether the CCN proteins are 22 SteVen CL, Ball-Mirth DK, Harding PA, et al. Characteri- involved, directly or indirectly, in tumour sation of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells development have demonstrated the extreme in vitro. Growth Factors 1998;15:199–213. complexity and diversity of CCN gene expres- 23 Igarashi A, Okochi H, Bradham DM, et al. Regulation of connective tissue growth factor gene expression in human sion patterns in the various systems analysed. skin fibroblasts and during wound repair. Mol Cell Biol http://mp.bmj.com/ Again, comparative studies would certainly be 1993;4:637–45. 24 Hishikawa K, Oemar BS, Tanner FC, et al. Overexpression very informative and would probably allow us of connective tissue growth factor gene induces apoptosis to establish a solid basis for the understanding in human aortic smooth muscle cells. Circulation 1999;100: of the biological properties of the CCN 2108–112. 25 Hishikawa K, Oemar BS, Tanner FC, et al. Connective proteins and to determine whether they can be tissue growth factor induces apoptosis in human breast used as tools for future molecular medicine. cancer cell line MCF-7. J Biol Chem 1999;274:37461–6. 26 Hishikawa K, Nakaki T, Fujii T. Connective tissue growth

factor induces apoptosis via caspase 3 in cultured human on September 29, 2021 by guest. Protected copyright. I wish to thank particularly my wife Annick for her help and aortic smooth muscle cells. Eur J Pharmacol 2000;392:19– constant support. Thanks are also due to S Gabaron for reading 22. the manuscript. The research performed in my laboratory was 27 Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the funded by grants from the Ligue Nationale Contre le Cancer CCN gene family member WISP3 cause progressive pseu- (Comités National, du Cher et de l’Indre), Association dorheumatoid dysplasia. Nat Genet 1999;23:94–8. Française contre les Myopathies (AFM), Matra-Hachettte, 28 Kim HS, Nagalla SR, Oh Y, et al. Identification of a family Fondation pour la Recherche Médicale (FRM), and Association of low-aYnity insulin-like growth factor binding proteins pour la Recherche contre le Cancer (ARC). I am grateful to (IGFBPs): characterisation of connective tissue growth Professor B Roizman for his support and for providing the facil- factor as a member of the IGFBP super family. Proc Natl ity to perform experiments with herpes simplex virus and the Acad Sci U S A 1997;94:12981–6. two hybrid system. 29 Grotendorst G, Lau L, Perbal B. CCN proteins are distinct from and should not be considered members of the 1 Bork P. The modular architecture of a new family of growth insulin-like growth factor-binding protein superfamily. regulators related to connective tissue growth factor. FEBS Endocrinologie 2000;141:2254–6. Lett 1993;327:125–30. 30 Perbal B. Nuclear localization of NOV protein: a potential 2 Lau LF, Lam SC. The CCN family of angiogenic role for nov in the regulation of gene expression. J Clin regulators: the connection. Exp Cell Res 1999; 248:44–57. Pathol: Mol Pathol 1999;52:84–91. 3 Brigstock DR. The connective tissue growth factor/cysteine- 31 Roizman B, Sears A. Herpes simplex viruses and their repli- rich 61/nephroblastoma overexpressed (CCN) family. cation. In: Fields BN, Knipe DM, eds. Fields virology,Vol. Endocr Rev 1999;20:189–206. 2. New York: Raven Press, 1990:1795–841. 4 Essam ED, Moussad A, Brigstock D. Connective tissue 32 Mahony D, Kalionis B, Antalis TM. Plasminogen activator growth factor: what’s in a name. Mol Genet Metab 2000;71: inhibitor type-2 (PAI-2) gene transcription requires a novel 276–92. NF-KappaB-like transcriptional regulatory motif. Eur J 5 Lau LF, Nathans D. Expression of a set of growth-related Biochem 1999;263:765–72. immediate early genes in BALB/c 3T3 cells: coordinate 33 Perbal B, Martinerie C, Sainson R, et al. The C-terminal regulation with c-fos or c-myc. Proc Natl Acad Sci USA 1987; :1182–6. domain of the regulatory protein NOVH is suYcient to 84 promote interaction with fibulin 1C: a clue for a role of 6 Almendral J, Sommer D, Mac Donald-Bravo H, et al. Com- plexity of the early genetic response to growth factors in NOVH in cell-adhesion signaling. Proc Natl Acad Sci U S A 1999; :869–74. mouse fibroblasts. Mol Cell Biol 1998;8:2140–8. 96 7 Brunner A, Chinn C, Neubauer M, et al. Identification of a 34 Ball DK, Surveyor GA, Diehl JR, et al. Characterisation of gene family regulated by transforming growth factor-â. 16-to 20-kilodalton (kDa) connective tissue growth factors DNA Cell Biol 1991;10:293–300. (CTGFs) and demonstration of proteolytic activity for 8 Matsunaga E. Genetics of Wilms’ tumor. Hum Genet 1981; 38-kDa CTGF in pig uterine luminal flushings. Biol Reprod 57:231–46. 1998;59:828–35. 9 Perbal B. Contribution of MAV-1-induced nephroblastoma 35 Chevalier G, Yeger H, Martinerie C, et al. NovH: diVerential to the study of genes involved in human Wilms’ tumor expression in developing kidney and Wilms’ tumors. Am J development. Crit Rev Oncog 1994;5:589–613. Pathol 1998;152:1563–75.

www.molpath.com NOV and the CCN family of genes 79

36 Burren C, Wilson E, Hwa V, et al. Binding properties and 54 Nishida T, Nakanishi t, Shimo T, et al. Demonstration of distribution of insulin-like growth factor binding protein- receptors specific for connective tissue growth factor on a related protein 3 (IGFBP-rP3/NovH), an additional mem- human chondrocytic cell line (HCS-2/8). Biochem Biophys ber of the IGFBP superfamily. J Clin Endocrinol Metab Res Commun 1998;247:905–9. Mol Path: first published as 10.1136/mp.54.2.57 on 1 April 2001. Downloaded from 1999;84:1096–103. 55 Nishida T, Nakanishi T, Asano M, et al.EVects of 37 Latinkic B, O’Brien P, Lau L. Promoter function and struc- CTGF/Hcs24, a hypertrophic chondrocyte-specific gene ture of the growth factor-inducible immediate early gene product, on the proliferation and diVerentiation of osteob- cyr61. Nucleic Acids Res 1991;19:3261–7. lastic cells in vitro. J Cell Physiol 2000;184:197–206. 38 Grotendorst G, Okochi H, Hayashi N. A novel transforming 56 Barbot W. DEA biologie cellulaire et moleculaire. Université growth factor â response element controls the expression of Paris VI. 1997. the connective tissue growth factor gene. Cell Growth DiVer 57 Perbal B. Caractérisation et expression du proto-oncogène 1996;7:469–80. nov humain dans les tumeurs de Wilms. Bull Cancer (Paris) 39 Martinerie C, Chevalier G , Rauscher F, Jr, et al. Regulation 1994;81:957–61. of nov by WT1: a potential role for nov in nephrogenesis. 58 Yang GP, Lau LF. Cyr61, product of a growth factor- Oncogene 1996;12:1479–92. inducible immediate early gene, is associated with the 40 Schutze N, Lechner A, Groll C, et al. The human analog of extracellular matrix and the cell surface. Cell Growth DiVer murine cysteine-rich protein 61 is a 1á,25- 1991;2:351–7. dihydroxyvitamin D3 responsive immediate early gene in 59 Martinerie C, HuV V, Joubert I, et al. Structural analysis of human fetal osteoblasts: regulation by cytokines, growth the human nov proto-oncogene and expression in Wilms factors, and serum. Endocrinology 1998;139:1761–70. tumor. Oncogene 1994;9:2729–32. 41 Rivera-Gonzalez R, Petersen DN, Tkalcevic G, et al. 60 Lopez-Bermejo A, Buckway CK, Devi GR, et al. Characteri- Estrogen-induced genes in the uterus of ovariectomized zation of insulin-like growth factor-binding protein-related rats and their regulation by droloxifene and tamoxifen.J proteins (IGFBP-rPs) 1, 2 and 3 in human prostate epithelial Steroid Biochem Mol Biol 1998;64:13–24. cells: potential roles for IGFBP-rP1 and 2 in senescence of 42 Chung KC, Ahn YS. Expression of immediate early gene the prostatic epithelium. Endocrinology 2000;141:4072–80. cyr61 during the diVerentiation of immortalized embryonic 61 Li WC, Martinerie C, Zumkeller W, et al.DiVerential hippocampal neuronal cells. Neurosci Lett 1998;255:155–8. expression of novH and CTGF in human glioma cell lines. 43 Stanhope-Baker P, Williams BR. Identification of connec- J Clin Pathol: Mol Pathol 1996;49:M91–7. tive tissue growth factor as a target of WT1 transcriptional 62 Babic A, Kireeva M, Kolesnikova T, et al. CYR61, a product regulation. J Biol Chem 2000;275:38139–50. of a growth factor-inducible immediate early gene, 44 Kondo Y, Nakanishi T, Takigawa M, et al. Immuno- promotes angiogenesis and tumor growth. Proc Natl Acad histochemical localization of connective tissue growth SciUSA1998;95:6355–60. factor in the rat central nervous system. Brain Res 1999; 63 Babic A, Chen CC, Lau L. Fisp 12/mouse connective tissue 834:146–51. growth factor mediates endothelial cell adhesion and 45 O’Brien TP, Yang GP, Sanders L, et al. Expression of cyr61, migration through integrin ávâ3, promotes endothelial cell a growth factor-inducible immediate–early gene. Mol Cell survival, and induces angiogenesis in vivo. Mol Cell Biol Biol 1990;10:3569–77. 1999;19:2958–66. 46 Su BY, Cai WQ, Zhang CG, . A developmental study of et al 64 Martinerie C, Viegas-Pequignot E, Van-Cong N, et al. novH gene expression in human central nervous system. C Chromosomal mapping and expression of the human 1998;321:883–92. R Acad Sci Paris CYR61 gene in tumor cells from the nervous system. J Clin 47 Kireeva M, Latinkic B, Kolesnikova T, et al. Cyr61 and 1997; :310–17. Fisp12 are both ECM-associated signaling molecules: Pathol: Mol Pathol 50 activities, metabolism, and localization during develop- 65 Gemini M, Schwalbe P, Scholl FA, Schufer BW. Isolation of genes diVerentially expressed in human primary myoblasts ment. Exp Cell Res 1997;233:63–77. 48 O’Brien TP, Lau LF. Expression of the growth factor- and embryonal rhabdomyosarcoma. Int J Cancer 1996;66: inducible immediate early gene cyr61 correlates with chon- 571–7. drogenesis during mouse embryonic development. Cell 66 Pilarsky C, Schmidt U, Eissrich C, et al. Expression of the Growth DiVer 1992;3:645–54. extracellular matrix signaling molecule Cyr61 is downregu- 49 Wong M, Kireeva M, Kolesnikova T, et al. Cyr61, product of lated in prostate cancer. Prostate 1998;36:85–91. a growth factor-inducible immediate–early gene, regulates 67 Tsai MS, Hornby AE, Lakins J, et al. Expression and func- chondrogenesis in mouse limb bud mesenchymal cells. Dev tion of CYR61, an angiogenic factor, in breast cancer cell Biol 1997;192:492–508. lines and tumor biopsies. Cancer Res 2000;60:5603–7. 50 Hadjiargyrou M, Ahrens W, Rubin CT. Temporal expres- 68 Frazier K, Grotendorst G. Expression of connective tissue sion of the chondrogenic and angiogenic growth factor growth factor mRNA in the fibrous stroma of mammary

CYR61 during fracture repair. J Bone Miner Res 2000;15: tumors. Int J Biochem Cell Biol 1997;29:153–61. http://mp.bmj.com/ 1014–23. 69 Igarashi A, Hayashi N, Nashiro K, et al.DiVerential expres- 51 Nakanishi T, Kimura Y, Tamura T, et al. Cloning of a sion of connective tissue growth factor gene in cutaneous mRNA preferentially expressed in chondrocytes by diVer- ﬁbrohistiocytic and vascular tumor. J Cutan Pathol ential display-PCR from a human chondrocytic cell line 1998;25:143–8. that is identical with connective tissue growth factor 70 Wenger C, Ellenrieder V, Alber B, et al. Expression and dif- (CTGF) mRNA. Biochem Biophys Res Commun 1997;234: ferential regulation of connective tissue growth factor in 206–10. pancreatic cancer cells. Oncogene 1999;18:1073–80. 52 Shimo T, Nakanishi T, Kimura Y, et al. Inhibition of endog- 71 Shakunaga T, Ozaki T, Ohara N, et al. Expression of enous expression of connective tissue growth factor by its connective tissue growth factor in cartilaginous tumors. antisense oligonucleotide and antisense RNA suppresses Cancer 2000;89:1466–73.

proliferation and migration of vascular endothelial cells. J 72 Kumar S, Hand AT, Connor JR, et al. Identiﬁcation and on September 29, 2021 by guest. Protected copyright. Biochem 1998;124:130–40. cloning of a connective tissue growth factor-like cDNA 53 Nakanishi T, Nishida T, Shimo T, et al.EVects of from human osteoblasts encoding a novel regulator of oste- CTGF/Hcs24, a product of a hypertrophic chondro- oblast functions. J Biol Chem 1999;274:17123–31. cyte-speciﬁc gene, on the proliferation and diVerentiation 73 Fan WH, Karnovsky MJ. Activation of protein kinase C of chondrocytes in culture. Endocrinology 2000;141:264– inhibits the expression of connective tissue growth factor. 73. Biochem Biophys Res Commun 2000;275:312–21.