help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thibout, H.
Right arrow Articles by Laurent, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thibout, H.
Right arrow Articles by Laurent, M.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 1 327-336
Copyright © 2003 by The Endocrine Society

Original Article

Characterization of Human NOV in Biological Fluids: An Enzyme Immunoassay for the Quantification of Human NOV in Sera from Patients with Diseases of the Adrenal Gland and of the Nervous System

H. Thibout, C. Martinerie, C. Créminon, F. Godeau, P. Boudou, Y. Le Bouc and M. Laurent

Institut National de la Santé et de la Recherche Médical, U-515, Prolifération, Différenciation et Processus Tumoraux, Bâtiment Kourilsky, Hôpital St. Antoine (H.T., C.M., F.G., Y.L.B., M.L.), 75012 Paris, France; Commissariat Energie Atomique (CEA), Service de Pharmacologie et d’Immunologie, CEA Saclay (C.C.), 91190 Gif-sur-Yvette, France; and Department of Hormonal Biology, St. Louis Hospital (P.B.), 75010 Paris, France

Address all correspondence and requests for reprints to: Dr. M. Laurent, INSERM, U-515, Prolifération, Différenciation et Processus Tumoraux, Bâtiment Kourilsky, Hôpital St. Antoine, 184 rue du Fbg St. Antoine, 75012 Paris, France. E-mail: laurent{at}st-antoine.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunochromatography has shown that human NOV (NOVH), a member of the CCN (CTGF/CYR61/NOV) family, forms a physiological complex with fibulin-1 in blood. We developed an enzyme immunoassay specific for NOVH and showed for the first time that the concentration of NOVH differs in each of these biological fluids. The normal concentration of NOVH circulating in the blood is 350–400 ng/ml, but this concentration varies with age. By using sera from patients with adrenal gland diseases we found that in vivo ACTH or glucocorticoids are not responsible for the high concentration of NOVH in this endocrine gland. However, the NOVH concentration was significantly modified in malignant adrenocortical tumors, but not in benign adrenocortical tumors. The concentration of NOVH was significantly decreased in patients suffering from astrocytomas or multiple sclerosis, two diseases of the nervous system. Thus, NOVH is a potentially useful marker for the diagnosis of these diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE nov GENE, encoding a cysteine-rich glycoprotein (NOV), was first identified in avian nephroblastoma as an integration site of the avian myeloblastosis-associated virus 1-N (1, 2). The nov gene, well conserved during evolution, has been cloned from chickens (novC) (1), humans (novH) (3), mice (novM) (4), and Xenopus laevis (5). NOV, Cyr61 (6), and connective tissue growth factor (CTGF; Ref.7) were the first members of the new CCN protein family (8), which also includes elm-1 (Wisp-1) (9, 10), R-Cop (Wisp-2) (9, 11, 12), and Wisp-3 (9). These proteins share the same multimodular structure and are constituted of an IGF-binding protein, a von Willebrand factor type C domain, a thrombospondin type I repeat domain, and a cystine knot domain. Members of the CCN family are involved in the control of cell proliferation, cell adhesion, apoptosis, and chemotaxis. Several in vivo studies have strongly suggested that they are involved in implantation, skeletal formation, and embryonic development. These proteins are also involved in various diseases as well as in fibrosis, wound healing, and tumors (13).

In normal tissues the NOVH protein can be detected at different levels in kidneys, muscles, cartilage, brain, lungs, ovaries, and heart (1, 14, 15). We recently showed that the adrenal cortex is a major endocrine site of novH expression in adults and during embryogenesis (16). The concentrations of novH in some tissues, such as lungs and heart, differ between species. In several tissues the expression of novH is also developmentally regulated. In chickens, novC mRNA is only detected in the heart during the embryonic stage, whereas it is found in the brain in both adults and embryos (1). In chick embryos, during wing and leg development, the expression of novC is maximal between d 6 and 8 of incubation, and the distribution of NOVC is consistent with it having a role in the formation of cartilage (our personal observations). In humans and rats, the nov gene is regulated during development of the central nervous system (17, 18, 19). The developmental regulation of nov suggests that this gene is implied in the embryonic development of several tissues, even though the function of NOV has not been clearly established.

In tumors derived from several sites of novH expression, the regulation of novH is altered. In renal carcinomas, human prostate cell lines, and prostatic tumors, the novH gene is overexpressed (20, 21, 22). In Wilms’ tumors, the expression of novH depends on the histology of the tumors (3); novH is underexpressed in undifferentiated tumors and up-regulated in differentiated tumors. In differentiated tumors, the NOVH protein is associated with tumoral blastemas and heterotypic differentiated tissues, such as cartilage and muscle, which suggests that novH is a marker of differentiated Wilms’ tumors. The recent analysis of benign and malignant adrenocortical tumors showed quantitative and qualitative alterations of NOVH (16). Although the localization of NOVH did not change in the tumors compared with that in normal tissue, the N-glycosylation of NOVH greatly differed in benign and malignant tumors in normal glands. The level of novH expression is higher in benign tumors and lower in malignant tumors. These abnormalities suggested that novH is involved in the development of adrenocortical tumors. In malignant adrenocortical tumors, the decreased expression of NOVH could play a role in cell invasiveness. Alternatively, in benign tumors a high amount of NOVH may be involved in the benign phenotype by increasing cell adhesion. NOVH is also produced at different levels in tumor cell lines derived from central nervous system. The inverse correlation between the amount of NOVH and the tumorigenicity of the cells (23) suggests that NOVH is also involved in brain tumors. NOVH, like all proteins of the CCN family, contains a signal peptide and is efficiently secreted as a recombinant protein produced by insect SF9 cells or as an endogenously protein produced by Madin Darby Canine Kidney-transfected cells (14) and by several human cancer cell lines: glioblastoma (G22) cells (our unpublished results), human breast cancer (HS 578T) cells, human prostate carcinoma (PC3) cells. and lymph node metastases of prostate cancer (LNCap) cells (15).

In this study we determined whether NOVH is released into biological fluids from expressing tissues. Given the sites of novH expression, we looked for NOVH in serum, urine, and cerebrospinal and amniotic fluids. We developed an enzyme immunoassay for NOVH and used it to evaluate the amounts of circulating NOVH-immunoreactive proteins in these fluids and to determine whether these concentrations are associated with development and with diseases affecting the tissues expressing novH. We first measured NOVH concentrations in serum from healthy adult blood donors and from umbilical cords. The high level of novH expressed in the adrenal glands may be the result of an in vivo up-regulation of novH by glucocorticoid or ACTH. To test this hypothesis, we measured the concentrations of NOVH-immunoreactive proteins in serum from patients treated with glucocorticoids for inflammatory diseases and from patients with Cushing’s syndrome. Cushing’s disease is a hormonal disorder caused by the overexpression of pituitary ACTH and prolonged exposure to high levels of cortisol.

To test whether NOVH could act as a marker for diseases affecting the adrenal gland and brain, two major sites of novH expression, serum samples from patients followed for adrenocortical tumors were tested, and circulating NOVH was measured in patients with astrocytoma and multiple sclerosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

The anti-NOVH K19M polyclonal antibody directed against the K19M peptide (339–357 amino acids) has been described previously (14). The K19M IgG was purified using standard methods on a K19M peptide affinity column. The antibody directed against the full-length NOVH provided by R. Rosenfeld has been previously described (15). The antifibulin-1 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Tebu, Le Perray en Yvelines, France). The mouse monoclonal antirabbit IgG was purchased from Spi Bio (Massy, France).

NOVH immunoaffinity column

Rabbit K19M IgG and normal rabbit IgG were purified by adding 0.5 M caprylic acid to 2 ml serum (24). The mixture was then incubated with cyanogen bromide-Sepharose (Amersham Pharmacia Biotech, Orsay, France) for 2 h at room temperature. Biological fluids were applied to the column, and the NOVH protein was eluted with 200 mM glycine buffer (pH 2.7), and immediately neutralized with 1 M Tris HCl (pH 8).

Biotinylation of NOVH and human fibulin-1 IgG

Rabbit K19M and human fibulin-1 IgG were purified with caprylic acid and biotinylated with 10 mg/ml biotinamidocaproate N-hydroxysuccinimide in 0.1 M carbonate buffer (pH 9) for 1 h at room temperature (25). The biotinylated products were dialyzed against 100 mM potassium phosphate (pH 7.4), 0.16 M NaCl, 0.1% BSA, and 0.001% azide. The biotinylated K19M and human fibulin-1 IgG were detected with either the streptavidin acetylcholinesterase (AchE) or the streptavidin peroxidase conjugates. AchE activity was measured by Ellman’s method by adding 200 µl Ellman’s medium (26).

Immunoprecipitation

The G59 glioblastoma-derived cell line (27), which expresses the fibulin-1C gene but not novH (28), was stably transfected with a plasmid expressing the sense NOVH cDNA under control of the cytomegalovirus promoter (14). Confluent transfected cells were cultured in DMEM in the absence of fetal calf serum (FCS) for 24 h. Protein A-Sepharose beads were added to the conditioned medium. After centrifugation the supernatant was incubated with the preformed protein A-K19M antibody complex. The negative controls consisted of incubating the supernatant with nonimmune serum, protein A-Sepharose beads alone, or K19M antibody in the presence of the K19M immunogenic peptide. After washing in 20 mM Tris (pH 8), 150 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, the immunoprecipitated proteins were eluted into Laemmli buffer (29) and separated by SDS-PAGE on 10% or 12% polyacrylamide gels. They were then analyzed by Western blotting using the K19M anti-NOVH and the polyclonal antihuman fibulin-1 antibodies.

Electrophoresis and Western blot analysis

Samples were separated by SDS-PAGE in 10% or 12% polyacrylamide gel and silver stained according to the conventional procedure. For Western blot analysis, the proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond P, Pharmacia-Amersham, Orsay, France). The membranes were washed in methanol for 30 sec and dried before being incubated for 1 h in 5% nonfat dried milk and 0.05% Tween 20 in PBS with the K19M polyclonal antibody diluted 1:500 or the antifibulin-1 antibody diluted 1:1000. After washing in 0.05% Tween 20 in PBS, the bound antibodies were detected with the antirabbit IgG horseradish peroxidase conjugate. Biotinylated IgG were detected with the streptavidin peroxidase conjugate. Peroxidase activity was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Trichloroacetic acid (TCA) precipitation

The proteins present in the conditioned medium were concentrated by the addition of 0.05% deoxycholate and 7% trichloroacetic acid. After centrifugation, the precipitated proteins were dried, neutralized with 1 N NaOH, and dissolved in Laemmli buffer.

Enzyme immunoassay (EIA) procedure

Microtiter plates (96-well; Maxisorp, Nunc, Roskilde, Denmark) were directly coated by incubating them in 200 µl 10 µg/ml mouse monoclonal antirabbit IgG diluted in 50 mM phosphate (pH 7.4) buffer at room temperature overnight and then saturating them with 1% BSA in PBS for 1 h at room temperature (26). After washing in 10 mM potassium phosphate (pH 7.4) and 0.05% Tween, serial dilutions were made of full-length and C-terminal recombinant NOVH proteins (2–250 ng/ml) and the K19M polyclonal antibody diluted 1:200,000, and the plates were incubated overnight at 4 C. The immunocomplexes were further incubated overnight at 4 C with the K19M immunogenic peptide covalently linked to AchE as described by Grassi et al. (26). The enzymatic activity was followed by Ellman’s reaction. After 1 h, absorbances were measured at 414 nm. The intraassay coefficients of variation were determined by repeating the same assay in duplicate six times using control serum from a healthy subject. Interassay coefficients of variation were determined in the same way by repeating these measurements on 3 different days.

Solid phase binding assay

The binding of NOVH and fibulin-1 in serum was assessed in solid phase. Microtiter plates (96 well; Maxisorp, Nunc) were coated at room temperature with 200 µl affinity-purified goat polyclonal antifibulin-1 antibodies (10 µg/ml; Tebu) or with the K19M antibody in 50 mM phosphate buffer (pH 7.4). The plates were then incubated overnight at 4 C with the proteins eluted from the NOVH immunoaffinity column diluted in EIA buffer. After washing, the NOVH protein bound to fibulin-1 was detected with the biotinylated K19M IgG, and the fibulin-1 bound to NOVH was detected with the biotinylated antifibulin-1 IgG. Biotinylated IgG were revealed by AchE linked to streptavidin. The specificity of the immunoreactivity was assessed by repeating the experiment using plates coated with nonimmune serum.

Purification of recombinant proteins

To generate a recombinant NOVH protein, a 1009-bp PCR fragment encoding the entire NOVH mature protein sequence (excluding its signal peptide) was generated using a plasmid encompassing the entire novH cDNA (3) as a template, 5'-ATTAATCGAAggccgtggggccAGCGCTGCCCTCCCCAG-3' comprising an SfiI restriction site (lower case letters) as a sense primer and 5'-GCCGctcgagTTACATTTTCCCTCTGGTAGTCTT-3' comprising an XhoI restriction site (lower case letters) as an antisense primer. After appropriate restriction cleavages, the corresponding DNA fragment was ligated into the pDB-3s-H6 transfer vector, which encodes the human placental alkaline phosphatase signal peptide, the first two Ile residues located at the N terminus, and the linker sequence, LVPRGSHHHHHHIEGRGGH, upstream from the SfiI cloning site. The construction of pDB-3s-H6 plasmid, derived from pMJ-Seap vector (30), will be described in detail elsewhere (Chabas, R., and F. Godeau, manuscript in preparation).

A PCR fragment encoding the C-terminal region of NOVH (28) was subcloned into the pDB-3s-H6 transfer vector after amplification using 5'-ATTAATCGAAggccgtgggggCAGGCTTACAGGCCAGAAGCC-3' and 5'-GCCGctcgagTTACATTTTCCCTCTGGTAGTCTT-3' oligonucleotides as sense and antisense primers, respectively. Sf9 cells were transfected with the plasmids, selected by restriction mapping and nucleotide sequencing, and baculoviral DNA, yielding the corresponding NOVH recombinant baculoviruses, which were then purified as previously described (31).

For protein production, medium containing 1% FCS that had been conditioned by Sf9 cells infected with NOVH recombinant baculoviruses for 72 h was clarified by centrifugation. The recombinant NOVH proteins were precipitated twice with (NH4)2SO4 at 75% saturation and then purified by nickel nitriloacetic acid (Ni-NTA)-agarose affinity chromatography as previously described (32).

Recombinant NOVH proteins were eluted in 125 mM imidazole (pH 7.5), pooled, and further purified by chromatography on Cibacron-Blue TSK columns (Tosohaas, Merck \|[amp ]\| Co., Inc., Eurolab, Fontenay sous Bois, France) previously equilibrated in 100 mM phosphate buffer (pH 7.2). The column was washed in the same buffer supplemented with 0.3 M NaCl, and the protein was eluted in the same buffer supplemented with 0.8 M NaCl. Fractions containing NOVH were desalted on a PD10 column (Amersham Pharmacia Biotech) equilibrated with 30 mM ammonium acetate. Eluted proteins were lyophilized, resuspended in PBS containing 10% glycerol, and kept at -80 C. The purity of the recombinant NOVH proteins was assessed by SDS-PAGE and silver staining.

Patients

The amounts of NOVH in serum from 7 umbilical cords and from 39 healthy volunteers were measured. The first group consisted of 10 men, aged 20 to 40 yr; the second group consisted of 18 women, aged 20–40 yr; and the third group consisted of 11 subjects, aged 60–79 yr. We also analyzed 5 patients, aged 39–57 yr, and 18 children, aged 3 months to 14 yr, treated with corticoids for inflammatory diseases. The children with inflammatory diseases were given 1 or 250 µg Synacthen (Novartis Pharma SA, Rueil-Malmaison, France), and the serum NOVH concentration was measured before and 20 or 60 min after Synacthen administration. Serum samples from patients with Cushing’s disease were also tested for NOVH. Serum samples from 10 patients, aged 39–65 yr, and 3 patients, aged 13–22 yr, were analyzed before any treatment, and serum samples from 7 treated patients (4 patients aged 39–65 yr, and 3 patients aged 13–22 yr) were analyzed. A group with adrenocortical tumors was also examined. This group consisted of patients with benign tumors (5 preoperative and 6 after surgical removal of the tumors) and malignant tumors (5 preoperative and 8 after surgical removal of the tumors). The tumors were defined according to histological criteria (Weiss score) and TNM classification. The amount of NOVH was also evaluated in cerebrospinal fluids and serum samples from 5 patients with astrocytoma tumors and 8 patients with multiple sclerosis.

Statistical analysis

Data were analyzed using StatView software (Abacus Concept Inc., Berkeley, CA) with the Mann-Whitney Utest. All P values are from two-sided tests, and only P value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NOVH is present in biological fluids

NOVH was isolated from 2-ml serum samples applied to a NOVH immunoaffinity column and to an IgG column. Western blotting, using the purified K19M IgG antibody, was used to detect NOVH in the unbound and bound states. In each fraction eluted from the NOVH affinity column, the purified K19M IgG detected two main proteins: a strong band at 52 kDa and a faint band at 32 kDa (Fig. 1Go, lane 4). The low molecular mass form of NOVH, previously observed in Wilms’ tumors and adrenocortical tumors (14, 16), is probably derived from the full-length NOVH protein by a natural proteolytic cleavage (28). No bands could be detected in the either fraction from the IgG column (data not shown). The 52- and 32-kDa NOVH proteins were also detected by a polyclonal antibody directed against the full-length recombinant NOVH protein (15) (Fig. 1Go, lane 5). The identity of the 52-kDa band was confirmed by use of a biotinylated-K19M antibody (Fig. 1Go, lane 3). The normal rabbit-IgG (Fig. 1Go, lane 6) weakly reacted with a protein that migrated to a similar position as the full-length NOVH. This protein, which also reacted with an antirabbit IgG horseradish peroxidase conjugate (data not shown), probably corresponded to K19M IgG released from the immunoaffinity column by the glycine buffer. The full-length NOVH isolated from blood did not migrate to the predicted 39-kDa molecular mass; thus, the NOVH present in serum undergoes additional posttranscriptional modifications, as is the case for the NOVH secreted by Madin Darby Canine Kidney-transfected cells (14) and from adrenocortical tumors (16).



View larger version (48K):
[in this window]
[in a new window]
  		Figure 1. Isolation of NOVH from serum by immunoadsorption on anti-NOVH Sepharose. Serum from control subject (2 ml) was precleared by incubation for 2 h at room temperature with protein G-Sepharose and applied to the anti-NOVH Sepharose column. Aliquots of unbound material (lane 2) as well as the proteins eluted from human anti-NOVH Sepharose (lanes 3–6) were concentrated with TCA and subjected to SDS-PAGE in 12% polyacrylamide gels. They were then transferred onto a PVDF membrane and probed with the biotinylated K19M IgG (lane 3), nonbiotinylated K19M IgG (lane 4), a polyclonal antibody directed against the full-length NOVH protein (lane 5), and the horseradish peroxidase conjugate (lane 6). Lane 1, Recombinant NOVH.

 
The 70-kDa band detected (Fig. 1Go, lane 2) in the unbound fraction corresponds to a nonspecific reaction of the human serum albumin with the K19M IgG in Western blot, as previously reported (14). The two NOVH isoforms (52 and 32 kDa) were also identified in amniotic fluid after immunoaffinity chromatography (Fig. 2Go, lane 3), whereas no NOVH was detected in 2 ml urine analyzed in same way (data not shown). NOVH proteins were also identified in cerebrospinal fluid. We used immunoaffinity and heparin-Sepharose chromatography to examine the profiles of NOVH in this fluid (Fig. 3AGo, lanes 2 and 3). With both methods, the K19M antibody mainly reacted with the 52-kDa full-length NOVH and slightly reacted with a 70-kDa protein. The 52-kDa NOVH protein did not react with the K19M antibody preabsorbed with the K19M immunogen peptide in contrast to the nonspecific 70-kDa protein (Fig. 3AGo, lane 4). In this cerebrospinal fluid sample, the C-terminal NOVH isoform was not characterized.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 2. Isolation of NOVH from amniotic fluid by immunoadsorption on anti-NOVH Sepharose. Amniotic fluid (2 ml) was incubated with anti-NOVH Sepharose. Aliquots of unbound material (lane 1) and the proteins eluted from human anti-NOVH Sepharose (lane 3) were concentrated by TCA and subjected to SDS-PAGE in 12% polyacrylamide gels. They were transferred onto a PVDF membrane and probed with the purified K19M antibody at a 1:500 dilution. Lane 2, Recombinant NOVH.

 


View larger version (88K):
[in this window]
[in a new window]
  		Figure 3. Analysis of NOVH in cerebrospinal fluids. Aliquot of cerebrospinal fluid was applied to the anti-NOVH Sepharose column or incubated overnight at 4 C with heparin-Sepharose beads. After washing, NOVH was eluted from the human anti-NOVH Sepharose (lane 2) and from the heparin-Sepharose (lane 3) and analyzed by Western blotting with the purified K19M IgG (lanes 2 and 3) or with the K19M antibody preabsorbed with the K19M immunogen peptide (lane 4). Lane 1, recombinant NOVH.

 
Fibulin-1 copurifies with NOVH isolated from serum

The two-hybrid system and glutathione-S-transferase pull-down techniques have shown that NOVH interacts with fibulin-1C (28). The G59 glioblastoma cell line, which expresses fibulin-1C but not the novH gene, was transfected with a NOVH recombinant plasmid, and the conditioned medium was tested for NOVH and fibulin-1 by Western blotting. Fibulin-1 appeared as a 100-kDa band, and NOVH as a 52-kDa band (Fig. 4Go, lane 1). The conditioned medium was then incubated with the K19M antibody. The immunoprecipitated proteins were separated by electrophoresis and analyzed for the presence of NOVH and fibulin-1 by Western blotting. A 100-kDa human fibulin-1 protein was detected by the antifibulin-1 antibody, and a 52-kDa NOVH isoform was detected by K19M antibody (lane 4). One unspecific band was identified by NOVH antibody, but not fibulin-1 antibody, when the proteins were immunoprecipitated with K19M antibody in the presence of the K19M immunogenic peptide, with a nonimmune serum, or with protein A-Sepharose beads alone (lanes 2, 3, and 5). Thus, NOVH and fibulin-1 can also interact in ex vivo systems.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 4. Coimmunoprecipitation of NOVH and fibulin-1 from medium conditioned by G59-transfected glioblastoma cells. After electrophoresis of proteins from culture medium of G59-transfected glioblastoma cells, NOVH and fibulin-1 were detected with the K19M and the fibulin-1 (lane 1) antibodies. Proteins were immunoprecipitated from conditioned medium with the K19M antibody and probed with the biotinylated K19M IgG or the fibulin-1 antibodies (lane 4). For the negative controls, proteins were immunoprecipitated with nonimmune serum (lane 2), the K19M antibody and the K19M peptide (lane 3), and protein A-Sepharose alone (lane 5).

 
Fibulin-1 is a blood glycoprotein present at a concentration of 30–50 µg/ml. Therefore, we used serum to determine whether NOVH can physiologically interact with fibulin-1. As fibulin-1 and NOVH could not be coimmunoprecipitated from blood, the fractions eluted from the NOVH affinity column were tested for fibulin-1 by Western blotting. A 100-kDa polypeptide was detected by fibulin-1 antibody in the fractions containing NOVH (Fig. 5Go, lanes 1–4). Fibulin-1 was also detected by a specific fibulin-1 polyclonal antibody provided by R. Timpl (data not shown), but not when the immunodetection was performed with a nonimmune serum. These results indicate that in serum, fibulin-1 copurifies with NOVH.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Characterization of NOVH/fibulin-1 interaction in serum. A, The proteins from the fractions eluted from human anti-NOVH Sepharose (lanes 1–4) were concentrated with TCA and probed with the K19M and human fibulin-1 polyclonal antibodies. B, Plates coated with either the K19M or fibulin-1 antibodies were incubated overnight at 4 C with the proteins eluted from the human anti-NOVH Sepharose column. After washing, the fibulin-1-coated plates were further incubated with the biotinylated K19M antibody, and the K19M-coated wells were incubated with the biotinylated fibulin-1 antibody. The bound antibodies were revealed with the AchE-streptavidin conjugate. The negative control consisted of using plates coated with nonimmune serum.

 
The NOVH/fibulin-1 interaction was then tested by a solid phase assay. The proteins eluted from the NOVH affinity column and the unbound proteins were incubated overnight at 4 C in microtiter plates coated with human fibulin-1, K19M antibodies, or nonimmune serum. After washing, the plates coated with the antifibulin-1 were incubated with the biotinylated K19M antibody to detect bound NOVH. The plates coated with K19M were incubating with the biotinylated antifibulin-1 to detect bound fibulin-1. A high level of AchE activity was measured when the wells were coated with fibulin-1 or NOVH antibodies and incubated with the proteins eluted from the NOVH affinity column, but not with the unbound material. No AchE activity could be measured in wells coated with nonimmune serum. These data strongly suggest that in blood, fibulin-1 physiologically interacts with NOVH.

Sensitivity and specificity of the NOVH enzyme immunoassay

To measure the amounts of NOVH protein in biological fluids, we developed a competitive EIA using the K19M polyclonal antibody and recombinant NOVH (the full-length and the C-terminal regions). The amino-terminal ends of these recombinant proteins were tagged with a six-histidine tag, purified by chromatography on an Ni-NTA column followed by a Cibacron Blue column, and silver stained. Silver staining revealed two major bands at 27 and at 44 kDa (Fig. 6Go, lanes 1–3). Western blotting showed that these two proteins reacted with the K19M antibody (lanes 2–4) and corresponded to the C-terminal fragment and the full-length recombinant NOVH proteins, respectively. Protein concentrations were determined, and the purified recombinant proteins were used to establish the standard curves for the NOVH EIA. At 50% inhibition, the C-terminal and full-length NOVHs were detected at concentrations of 1.2 and 0.5 pmol/ml, respectively (Fig. 6BGo). When the C-terminal fragment and the full-length NOVH proteins were combined in a 3:1 or a 1:3 ratio, 0.89 and 0.56 pmol NOVH-immunoreactive proteins were detected, respectively. This indicates that the K19M antibody has different affinities for the two NOVH isoforms. To measure the total amount of NOVH proteins in serum, NOVH profiles were examined in several serum samples from adults, healthy elderly volunteers, umbilical cords, and patients with Cushing’s syndrome, inflammatory diseases, and adrenocortical tumors. Western blot analysis of proteins bound to NOVH affinity beads revealed that the NOVH profiles were similar in all samples. As in the control serum, the full-length NOVH was the main component, and only small amounts of the C-terminal 32-kDa isoform were present (data not shown). Therefore, this EIA assay was used as a preliminary approach to determine whether the total amount of immunoreactive NOVH proteins in serum varied in these populations.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 6. Characteristics of the NOVH enzyme immunoassay and analysis of the recombinant NOVH proteins produced in the Baculovirus system. A, The recombinant proteins, the 27-kDa C-terminal isoform and the whole 44-kDa NOVH, were extracted from medium conditioned by Sf9 insect cells and purified by chromatography on an Ni-NTA column, followed by a Cibacron Blue column. After electrophoresis, the 27- and 44-kDa NOVH proteins were silver-stained (lanes 1 and 3) and characterized by Western immunoblotting (lanes 2 and 4). B, Standard curves were established for the C-terminal region of NOVH (a), a 3:1 or a 1:3 ratio of the C-terminal region of NOVH and the full-length NOVH (b and c), and the full-length NOVH (d). Results are means from a representative experiment performed in triplicate.

 
The intraassay coefficient of variation was determined on six repetitions using serum from a healthy control. For the interassay coefficient of variation, the assay was repeated three times. The intra- and the interassay coefficients of variation were, respectively, 4% and 11%. To test whether the EIA could be used to detect NOVH in serum samples, we measured the interference of serum proteins in the presence or absence of FCS; 25 ng/ml NOVH was detected at 50% inhibition (data not shown).

This assay showed that the control human adult serum sample contained 350 ng/ml NOVH-immunoreactive protein. To test the specificity of the assay, the control serum was applied to the NOVH immunoaffinity column. No AchE activity was detected in the unbound material, confirming the absence of cross-reactivity between the assay and serum proteins. As expected, the small amount of K19M IgG eluted from the NOVH affinity column (Fig. 1Go, lane 6) interfered with the EIA and prevented determination of the amount of NOVH-immunoreactive proteins bound to this column.

To test whether in the assay all of the recombinant protein and NOVH in the serum is captured, the supernatant of an overnight capture of recombinant NOVH and NOVH from serum was transferred to other similar wells, and no NOVH was detected.

In newly formed thrombi, fibulin-1 is one component of fibrin clots (33). To test whether the NOVH bound to fibulin-1 can be incorporated into fibrin clots, the amount of NOVH was evaluated in serum and plasma samples. The amount of NOVH was identical in serum and plasma. This suggests that NOVH is not incorporated into the fibrin clots.

The EIA was then used to analyze amniotic fluid, urine, and cerebrospinal fluids. The amounts of NOVH in amniotic and cerebrospinal fluids were less than that in serum. The amount of NOVH was 70 ± 0.06 ng/ml in amniotic fluid and 45 ± 2.8 ng/ml in cerebrospinal fluid. However, the presence of different NOVH-immunoreactive proteins in several samples of cerebrospinal fluid (data not shown) may affect the value of NOVH measured in that fluid. No NOVH could be detected in urine.

Serum NOVH concentrations in healthy subjects and in patients with various diseases of the adrenal gland and brain

The amount of immunoreactive NOVH proteins in serum from 39 healthy volunteers was determined. We examined a group of 10 healthy male adults and a group of 18 healthy female adults. The concentrations were 410 ± 86 ng/ml in the men and 391 ± 77 ng/ml in the women. The data were normally distributed; the highest concentration was 551 ng/ml in the males and 620 ng/ml in the females, and the lowest values were 314 and 299 ng/ml, respectively. The Mann-Whitney Utest did not find any significant difference between the two populations. The two populations were therefore pooled and are referred to as a control population in the subsequent analysis. The immunoreactive NOVH concentrations were then evaluated in serum from umbilical cords, from healthy elderly subjects (67–79 yr old), and from patients with various diseases (Fig. 7Go). The serum concentrations were similar in elderly and control subjects (P = 0.14; Fig. 7AGo). In contrast, there was 30% less NOVH-immunoreactive proteins in serum from umbilical cords than in control adult serum (P = 0.0001).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Analysis of NOVH in serum from healthy subjects and patients. Quantification of NOVH in serum from healthy volunteers and umbilical cords (A); patients with Cushing’s disease, adults and children treated with glucocorticoid for inflammatory diseases, and children evaluated by the ACTH test (Synacthen, B); patients with benign and malignant adrenocortical tumors (C); and patients with astrocytomas or multiple sclerosis (D). All values were standardized with respect to the NOVH concentration of a control serum sample included in each assay. The values from each population were compared with the Mann-Whitney Utest. The NOVH concentrations in each population were evaluated in duplicate, and each assay was repeated twice. The values, expressed as nanograms per milliliter, were standardized with respect to a control serum sample, which was used as an internal standard in each assay.

 
To determine whether the high amount of NOVH observed in the adrenal gland (16) was due to the regulation of novH by cortisol or ACTH, we analyzed serum from patients with Cushing’s syndrome (Fig. 7BGo). The amounts of NOVH-immunoreactive proteins were similar in the treated and nontreated patients, aged 41–64 yr, and in the control patients (P = 0.63). However, the serum from the three patients aged 13–22 yr contained significantly less NOVH-immunoreactive proteins than control adult serum (P = 0.04).

In the population of patients with inflammatory diseases and receiving glucocorticoid treatment, serum samples from children (aged 3 months to 15 yr) contained less NOVH-immunoreactive protein (P = 0.0003) than control adult serum. In those patients the serum NOVH concentration was not affected by the increase in cortisol, evaluated 20 or 60 min after the injection of 1 or 250 µg Synacthen. In the adults, serum samples contained similar amounts of NOVH-immunoreactive proteins as those from control patients (P = 0.8; Fig. 7BGo). The serum from patients with benign tumors (Fig. 7CGo) contained similar amounts of NOVH-immunoreactive proteins as that from healthy volunteers of the same mean age. Similarly, after surgical removal of these benign tumors, the serum NOVH concentrations of this population did not differ from those of the control population. In contrast, serum samples from patients with malignant adrenocortical tumors contained significantly more NOVH (P = 0.05) than control serum samples, whereas after surgery the levels of NOVH were not statistically different in the two groups (P = 0.38). Serum samples from patients with astrocytomas or multiple sclerosis contained significantly (P = 0.012 and P = 0.005) less NOVH-immunoreactive proteins than the control population (Fig. 7DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We showed that NOVH is present in the biological fluids amniotic liquid, cerebrospinal fluid, and human serum; in this latter fluid, NOVH forms a physiological complex with fibulin-1. We also designed the first immunoassay specific for NOVH. This assay showed that the concentration of NOVH-immunoreactive proteins differs in the different biological fluids, and in serum the values vary in normal and pathological situations. Immunopurification of the proteins from 60 µl serum on a NOVH affinity column revealed two protein species. The intact 52-kDa protein was the main form circulating in serum, whereas only small amounts of the C-terminal 32-kDa isoform were present. In only 0.2 µl human serum, Burren et al. (15) detected several NOVH isoforms with slightly different molecular masses, and the ratios of the isoforms were different from those observed in our analysis. NOVH is not the only member of the CCN family present in biological fluids. CTGF has also been found in follicular, peritoneal, cerebrospinal, and amniotic fluids and in human serum and urine (34). The relevance of the presence of two members of the CCN family in a variety of human biological fluids is not known, and it is not known whether the two interact with each other in blood. To further investigate the significance of the presence of NOVH in serum, we showed that NOVH binds to fibulin-1 in human serum. The fibulins are a family of extracellular matrix and blood proteins. Fibulin-1 is the predominant fibulin in blood (35, 36, 37). Fibulin-1 was found to copurify with NOVH and a NOVH/fibulin-1 complex adsorbed onto microtiter wells coated with NOVH or human fibulin-1 antibodies. Thus, NOVH and fibulin-1 interact in a physiological manner. Fibulin-1 is present at a concentration of 30–40 µg/ml, whereas NOVH is present at a concentration of 350–400 ng/ml. The amount of NOVH in normal human serum is therefore much higher than that of CTGF (38). If fibulin-1 and NOVH form a 1:1 molar complex, all of the NOVH circulating in blood would form a complex with fibulin-1. The biological functions of fibulin-1 have been investigated by disrupting the fibulin-1 gene in mice. This resulted in the death of nearly all of the homozygous embryos at birth (39). Deficient mice presented abnormalities in small vessels, malformations of glomeruli, and disorganization of podocytes. The nov gene, first identified in avian nephroblastoma (1), is also expressed in normal human kidneys (14). The corresponding mRNA and protein are colocalized in kidney structures, such as immature tubules, podocytes of glomeruli, and collecting ducts. Therefore, NOVH and fibulin-1 may interact in these kidney structures, allowing NOVH to participate in glomeruli formation and the differentiation of podocytes. This hypothesis is presently being investigated.

The first assay was designed to quantify immunoreactive NOVH proteins. This assay is based on the K19M NOVH antibody directed against the 19 amino acids at the C-terminal end of NOVH. The full-length and the C-terminal fragment of NOVH are recognized differently, and thus each NOVH isoform cannot be accurately quantified in the assay. However, in serum the full-length NOVH is the main NOVH component, and no variations in the ratios between the full-length and C-terminal NOVH proteins were observed in several samples from patients with different diseases. Therefore, this assay was used as a first step to evaluate the amount of immunoreactive NOVH proteins in serum and to investigate whether variations in the total amount of immunoreactive proteins were associated with diseases of the adrenal gland and the central nervous system, two major sites of novH expression.

The concentration of NOVH is high in glomeruli (14), but NOVH could not be detected in urine either after immunochromatography on a NOVH-Sepharose column or with our EIA. This suggests that NOVH is not filtrated, reabsorbed, or degraded in the tubules so that no or very low concentrations of NOVH are present in the urine of healthy subjects. However, in some pathological situations NOVH could be released in urine. In Wilms’ tumors, the expression of novH is deregulated, and the concentration of NOVH in these tumors depends on the histology of the tumors (3). Therefore, urine samples from patients with Wilms’ tumor or with nephropathies may contain NOVH, as is the case for CTGF, the expression of which is up-regulated in in vivo and in vitro models of diabetic nephropathy (40, 41).

In normal tissues, novH is mostly expressed in the adrenal gland. This high novH expression may be the result of an in vivo regulation by glucocorticoids, as dexamethasone up-regulates expression of the nov gene in rat and human glioblastoma cell lines and in mouse astrocytes (42) (our unpublished data). Adults and children with Cushing’s disease had higher serum NOVH-immunoreactive protein concentrations before treatment than after treatment, but the values were in the physiological range. Adults and children receiving chronic corticotherapy for the treatment of various inflammatory diseases also had normal levels of NOVH. In addition, the increased serum levels of glucocorticoid measured 20 and 60 min after acute administration of Synacthen did not result in modification of the serum NOVH concentration. These data indicate that in vivo neither ACTH nor glucocorticoids have a major effect on novH gene expression or are responsible for the high concentration of NOVH detected in the adrenal gland. However, it is noteworthy that children with normal and high levels of cortisol had significantly lower NOVH concentrations than healthy adults, and blood from umbilical cords contained less NOVH than blood from healthy adults. Thus, age may affect the amount of NOVH in blood.

We previously reported quantitative alterations of NOVH in adrenocortical tumors, with the level of novH expression being high in benign tumors and low in malignant tumors (16). This study shows that patients with benign adrenal gland tumors and control patients had similar serum concentrations of immunoreactive NOVH proteins and that patients with malignant tumors had values in the normal range, but slightly higher than controls. For some patients the NOVH content has been analyzed in both tumors (16) and serum (this study). The data showed that two patients with malignant tumors had normal levels of circulating NOVH and no detectable NOVH in the tumors and that two patients with benign tumors had high intratumoral concentrations of NOVH and the highest serum values. This indicates that only some of the circulating NOVH originated from the tumor and that several additional factors affect the levels of circulating NOVH. The novH gene is also expressed in several tissues, in particular cartilage and muscles, which may contribute to the circulating NOVH.

The novH gene is also highly expressed in the brain and nervous system, where it is switched on during embryonic development. An inverse correlation has been found between the expression of novH and the tumorigenicity of glioblastoma cell lines (23). This suggests that NOVH is involved in the formation and development of brain tumors. We found that patients with astrocytomas or multiple sclerosis have significantly lower blood concentrations of NOVH than controls. It is not clear whether these differences have specific biological activities or if they are involved in the progression of the disease. Further studies are necessary to determine whether circulating NOVH can be used as a parameter for the diagnosis of multiple sclerosis. Our data indicate that the amount of circulating immunoreactive NOVH protein varies with age and in some tumoral and nontumoral diseases. Further investigations are necessary to develop a specific assay for each NOVH isoform to enable us to evaluate their concentrations in these diseases.


   Acknowledgments
 
We thank Dr. J. Grassi for helpful discussion during the establishment of the EIA assay, P. Fettier (CEA, Saclay, Gif-sur-Yvette, France) for technical assistance, Prof. R. Liblau (INSERM, U-546, Hôpital Pitié-Salpétrière, Paris, France) for the study of the cerebrospinal fluids, Dr. Raux-Demay (Hôpital Trousseau, Paris, France) for study of patient serum, Prof. M. Westphal (Hospital Eppendorf, Hamburg, Germany) for providing the glioblastoma cell line, and Dr. Y. Blouquit (Institut Curie, Orsay, France) for help with protein purification.


   Footnotes
 
Abbreviations: AchE, Acetylcholinesterase; CTGF, connective tissue growth factor; EIA, enzyme immunoassay; FCS, fetal calf serum; Ni-NTA, nickel nitrilotriacetic acid; NOVH, human NOV; PVDF, polyvinylidene difluoride; TCA, trichloroacetic acid.

Received February 26, 2002.

Accepted October 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Joliot V, Martinerie C, Dambrine G, Plassiart G, Brisac M, Crochet J, Perbal B 1992 Proviral rearrangements and overexpression of a new cellular gene (nov) in myeloblastosis-associated virus type 1-induced nephroblastomas. Mol Cell Biol 12:10–21[Abstract/Free Full Text]
  2. Martinerie C, Perbal B 1991 Expression of a gene encoding a novel IGF binding protein in human tissues. C R Acad Sci [D] (Paris) 313:345–351
  3. Martinerie C, Huff V, Joubert I, Badzioch M, Saunders G, Strong L, Perbal B 1994 Structural analysis of the human nov proto-oncogene and expression in Wilms tumor. Oncogene 9:2729–2732[Medline]
  4. Snaith M, Natarajan D, Taylor L, Choi C, Martinerie C, Perbal B, Schofield P, Boutler C 1996 Genomic structure and chromosomal mapping of the mouse nov gene. Genomics 38:425–428[CrossRef][Medline]
  5. Ying Z, Ling ML 1996 Isolation and characterization of xnov, a Xenopus laevis ortholog of the chicken nov gene. Gene 171:243–248[CrossRef][Medline]
  6. Lau L, Nathans D 1985 Expression of a set of growth-regulated immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc Natl Acad Sci USA 84:1182–1186
  7. Bradham DM, Igarashi A, Potter RL, Grotendorst GR 1991 Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 114:1285–1294[Abstract/Free Full Text]
  8. Bork P 1993 The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 327:125–130[CrossRef][Medline]
  9. Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence DA, Lee J, Brush J, Taneyhill LA, Deuel B, Lew M, Watanabe C, Cohen RL, Melhem MF, Finley GG, Quirke P, Goddard AD, Hillan KJ, Gurney AL, Botstein D, Levine AJ 1998 WISP genes are members of the connective tissue growth factor family that are up-regulated in human colon tumors. Proc Natl Acad Sci USA 95:14717–14722[Abstract/Free Full Text]
  10. Hashimoto Y, Shindo-Okada N, Tani M, Nagamashi Y, Takeuchi K, Shiroishi T, Toma H, Yokota J 1998 Expression of the Elm-1 gene, a novel gene of the CCN (CTGF, Cyr61/Cef10 and nov) family, suppress in vivo growth and metastasis of K-1735 murine melanoma cells. J Exp Med 187:289–296[Abstract/Free Full Text]
  11. Kumar S, Hand AT, Connor JR, Dodds RA, Ryan PJ, Trill JJ, Fisher SM, Nuttall ME, Lipshutz DB, Zou C, Hwang SM, Votta BJ, James IE, Rieman DJ, Gowen M, Lee JC 1999 Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions. J Biol Chem 274:17123–17131[Abstract/Free Full Text]
  12. Zhang R, Averboukh L, Zhu W, Zhang H, Jo H, Dempsey PJ, Coffey RJ, Pardee AB, Liang P 1998 Identification of rCop-1, a new mamber of the CCN protein family, as a negative regulator for cell transformation. Mol Cell Biol 18:6131–6141[Abstract/Free Full Text]
  13. Brigstock DR 1999 The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 20:189–206[Abstract/Free Full Text]
  14. Chevalier G, Yeger H, Martinerie C, Laurent M, Alami J, Schofield PN, Perbal B 1998 nov H: Differential expression in developing kidney and in Wilms’ tumors. Am J Pathol 152:1563–1575[Abstract]
  15. Burren CP, Wilson EM, Hwa V, Oh Y, Rosenfeld RG 1999 Binding properties and distribution of insulin-like growth factor binding protein-related protein 3 (IGFBP-rP3/NovH), an additional member of the IGFBP superfamily. J Clin Endocrinol Metab 84:1096–1103[Abstract/Free Full Text]
  16. Martinerie C, Gicquel C, Louvel A, Laurent M, Schofield P, LeBouc Y 2001 Altered expression of NovH is asociated with human adrenocortical tumorigenesis. J Clin Endocrinal Metab 86:3929–3940[Abstract/Free Full Text]
  17. Su BY, Cai WQ, Zhang CG, Su HC, Perbal B 1998 A developmental study of novH gene expression in human central nervous system. C R Acad Sci III 321:883–892[Medline]
  18. Su B, Cai WQ, Zhang CG, Martinez V, Lombet A, Perbal B 2001 The expression of ccn3 (nov) RNA and protein in the rat central nervous system is developmentally regulated. Mol Pathol 54:184–191[Abstract/Free Full Text]
  19. Kocialkowski S, Yeger H, Kingdom J, Perbal B, Schofield PN2001 Expression of the human NOV gene in first trimester fetal tissues. Anat Embryol 203:417–427
  20. Lopez-Bermejo A, Buckway C, Devi G, Hwa V, Plymate S, Oh Y, Rosenfeld R 2000 Characterization of insulin-like growth factor-binding protein-related proteins (IGFBP-rPs) 1, 2, and 3 in human prostate epithelial cells: potential roles for IGFBP-rP1 and 2 in senescence of the prostatic epithelium. Endocrinology 141:4072–4080[Abstract/Free Full Text]
  21. Glukhova L, Angevin E, Lavialle C, Cadot B, Terrier-Lacombe MJ, Perbal B, Bernheim A, Goguel AF 2001 Patterns of specific genomic alterations associated with poor prognosis in high-grade renal cell carcinomas. Cancer Genet Cytogenet 15:105–110
  22. Maillard M, Cadot B, Ball RY, Sethia K, Edwards DR, Perbal B, Tatoud R 2001 Differential expression of the ccn3 (nov) proto-oncogene in human prostate cell lines and tissues. Mol Pathol 54:275–280[Abstract/Free Full Text]
  23. Wen-Xin L, Martinerie C, Zumkeller W, Westphal M, Perbal B 1996 Differential expression of novH and CTGF in human glioma cell lines. J Clin Pathol Mol Pathol 49:M91–M97
  24. Russ G 1983 Preparation and use of monoclonal antibodies. Bratisl Lek Listy 80:257–266[Medline]
  25. Bayer E, Wilchek M 1980 The use of the avidin-biotin complex as a tool in molecular biology. Methods Biochem Anal 26:1–45[Medline]
  26. Grassi J, Frobert Y, Pradelles P, Chercuitte F, Gruaz D, Dayer J, Poubelle P 1989 Production of monoclonal antibodies against interleukin-1{alpha} and -1ß. Development of two enzyme immunometric assays (EIA) using acetylcholinesterase and their application to biological media. J Immunol Methods 123:193–210[CrossRef][Medline]
  27. Westphal M, Hansel M, Brunken M, Konig A, Koppen JA, Herrmann HD 1987 Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells. Exp Cell Biol 55:152–163[Medline]
  28. Perbal B, Martinerie C, Sainson R, Werner M, He B, Roizman B 1999 The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: a clue for a role of NOVH in cell-adhesion signaling. Proc Natl Acad Sci USA 96:869–874[Abstract/Free Full Text]
  29. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 15:680–685
  30. Bara J, Chastre E, Mahiou J, Singh R, Forgue-Lafitte M, Hollande E, Godeau F 1998 Gastric M1 mucin, an early oncofetal marker of colon carcinogenesis, is encoded by the MUC5AC gene. Int J Cancer 75:767–773[CrossRef][Medline]
  31. Godeau F, Saucier C, Kourilsky P1992 Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses. Nucleic Acids Res 20:6239–6246
  32. Mahiou J, Abastado J, Cabanie L, Godeau F1998 Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system. Biochem J 330:1051–1058
  33. Tran H, Tanaka A, Litvinovich SV, Medved LV, Haudenschild CC, Argraves WS 1995 The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis. J Biol Chem 270:19458–19464[Abstract/Free Full Text]
  34. Yang DH, Kim HS, Wilson EM, Rosenfeld RG, Oh Y1998 Identification of glycosylated 38-kDa connective tissue growth factor (IGFBP-related protein 2) and proteolytic fragments in human biological fluids, and up-regulation of IGFBP-rP2 expression by TGF-ß in Hs578T human breast cancer cells. J Clin Endocrinol Metab 83:2593–2596
  35. Argraves WS, Tran H, Burgess WH, Dickerson K 1990 Fibulin is an extracellular matrix and plasma glycoprotein with repeated domain structure. J Cell Biol 111:3155–3164[Abstract/Free Full Text]
  36. Argraves WS, Dickerson K, Burgess WH, Ruoslahti E 1989 Fibulin, a novel protein that interacts with the fibronectin receptor ß subunit cytoplasmic domain. Cell 58:623–629[CrossRef][Medline]
  37. Pan TC, Kluge M, Zhang RZ, Mayer U, Timpl R, Chu ML 1993 Sequence of extracellular mouse protein BM-90/fibulin and its calcium-dependent binding to other basement-membrane ligands. Eur J Biochem 215:733–740[Medline]
  38. Tamatani T, Kobayashi H, Tezuka K, Sakamoto S, Suzuki K, Nakanishik T, Takigawa M, Miyano T 1998 Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia. Biochem Biophys Res Commun 29:748–752
  39. Kostka G, Giltay, R. Bloch, W. Addicks K, Timpl R, Fassler R, Chu ML 2001 Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice. Mol Cell Biol 21:7025–7034[Abstract/Free Full Text]
  40. Clarkson MR, Gupta SM, Martin FG, Brady HR 1999 Connective tissue growth factor: a potential stimulus for glomerulosclerosis and tubulointerstitial fibrosis in progressive renal disease [Review]. Curr Opin Nephrol Hypertens 8:543–548[CrossRef][Medline]
  41. Murphy M, Godson C, Cannon S, Kato S, Mackenzie HSM, F, Brady HR 1999 Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 26:5830–5834
  42. Liu C, Liu XJ, Crowe PD, Kelner GS, Fan J, Barry G, Manu F, Ling N, De Souza EB, Maki RA 1999 Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation. Gene 238:471–478[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
M. Doghman, M. Arhatte, H. Thibout, G. Rodrigues, J. De Moura, S. Grosso, A. N. West, M. Laurent, J.-C. Mas, A. Bongain, et al.
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
J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3253 - 3260.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
R. Gupta, D. Hong, F. Iborra, S. Sarno, and T. Enver
NOV (CCN3) Functions as a Regulator of Human Hematopoietic Stem or Progenitor Cells
Science, April 27, 2007; 316(5824): 590 - 593.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. T. Fu, J. F. Bechberger, M. A. Ozog, B. Perbal, and C. C. Naus
CCN3 (NOV) Interacts with Connexin43 in C6 Glioma Cells: POSSIBLE MECHANISM OF CONNEXIN-MEDIATED GROWTH SUPPRESSION
J. Biol. Chem., August 27, 2004; 279(35): 36943 - 36950.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thibout, H.
Right arrow Articles by Laurent, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thibout, H.
Right arrow Articles by Laurent, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals