NOV a Selective Adrenocortical__ Cell Proapoptotic Factor

Nephroblastoma Overexpressed/Cysteine-Rich Protein 61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed Gene-3(NOV/CCN3),a Selective Adrenocortical Cell Proapoptotic Factor,Is Down-Regulated in Childhood Adrenocortical Tumors

Mabrouka Doghman,Malika Arhatte,He ′le `ne Thibout,Giovanna Rodrigues,Juliana De Moura,Se ′bastien Grosso,Alina Nico West,Maryvonne Laurent,Jean-Christophe Mas,Andre ′Bongain,Gerard P.Zambetti,Bonald C.Figueiredo,Patrick Auberger,Ce ′cile Martinerie,and Enzo Lalli

Institut de Pharmacologie Mole ′culaire et Cellulaire Centre National de la Recherche Scientifique Unite ′Mixte de Recherche 6097and Universite ′de Nice Sophia Antipolis (M.D.,M.A.,G.R.,J.D.M.,E.L.),06560Valbonne,France;Institut National de la Sante ′et de la Recherche Me ′dicale U515(H.T.,M.L.,C.M.),Ho ?pital St.Antoine,UPMC,Paris 6,75012Paris,France;Institut National de la Sante ′et de la Recherche Me ′dicale U526(S.G.,P.A.),Faculte ′de Me ′decine,06107Nice,France;

Department of Biochemistry (A.N.W.,G.P.Z.),St.Jude Children’s Research Hospital,Memphis,Tennessee 38105;Service de Pe ′diatrie (J.-C.M.)and Service de Gyne ′cologie Obste ′trique (A.B.),Ho ?pital de l’Archet,Centre Hospitalier Universitaire,06200Nice,France;and Instituto de Pesquisa Pele ′Pequeno Principe and Centro de Gene ′tica Molecular e Pesquisa do Ca ?ncer em Crianc ?as (B.C.F.),Curitiba,80.060-110Parana ′,Brazil

Context:Childhood adrenocortical tumors (ACTs)have a fetal ad-renal phenotype and overexpress steroidogenic factor-1(SF-1).Nephroblastoma overexpressed (NOV)/cysteine-rich protein 61/con-nective tissue growth factor/nephroblastoma overexpressed gene-3mRNA is significantly down-regulated in childhood ACTs.

Objective:The objective of the study was to measure NOV protein levels in childhood ACTs and characterize NOV expression regulation and biological function in human adrenocortical cells.

Design and Setting:Protein extracts from ACT and normal adrenal cortex samples,human adrenocortical carcinoma H295R,primary adrenocortical tumors and fetal adrenal cultures,tissue culture su-pernatants,and cell lysates from H295R cells overexpressing SF-1in an inducible fashion were used.

Main Outcome Measures:NOV protein levels were measured by enzyme-linked immunoassay and immunoblot.Transient transfec-tion assays were used to study the activity of NOV promoter.Terminal

deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling,caspase assays,and flow cytometry were used to assess the proapoptotic activity of NOV on cells in culture.

Results:NOV mRNA and protein expression is lower in childhood ACTs than in normal adrenal cortex.No significant difference was observed between adenomas and carcinomas.SF-1overexpression down-regulates NOV at the transcriptional level.NOV has a selective proapoptotic activity toward human adrenocortical cells.The C-ter-minal domain of NOV is responsible for its proapoptotic effect.NOV protein is expressed in DAX-1-positive human fetal adrenal cells.Conclusions:NOV is a selective proapoptotic factor for human ad-renocortical cells.Reduced expression of NOV in ACTs may play an important role in the process of childhood ACT tumorigenesis,ac-counting at least in part for the defect of apoptotic regression of the fetal adrenal that has been proposed to be responsible for tumor formation.(J Clin Endocrinol Metab 92:3253–3260,2007)

T

HE STRUCTURE OF the human adrenal cortex is dif-ferent during fetal life and after birth.In fact,during intrauterine life,most of the inner part of the adrenal cortex (fetal zone)is occupied by large,steroid-secreting cells,

which are differentiated to synthesize large amounts of de-hydroepiandrosterone and its sulfate derivative.Conversely,a few layers of cells lying in an outer,subcapsular position (definitive zone)are nonsteroidogenic and constitute a pro-liferative compartment in which most probably the precur-sors of fetal zone cells are generated (1–3).An additional zone (transitional zone)develops between the definitive and the fetal zone late in pregnancy and synthesizes cortisol.The fetal adrenal undergoes a process of rapid involution and remodeling after birth (1,4).The fetal zone regresses by apoptosis,whereas cells of the definitive zone proliferate and differentiate to form the glomerulosa,fasciculata,and re-ticularis zones of the adult adrenal cortex.Childhood adre-nocortical tumors (ACTs)are thought to be derived from the fetal adrenal,probably through defective apoptosis because of their age distribution,their pattern of hormone secretion,

First Published Online June 19,2007

Abbreviations:ACT,Adrenocortical tumors;CCN,connective tissue growth factor;DAPI,4?,6?-diamidino-2-phenylindole;DAX-1,dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X chromosome-gene 1;FITC,fluorescein isothiocyanate;IGFBP,IGF bind-ing protein;NOV,nephroblastoma overexpressed;P450scc,P450side-chain cleavage;SF-1,steroidogenic factor-1;TUNEL,terminal deoxy-nucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

JCEM is published monthly by The Endocrine Society (https://www.wendangku.net/doc/379637802.html,),the foremost professional society serving the en-docrine community.

0021-972X/07/$15.00/0The Journal of Clinical Endocrinology &Metabolism 92(8):3253–3260

Printed in U.S.A.

Copyright ?2007by The Endocrine Society

doi:10.1210/jc.2007-0342

3253

and their molecular phenotype(5,6).These tumors can arise isolated or in the context of genetic syndromes(for a review see Ref.7)and most commonly present with virilization due to production of androgenic steroids,which can be associ-ated with Cushing syndrome.A peculiar epidemiological feature of childhood ACTs is their high incidence in the states of Parana′and Sa?o Paulo in southern Brazil,where they are almost invariably found associated with a specific germline TP53mutation(R337H)(8).In addition,a role for the tran-scription factor steroidogenic factor-1(SF-1)in childhood ACT pathogenesis has been suggested based on its ampli-fication and overexpression in this type of cancer(9,10).It is remarkable that an increase in SF-1dosage is by itself sufficient to increase proliferation of human tumor adreno-cortical cells and to cause adrenocortical tumors in mice (Doghman,M.,T.Karpova,G.Rodrigues,M.Arhatte,J.De Moura,L.R.Cavalli,,V.Virolle,P.Barby,G.P.Zambetti,B.

C.Figueiredo,L.L.Heckert,https://www.wendangku.net/doc/379637802.html,lli,submitted for publication).

A gene expression profiling study performed in childhood ACTs has recently shown that distinct patterns of gene ex-pression distinguish tumors from normal adrenal cortex. Nephroblastoma overexpressed(NOV)/cysteine-rich pro-tein61/connective tissue growth factor/nephroblastoma overexpressed gene(CCN)-3is one of the transcripts that is most powerfully down-regulated in childhood ACTs,inde-pendently from their degree of malignancy(11).Conversely, it was previously shown that loss of NOV expression is associated with a poor prognosis in adult adrenocortical tumors(12).NOV is a multidomain-secreted protein,mem-ber of the CCN family,whose expression was described to be mostly restricted to the definitive zone of the fetal adrenal cortex(13).Its known functions are related to cell adhesion and angiogenesis,and its expression is negatively correlated to proliferation and associated with differentiation in many cellular systems(for review see Ref.14).Here we show that NOV is strikingly down-regulated at the protein level in childhood ACTs and in human adrenocortical cells in a man-ner dependent on SF-1dosage and characterize its selective proapoptotic activity for human adrenocortical cells.

Subjects and Methods

Tumor samples

Twenty-four childhood ACTs(five adenomas,18carcinomas,and one of undetermined histological type)and seven normal adrenal cortex samples were used for mRNA studies.These were the same samples that were used for gene expression profiling using Affymetrix microarrays (11).For protein expression studies,tumor samples were obtained at the time of surgery from eight children with ACTs(three boys and five girls). Five patients presented with virilization and three with virilization associated with Cushing’s syndrome.Histologically,four tumors were classified as adenomas and four as carcinomas.Eight normal adrenal glands resected from age-matched children undergoing surgery for Wilms’tumor were used as controls.Samples were immediately snap frozen in liquid nitrogen before processing for protein extraction.In one case of adrenocortical adenoma in a5-yr-old child,tumor cells were dissociated with collagenase and cultured in OptiMEM(Invitrogen, Carlsbad,CA)containing2%fetal calf serum,following an established protocol(15).All patients and control subjects were included in the study after one of the parents or legal representatives signed an informed consent form approved by the Ethics Committee of the Hospital de Cl?′nicas of the Federal University of Parana′.Human fetal adrenal tissue preparation

Adrenal glands were obtained from human fetuses aged10–14wk after elective termination of pregnancy.The research protocol was pre-viously submitted to the French national Agence de la Biome′decine and local Ethical Committee of the Nice University Hospital.Fetal adrenal cells were isolated and cultured as described(15).

Immunofluorescence

It was performed as described(16)using the following antibodies: rabbit anti-NOV(K19M);mouse anti-dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X chromosome-gene 1(DAX-1)(2F4);rabbit anti-P450side-chain cleavage(P450scc;gift of https://www.wendangku.net/doc/379637802.html,ler)(17);rabbit anti-SF-1(Upstate,Lake Placid,NY).As negative control for primary antibodies,species-matched IgG was used.Cell nuclei were counterstained with4?,6?-diamidino-2-phe-nylindole(DAPI).

Cell culture

H295R cells(American Type Culture Collection,Manassas,VA)were cultured in DMEM/F-12supplemented with2%NuSerum(Becton Dick-inson,Lincoln Park,NJ),1%ITS Plus(Becton Dickinson),and antibiotics. H295R cell clones overexpressing SF-1in a doxycycline-inducible fash-ion(H295R TR/SF-1)were produced and maintained as described (Doghman,M.,T.Karpova,G.Rodrigues,M.Arhatte,J.De Moura,L. R.Cavalli,V.Virolle,P.Barby,G.P.Zambetti,B.C.Figueiredo,L.L. Heckert,https://www.wendangku.net/doc/379637802.html,lli,submitted for publication).Proliferation assays were performed in the absence of NuSerum.HeLa cells were cultured in DMEM(1g/liter glucose)supplemented with10%fetal calf serum (Invitrogen)and antibiotics.COS cells were cultured in DMEM(4.5 g/liter glucose)supplemented with5%fetal calf serum(Invitrogen)and antibiotics.Jurkat cells were cultured in RPMI1640supplemented with 5%fetal calf serum(Invitrogen)and antibiotics.

cDNA amplification and real-time PCR analysis for NOV mRNA expression

This was performed as described(11).Results were normalized to ubiquitin expression levels using the??C t method

(18).

F IG.1.NOV is down-regulated in childhood ACTs at the transcript and protein level.A,NOV mRNA levels were measured in seven normal adrenals and24childhood ACT samples by quantitative RT-PCR.NOV mRNA levels are significantly down-regulated in tumors,compared with normal adrenal(Mann-Whitney U test;P?0.002).**,P?0.05.B, Immunoblot showing NOV protein expression in eight childhood ACTs (T1-T4adenomas;T5-T8carcinomas)and eight normal adrenals.?-Tu-bulin levels show equivalent protein loading of gel lanes.

3254J Clin Endocrinol Metab,August2007,92(8):3253–3260Doghman et al.?NOV/CCN3and Childhood Adrenocortical Tumors

NOV assays

Secreted NOV protein was quantified by an enzyme linked immu-noassay,as described (19)or extracted from culture supernatants by heparin-Sepharose chromatography,as described (20),and detected by immunoblot.

Immunoblots

The immunoblots were performed as described (10).Normal and tumor adrenal samples were homogenized in Laemmli buffer,and pro-

teins were separated on SDS-PAGE and blotted on a polyvinyl difluoride membrane (GE Healthcare,Indianapolis,IN).Antibodies used for im-munoblot were:rabbit anti-NOV (K19M),mouse anti-?-tubulin (Sigma,St.Louis,MO),and goat anti-IGF binding protein (IGFBP)-2(Santa Cruz Biotechnology,Santa Cruz,CA).

Transient transfection and luciferase assays

H295R cells were transfected with NOV promoter reporters (20)and pCH110as a ?-galactosidase expression vector using the jetPEI

reagent

F I

G .2.NOV production is negatively regulated by SF-1overexpression in H295R cells.A,NOV protein levels are decreased in the culture su-pernatant of H295R TR/SF-1cells treated with doxycycline,compared with untreated cells.Cells were incubated for 48h in the absence or presence of Dox,medium was collected,and NOV levels were assayed by a specific enzyme immunoassay and Western blot.Culture me-dium was replaced,and measurements were re-peated after 24h.Significant differences existed between NOV levels in the absence (white his-tograms )and presence (black histograms )of Dox at both 48and 72h (P ?0.0055and P ?0.0378,respectively,t test).Conversely,Dox treatment did not modify the levels of the secreted protein IGFBP2in the supernatant of H295R TR/SF-1cells at both 48and 72h.*,P ?0.01;**,P ?0.05.B,SF-1overexpression reduces NOV promoter activity.NOV promoter ?2540,?625,and ?492luciferase reporters were transfected in H295R TR/SF-1or parental H295R TR cells.Cells were subsequently cultured for 48h in the presence or absence of Dox.For each promoter fragment,luciferase activity in Dox-treated cells (white histograms )was compared with the activity of untreated cells (black histograms ).No effect of Dox treatment was detected in transfected parental cells.Data shown are the average of three independent transfection ex-periments,each one performed in

duplicate.

F I

G .3.NOV reduces H295R cell number in a dose-de-pendent manner.A,H295R cells were cultured in me-dium without NuSerum in the presence of protein buffer only (Ctrl)or with increasing doses [0.01(0.25n M ),0.05(1.25n M ),0.1(2.5n M ),and 0.5(12.5n M )?g/ml]of re-combinant NOV protein added.Cell numbers were counted after 4d.Data are normalized by the number of cells present in buffer-treated cultures.Data shown are the average of two experiments performed in duplicate.B,Cell cycle distribution of untreated H295R cells (left )and cells treated with 0.5?g/ml NOV protein for 4d (right ).

Doghman et al.?NOV/CCN3and Childhood Adrenocortical Tumors J Clin Endocrinol Metab,August 2007,92(8):3253–32603255

at a nitrogen to phosphate ratio of 5,following the manufacturer’s (Polyplus Transfection,Illkirch,France)instructions.Luciferase assays were realized using a Luminoskan Ascent (Thermo Labsystems,San Jose,CA)luminometer.Results were normalized by ?-galactosidase activities.

Recombinant NOV proteins

The recombinant NOV proteins were produced and purified as de-scribed (19).

Flow cytometry

H295R cells were fixed in 70%ethanol and then treated with RNase A (1?g/ml)for 30min at 37C.DNA was stained with propidium iodide (50?g/ml),and cells were analyzed for cell-cycle distribution with a FACScan instrument (Becton Dickinson).Results are expressed as pro-liferating index (sum of percentages of cells in S ?G 2/M phases).

Apoptosis and caspase activity assays

Apoptosis was assayed using the TdT-based in situ cell death detec-tion kit (Roche,Indianapolis,IN).For caspase activity assays,H295R cells were lysed for 30min at 4C in lysis buffer [50m m HEPES (pH 8),150m m NaCl,20m m EDTA,1m m phenylmethylsulfonyl fluoride,10?g/ml leupeptin,10?g/ml aprotinin,and 0.2%Triton X-100],and lysates were cleared at 10,000?g for 15min at 4C.Each assay (in triplicate)was performed using 20?g of protein lysate prepared from control or NOV-stimulated cells and complemented with 5m m dithio-threitol.Briefly,cellular extracts were incubated in a 96-well plate with 0.2m m of Ac-DEVD-AMC (caspase-3),Ac-LEHD-AMC (caspase-9),or

Ac-IETD-AMC (caspase-8)as substrates at 37C.Caspase activity was measured after absorbance at 460nm (excitation at 390nm)in the absence or presence of 1?m of Ac-DEVD-CHO,Ac-LEHD-CHO,and Ac-IETD-CHO,for caspase-3,-9,and -8,respectively.To assay for active caspase-3levels in Jurkat T cells,cells were treated for 8or 24h in the presence of either NOV (2.5?g/ml)or CH11anti-Fas antibody (50ng/ml),as a control of induction of apoptosis.Cells were then washed with PBS,fixed,and permeabilized.Subsequently cells were incubated for 30min with antiactive-caspase-3-fluorescein isothiocyanate (FITC)monoclonal antibody (BD Biosciences,Franklin Lakes,NJ).After wash-ing,samples were immediately analyzed with a FACScan (Becton Dickinson).

Results

NOV protein is down-regulated in childhood ACT

By microarray analysis we have shown that NOV is one of the transcripts that is more significantly down-regulated in a series of 24childhood ACTs,compared with normal ad-renal cortex (11).NOV mRNA levels measured by quanti-tative RT-PCR are significantly lower in childhood ACTs than normal adrenal cortex (Fig.1A).To verify whether NOV protein levels correlate with mRNA levels,we compared NOV protein expression in a series of eight tumors (four adenomas and four carcinomas)and eight samples of age-matched normal adrenal cortex.NOV protein levels are in-variably reduced in all tumor samples,compared with nor-mal tissue,for both adenomas and carcinomas (Fig.

1B).

F I

G .4.NOV is a selective proapoptotic factor for hu-man tumor adrenocortical cells.A,Structure of the NOV protein.The different protein domains are shown with different colors .SP,Signal peptide;VWC,von Willebrand factor type C domain;TSP1,thrombospondin type I repeat;CT,cysteine-knot do-main.A variable amino acid stretch connects do-mains von Willebrand factor type C domain and thrombospondin type I repeat.The extensions of the N-terminal (N-ter)and C-terminal (C-ter)domains expressed in recombinant form are indicated.B,H295R cells were treated with buffer (Ctrl)or NOV full-length (FL),N-(N-ter)and C-terminal (C-ter)fragments (2.5?g/ml each)for 24h,and the percent-age of apoptotic cells was measured by TUNEL stain-ing.Apoptosis was significantly increased in cells treated with full-length NOV and NOV C-terminal fragment,as compared with buffer-treated cells (P ?0.0026and P ?0.0398,Fisher’s exact test).On the right side,fluorescence microscopy view of DAPI staining (top panels )and TUNEL staining (bottom panels )of H295R cells treated with buffer (left pan-els )or full-length NOV (2.5?g/ml;right panels ).Size bar ,50?m.**,P ?0.05.C,H295R,HeLa,and COS cells were treated for 24h with buffer (white histo-grams )or 2.5?g/ml NOV (black histograms ),and percentage of apoptotic cells was measured by TUNEL staining.Data shown represent the average of two experiments performed in duplicate.

3256J Clin Endocrinol Metab,August 2007,92(8):3253–3260Doghman et al.?NOV/CCN3and Childhood Adrenocortical Tumors

?-Tubulin staining shows similar protein loading in each sample(Fig.1B).

Increase of SF-1dosage in H295R cells represses NOV expression at the transcriptional level

H295R cells are the only differentiated human adrenocor-tical cell line with a fetal adrenal phenotype currently avail-able(21).They express SF-1endogenously(16).We estab-lished cellular clones derived from H295R cells that overexpress SF-1in a doxycycline-inducible fashion(H295R TR/SF-1)to produce a cellular model for childhood ACT (Doghman,M.,T.Karpova,G.Rodrigues,M.Arhatte,J.De Moura,L.R.Cavalli,V.Virolle,P.Barby,G.P.Zambetti,B.

C.Figueiredo,L.L.Heckert,https://www.wendangku.net/doc/379637802.html,lli,submitted for publica-tion).Gene expression profiling of H295R TR/SF-1cells re-vealed that NOV is one of the transcripts that is more pow-erfully down-regulated when SF-1dosage is increased. Microarray results were confirmed by quantitative RT-PCR in two different H295R TR/SF-1clones(Doghman,M.,T. Karpova,G.Rodrigues,M.Arhatte,J.De Moura,L.R.Cav-alli,V.Virolle,P.Barby,G.P.Zambetti,B.C.Figueiredo,L. L.Heckert,https://www.wendangku.net/doc/379637802.html,lli,submitted for publication).NOV protein levels are significantly reduced in the supernatant of H295R TR/SF-1cells treated with doxycycline for either48or72h to induce SF-1overexpression(Fig.2A).The IGFBP2-se-creted protein harbors an IGF-binding domain similar to the one present in the N-terminal half of NOV and is readily detectable in the tissue culture supernatant of H295R cells (22).Remarkably,doxycycline treatment of H295R TR/SF-1 cells does not appreciably modulate IGFBP2levels in their culture supernatant(Fig.2A),showing the specificity of the effect of increased SF-1levels to negatively regulate NOV expression in H295R human adrenocortical cells. Increased SF-1dosage regulates NOV expression at the transcriptional level because doxycycline treatment reduced activity of NOV promoter in H295R TR/SF-1cells,whereas it had no effect on NOV promoter activity in the parental H295R TR clone(Fig.2B).Deletion analysis showed that the activity of the shortest NOV promoter fragment analyzed (?492)is still repressed by elevated SF-1levels in H295R TR/SF-1cells.In the minimal NOV promoter sequences sen-sitive to inhibition by increased SF-1levels,no putative SF-1 response element can be found by MatInspector(Genomatix, Munich,Germany)analysis.Furthermore,NOV promoter reporters are not sensitive to inhibition by SF-1after transient transfection in COS cells(data not shown).This data suggest that the specific effect of increased SF-1levels on NOV pro-moter activity in adrenocortical cells is mediated by other transcription factors whose levels,or activities,are regulated by SF-1dosage.

NOV induces apoptosis of human tumor adrenocortical cells through caspase activation

To study the biological effects of NOV on human adre-nocortical cells,we treated H295R cells with increasing doses of recombinant full-length NOV protein and monitored their growth over a4-d period.NOV decreases cell number in a dose-dependent manner(Fig.3A).This effect of NOV cannot be ascribed to block of cell-cycle progression because the proliferating index of H295R cells treated with the highest dose of NOV(0.5?g/ml)was not decreased,compared with untreated cells(36.7vs.34.8,respectively;Fig.3B).On the other hand,full-length NOV in nanomolar concentrations induces a significant increase in the percentage of apoptotic H295R cells,as detected by terminal deoxynucleotidyl trans-ferase-mediated deoxyuridine triphosphate nick end label-ing(TUNEL)staining,after a24-h treatment(Fig.4B).The C-terminal NOV fragment,encompassing the sequence

con-F IG.5.NOV induces apoptosis through caspase activation.A, Caspase inhibitors inhibit apoptosis induced by NOV.H295R cells were treated for24h with buffer(Ctrl)or recombinant full-length NOV protein(2.5?g/ml),alone or in the presence of caspase inhibitors Z-VAD(pan-caspase),Z-DEVD(caspase-3),Z-IETD(caspase-8),and Z-LEHD(caspase-9)(50?M each).Percentage of apoptotic cells was measured by TUNEL staining.Data shown represent the average of two experiments performed in duplicate.B,Assay of caspase-3(left), caspase-8(center),and caspase-9activities(right)in H295R cells treated with buffer(white histograms)or2.5?g/ml full-length NOV protein(black histograms)for24h.C,Jurkat T cells were treated for 8or24h with recombinant NOV protein or CH11anti-Fas antibody. Cells were stained with antiactive caspase-3FITC antibody,washed, and the percentage of FITC-positive cells measured by flow cytom-etry.

Doghman et al.?NOV/CCN3and Childhood Adrenocortical Tumors J Clin Endocrinol Metab,August2007,92(8):3253–32603257

taining the thrombospondin-type I repeat and the CT (Fig.4A),induces apoptosis of H295R cells nearly as efficiently as the full-length protein,whereas the N-terminal fragment,encompassing the IGFBP and von Willebrand factor type C domain domains,is inactive (Fig.4B).Remarkably,NOV effect appears to be relatively selective for adrenocortical cells because it has no proapoptotic activity on other cell lines tested (Hela,COS;Fig.4C).NOV-induced apoptosis is sen-sitive to the pan-caspase inhibitor Z-VAD and is also reduced by treatment with the selective caspase-3(Z-DEVD),caspase-8(Z-IETD),and caspase-9(Z-LEHD)inhibitors (Fig.5A).Consistent with these results,NOV induces a significant increase of caspase-3,caspase-8,and caspase-9activities in H295R cells (Fig.5B).Remarkably,NOV was unable to ac-tivate caspase-3in Jurkat T leukemic cells (Fig.5C),whereas this caspase was activated by anti-Fas treatment,as previ-ously described (23).Furthermore,NOV effect on apoptosis of primary tumor adrenocortical cells was measured.Adre-nocortical tumor cells in culture keep their differentiated phenotype,with the majority of cells expressing transcrip-tion factors SF-1and DAX-1(Fig.6A)and steroidogenic enzymes P450scc and P450C17(not shown).Similarly to the H295R cell line,apoptosis of primary adrenocortical tumor cells could be significantly elicited by recombinant NOV protein in SF-1-positive cells (Fig.6B and Table 1).

NOV protein is expressed in human fetal adrenal cells

Previous studies have shown that NOV mRNA is detect-able in the human fetal adrenal,mostly in the definitive zone (13).Adrenocortical fetal adrenal cells in culture isolated from fetuses aged 11–14wk after conception express the DAX-1transcription factor (24)and the first enzyme in the steroidogenic cascade,P450scc (Fig.7A).NOV protein is detected in the cytoplasm of the same cells that express

DAX-1(Fig.7B).Remarkably,DAX-1-negative cells,which are not steroidogenic (Fig.7A),do not express NOV.

Discussion

Members of the CCN family of proteins are multimodular factors associated with the extracellular matrix endowed with diverse physiological functions.Their biological activ-ities are often dependent on the specific cellular context,and they may even have opposing effect in different cell types (reviewed in Ref.25).Here we have shown that NOV/CCN3has a potent proapoptotic effect on human adrenocortical cells.Remarkably,NOV proapoptotic activity appears to be relatively selective for adrenocortical cells because other cell types are not sensitive to NOV.This is the first time that direct evidence is presented showing a proapoptotic effect of NOV through caspase activation.However,NOV has been associated with the inhibition of proliferation of several other cancer cell types (26–28).Together with data shown here,those reports indicate that the proapoptotic effect of NOV may target a restricted spectrum of tissues.Other

members

F I

G .6.Primary adrenocortical tumor cells express transcription factors SF-1and DAX-1and are sensitive to NOV pro-apoptotic effect.A,Immunofluorescence shows nuclear SF-1staining (green )and predominantly nuclear DAX-1staining (red )in tumor cells.Nuclei were stained with DAPI dye.Size bar ,20?m.B,Cul-tured primary adrenocortical tumor cells were treated with either protein buffer (Ctrl)or recombinant full-length NOV protein (10?g/ml)for 18h.Apoptotic cells were detected by TUNEL staining (bot-tom panels ).Nuclei were stained with DAPI dye (top panels ).Size bar ,50?m.

TABLE 1.Recombinant NOV protein triggers apoptosis of primary adrenocortical tumor cells

SF-1-positive apoptotic cells

Nonapoptotic SF-1-positive cells

14468Control cells

46419NOV-treated cells

Primary tumor adrenocortical cells isolated from one childhood ACT were isolated by collagenase treatment and cultured in eight-well chamber slides.Equivalent volumes of protein buffer (control)or recombinant full-length NOV protein (10?g/ml)were added.Num-bers of apoptotic (TUNEL staining)SF-1-positive cells in the cultures were counted after an 18-h incubation.The difference in the number of apoptotic cells between control and NOV-treated samples is sig-nificant (P ?0.0001,Fisher’s exact test).

3258J Clin Endocrinol Metab,August 2007,92(8):3253–3260Doghman et al.?NOV/CCN3and Childhood Adrenocortical Tumors

of the CCN family may function as growth factors or induce apoptosis,depending on the cell type (29–31).The molecular bases of those differential effects are still unknown,but they might be accounted for by differential expression of NOV receptors and/or coupling to different signaling pathways in different cell types.

It is remarkable that in childhood ACTs,NOV expression is invariably reduced at the transcript and protein level (Fig.1).Reduced expression of an endogenous proapoptotic factor may play an important role in the process of childhood ACT tumorigenesis,accounting at least in part for the defect of apoptotic regression of the fetal adrenal that has been pro-posed to be responsible for tumor formation (6).Signifi-cantly,in childhood ACTs,NOV levels are down-regulated in both the adenoma and carcinoma groups (Ref.11and our data).Importantly,increase of SF-1dosage in human tumor adrenocortical cells is sufficient to repress NOV expression at the transcriptional level (Fig.2).SF-1overexpression in childhood ACTs is also a widespread finding,independent from clinical outcome (10).This is at variance with data reported in adult ACT,in which NOV expression is lower in carcinomas,compared with adenomas,and associated with a more severe prognosis (12).Our results suggest that in childhood ACTs,SF-1overexpression and NOV down-reg-ulation may be essential for tumor formation but not for

progression to a malignant phenotype.Conversely,loss of NOV expression in adult adrenocortical carcinomas may reflect their loss of the differentiated adrenal cortex pheno-type,similarly to what has been shown for other transcripts that are expressed at high levels in normal adrenal cortex and down-regulated in carcinomas (32,33).

In cultured human fetal adrenal cells,NOV is exclusively expressed by cells that are also positive for DAX-1staining.Previous studies have shown that in the human fetal adrenal NOV mRNA is mainly expressed in the outer definitive zone (13).This zone represents the adrenal cortex proliferative compartment,in which the precursors of steroidogenic cells are generated.Because of its proapoptotic effect on adreno-cortical cells,it is tempting to speculate that in the physio-logical setting,NOV may play an important role in the dy-namic interplay of diffusible factors that regulate (1,2,34,35)the proliferation and differentiation of fetal adrenal cells.

Acknowledgments

We thank Dr.Nicole Gallo-Payet for advice about fetal adrenal cell culture,Dr.Walter https://www.wendangku.net/doc/379637802.html,ler for gift of the anti-P450scc antibody,and the personnel of the Service d’Orthoge ′nie of the Archet Hospital for collaboration.

Received February 14,2007.Accepted May 31,2007.

Address all correspondence and requests for reprints to:Enzo Lalli,M.D.,Institut de Pharmacologie Mole ′culaire et Cellulaire,Centre Na-tional de la Recherche Scientifique Unite ′Mixte de Recherche 6097,660Route des Lucioles,Sophia Antipolis,06560Valbonne,France.E-mail:ninino@https://www.wendangku.net/doc/379637802.html,rs.fr.

This work was supported by a Centre National de la Recherche Scientifique ATIP grant,contrat d’interface Institut National de la Sante ′et de la Recherche Me ′dicale-Centre Hospitalier Universitaire de Nice,CAPES-COFECUB (419/03),Fondation Recherche Me ′dicale,Associa-tion Recherche sur le Cancer (3142),and Princess Grace Foundation (to E.L.);and National Institutes of Health Grant CA63230(to G.P.Z.).M.D.is a recipient of a Fondation Recherche Me ′dicale fellowship.Disclosure Information:All authors have nothing to declare.

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