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The Journal of Clinical Endocrinology & Metabolism 86(8):3929 –3940
Copyright © 2001 by The Endocrine Society
Altered Expression of novH Is Associated with Human
Adrenocortical Tumorigenesis
CÉCILE MARTINERIE, CHRISTINE GICQUEL, ALBERT LOUVEL, MARYVONNE LAURENT,
PAUL N. SCHOFIELD, AND YVES LE BOUC
INSERM, U-515, Croissance, Differenciation et Processus Tumoraux, Hôpital Saint-Antoine (C.M., C.G., M.L., Y.L.B.),
75571 Paris, France; Service d’Anatomo-Pathologie, Hôpital Cochin (A.L.), Assistance Publique-Hôpitaux de Paris, 75014
Paris, France; and Department of Anatomy, University of Cambridge (P.N.S.), Cambridge, United Kingdom
NOVH belongs to the CCN (CTGF/CYR61/NOV) family of proteins, some of which have chemotactic, mitogenic, adhesive,
and angiogenic properties. Whereas ctgf and cyr61 are growth
factor-inducible, immediate-early genes, nov is expressed in
growth-arrested or quiescent cells. As nov expression has
been shown to be altered in both avian and human nephroblastomas and to be a target of WT1 regulation, NOV may play
important roles in normal nephrogenesis and the development of Wilms’ tumors.
The aim of this study was to determine whether changes in
novH expression were associated with tumorigenesis in tissues other than those of the kidney. We showed by Northern
blotting and immunohistochemistry that among human adult
endocrine tissues, the adrenal gland is a major site of novH
A
DRENOCORTICAL CARCINOMAS are rare tumors
with a poor prognosis (1, 2). Their pathogenesis is not
completely understood, but evidence is accumulating that
the insulin-like growth factor (IGF) system plays a major role
in adrenocortical tumorigenesis (2– 4). Alterations to the IGF
system have been demonstrated in malignant adrenocortical
tumors. The changes observed include imprinting mistakes
of the 11p15 region, overexpression of the IGF-II gene and of
its receptor IGF-I receptor and high levels of IGF-binding
protein-2 (IGFBP-2) (2, 3, 5, 6). In addition, IGF-II has been
shown to be involved in the auto/paracrine proliferation of
H295R cells, an in vitro model for adrenocortical carcinoma
(4). Imprinting mistakes of the 11p15 region are also responsible for the loss of expression of CK1 p57Kip2 and, consequently, for overactivity of G1/S phase cyclin-CDK complexes (7).
Dysregulation of imprinted growth regulatory genes
within the 11p15 region is also involved in the BeckwithWiedmann syndrome, an overgrowth syndrome predisposing patients to various tumors, including nephroblastoma
(Wilms’ tumor) and adrenocortical carcinoma (8). The avian
nephroblastoma induced by the myeloblastosis-associated
virus 1-N constitutes a unique animal model of Wilms’ tumor
(9). Molecular cloning of myeloblastosis-associated virus 1-N
integration sites in avian nephroblastoma resulted in identification of the nephroblastoma overexpressed (nov) gene
(10, 11). nov belongs to the recently discovered CCN [ctgf
Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase;
IGFBP-2, IGF-binding protein-2; poly(A)⫹, polyadenylated; VSMC, vascular smooth muscular cells.
expression, and that in adult and fetal adrenal tissue, novH is
primarily expressed in the adrenal cortex. Studies with 12
benign and 18 malignant adrenocortical tumors revealed that
the levels of novH mRNA and protein decreased significantly
(P < 0.004) with progression of adrenocortical tumors from a
benign to a malignant state. Although the localization of
NOVH did not change, the N-glycosylation profile of benign
and malignant tumors differed considerably from that of normal adrenocortical tissue, and these differences may affect
the biochemical properties of the molecule. The properties of
NOVH here provide the first evidence that this member of the
CCN family could be involved in adrenocortical tumor
development. (J Clin Endocrinol Metab 86: 3929 –3940, 2001)
(12), cyr 61, (13), and nov] family of genes (14), which also
includes elm1/wisp1 (15, 16), r-cop1/wisp2 (15, 17, 18), and
wisp3 (15). This family of genes has been previously included
in the IGFBP superfamily (19). nov has been cloned from
chicken, human, mouse, and Xenopus (10, 20 –22) and is well
conserved throughout evolution. The biological properties of
this family of genes include the regulation of cell proliferation, chemotaxis, angiogenic and adhesive activities, and
extracellular matrix formation. In vivo, the CCN family appears to be involved in both normal processes, such as implantation, placentation, embryogenesis differentiation, and
development, and in pathological situations, including
wound healing, fibrotic disorders, and tumors (for a review,
see Ref. 23).
nov expression is altered in both avian and human nephroblastomas (10, 20, 24). In Wilms’ tumors nov expression is
altered in the blastema and is associated with heterotypic
blastemal differentiation (24). In addition, we showed that
levels of nov and Wilms’ tumor suppressor gene (wt1)
mRNA were inversely correlated in several Wilms’ tumors
and that nov was down-regulated by WT1 proteins in ex vivo
assays and was therefore a potential target for wt1 regulation
(20, 25). High levels of IGF-II and IGF-I receptor have also
been described in these tumors (26 –28), and there is an inverse correlation between wt1 and IGF-I receptor (29). Moreover, WT1 proteins inhibit IGF-II transcription (30).
nov expression is not restricted to kidney and has been
detected in other tissues, such as brain, muscle, cartilage,
bone, and lung (10, 24, 31). It is also associated with the
development of the central nervous system in humans (32).
These observations raise the possibility that novH may be
3929
3930
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940
involved in diseases in organs other than the kidney. As
Wilms’ and adrenocortical tumors have some physiopathological and molecular alterations in common (33), we investigated novH expression in the adrenal cortex. We found that
among endocrine glands, this tissue was a major site of novH
expression in adults and during embryogenesis and that
quantitative and qualitative changes in novH expression correlated with the acquisition by adrenocortical tissue of a
tumoral phenotype. Thus, alterations of novH expression
may play a role in this tumorigenesis.
Subjects and Methods
Patients
Thirty patients with sporadic adrenocortical tumors, aged 16 –79 yr,
were included in this study. Hormonal status and stage of the tumor
were assessed as previously described (34). Histological features, including, high mitotic rate, atypical mitoses, high nuclear grade, low
percentage of clear cells, necrosis, diffuse architecture of tumor, capsular
invasion, sinusoidal invasion, and venous invasion, were carefully investigated. Tumors with none of these histological features were classified as benign. Localized tumors with one to three of these histological
features were classified as suspect. Tumors with more than three of these
features or a history of metastasis or recurrence were classified as malignant (35).
Two groups of tumors were considered based firstly on pathological
Martinerie et al. • Expression of novH in Adrenocortical Tumors
data and secondly on 11p15 molecular abnormalities: group 1 (n ⫽ 12),
all benign tumors and suspect tumors with no 11p15 abnormalities; and
group 2 (n ⫽ 18), all malignant tumors and suspect tumors with 11p15
abnormalities. Pathological, hormonal and molecular data are summarized in Table 1. Patients were numbered after entry in the study.
Human adrenocortical tumors
Tumor samples were obtained during surgery, carefully dissected by
the pathologist, and immediately frozen and kept at ⫺80 C.
Protein extractions
Frozen tissues (mean weight, 100 –300 mg) were quickly homogenized on ice in 3 ml lysis buffer [50 mm HEPES (pH 7), 250 mm NaCl,
5 mm EDTA, 0.1% Nonidet P-40, and 1 mm dithiothreitol] containing
proteases inhibitors (1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 50
␮g/ml phenylmethylsulfonylfluoride) and phosphatase inhibitors (1
mm orthovanadate and 2 mm sodium pyrophosphate), using a Polytron
(Brinkmann Instruments, Inc., Westbury NY). The homogenates were
incubated for 1 h at 0 C and centrifuged at 20,000 ⫻ g for 15 min at 4 C.
The supernatants were removed and frozen at ⫺80 C. Small aliquots of
the supernatants were used for protein determination (protein assays
from Bio-Rad Laboratories, Inc., Richmond, CA).
Immunoblotting
Protein samples (40 ␮g) were subjected to 12% SDS-PAGE under
reducing conditions and were transferred to polyvinylidene difluoride
TABLE 1. Clinical, hormonal, histological, and molecular data from patients with adrenocortical tumors
Patient
no.
Group 1
1
2
3
4
5
6
7
8
9
10
11
12
Group 2
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
Clinical data
Histological data
Weissb
Age
(yr)
Hormonal
patterna
Size
(cm)
56
40
32
41
36
57
66
48
40
61
55
41
GC
GC
GC ⫹ MC
GC
GC
GC
GC
GC
GC
NS
GC
GC
3
4
3.7
5.5
3
4
3.5
3
2
3
5
3
0
0
0
0
0
0
0
0
0
0
1
3
53
18
54
34
40
64
44
30
69
26
16
55
47
67
48
79
27
51
NS
A
GC ⫹ A
E ⫹ MC
GC ⫹ A
NS
GC ⫹ A
GC
NS
GC
GC ⫹ A
NS
GC ⫹ MC
GC ⫹ A
GC ⫹ A
GC ⫹ A
GC ⫹ E
GC
4.5
4
13
18
8
17
22
7.5
12
8
22
11.5
15
10
20
9
9
4.5
3
4
6
7
4
8
5
2
6
5
NA
8
5
5
5
NA
5
1
Histology
Molecular data
Tumor stage
at diagnosis
11p15
LOHc
Benign
Benign
Benign
Benign
Benign
Benign
Benign
Benign
Benign
Benign
Suspect
Suspect
Localized
Localized
Localized
Localized
Localized
Localized
Localized
Localized
Localized
Localized
Localized
Localized
No
No
No
No
No
No
No
No
No
No
No
No
Suspect
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Suspect
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Suspect
Localized
Localized
Metastases
Metastases
Localized
Localized
Localized
Localized
Localized
Localized
Metastases
Localized
Localized
Metastases
Localized
Metastases
Localized
Localized
LOH
LOH
No
LOH
LOH
LOH
LOH
LOH
LOH
LOH
No/LOI
LOH
LOH
LOH
LOH
LOH
LOH
LOH
IGF II mRNA
contentd
0.25
0.3
0.5
4.0
1.0
2.1
1.7
0.6
1.0
2.2
0.8
0.5
50
44
0.9
35.5
1.1
31.5
26
11
13
145
90
40.5
20
181
22
41
38
100
GC, Glucocorticoid secretion; NS, nonsecreting; A, androgen secreting; E, estrogen secreting; MC, mineralocorticoid secreting.
NA, nonavailable.
LOI, loss of imprinting.
d
Normal adult adrenals taken as references: mean ⫾ SD, 1 ⫾ 0.22 (range, 0.83–1.33; n ⫽ 4). LOH, LOI, and IGFII mRNA content were
determined as previously described (57).
b
c
Martinerie et al. • Expression of novH in Adrenocortical Tumors
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940 3931
membranes (Hybond P, Amersham Pharmacia Biotech, Orsay, France)
for immunological detection.
The K19M anti-NOVH polyclonal antibody has been described previously (24). A 1:500 dilution of K19M antibody was first incubated with
the membrane for 1 h at 37 C. Immunoreactive proteins were detected
by enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
N-glycosidase F treatment
Proteins were precipitated from extracts of adrenal tissue (40 ␮g) or
from conditioned medium (20 ␮l) from SF9 insect cells synthesizing
NOVH protein, by incubation at 4 C in the presence of 0.02% sodium
deoxycholate in 100 mm Tris-HCl, pH 8.5, and 20% trichloroacetic acid.
The precipitated protein was pelleted by centrifugation at 20,000 ⫻ g for
10 min and was then washed twice with 500 ␮l acidified acetone (10 mm
HCl) to extract and dissolve trichloroacetic acid and deoxycholate. Acetone was then eliminated with 500 ␮l diethyl ether, and the protein pellet
was dried for 5 min at 37 C. Proteins were then dissolved and denatured
in 100 ␮l N-glycosidase F buffer [50 mm sodium phosphate buffer (pH
7.2), 10 mm EDTA, 0.1% SDS, and 1% ␤-mercaptoethanol]. Nonidet P-40
(1%) was added to neutralize SDS, and protein samples were treated
with 5 U N-glycosidase F (1 U/␮l; Roche, Meylan, France) for 16 h at 37
C. Protein samples incubated for the same time at 37 C without enzyme
were used as controls.
Immunohistochemistry
Immunohistochemistry was performed on 4-␮m, formalin- or Bouinfixed, paraffin-embedded sections as previously described (36) using the
NOVH–specific K19M antibody at a 1:250 dilution. The peroxidase reaction was developed for 5 min in diaminobenzidine solution (DAKO
Corp., Glostrup, Denmark), and sections were counterstained with Mayer’s hematoxylin solution (Labonord, Villeneuve d’Asq, France), dehydrated, and mounted with Eukitt (Labonord). Controls were incubated
without the primary antibody, or the K19M antibody was first incubated
with 10 ␮g/ml NOVH-specific antigen. In neither control was any specific staining observed. The specificity of detection was also checked
with affinity-purified K19M polyclonal antibody with similar results.
Antichromogranin A monoclonal antibody (Biosoft,Varilhes, France)
was used at a 1:100 dilution. Anti-PS100 polyclonal antibody (DAKO
Corp.) was used at a 1:500 dilution. The alkaline phosphatase reaction
was developed in Fast Red solution (DAKO Corp.). After hematoxylineosin-saffron staining or after incubation with antibodies, the tissues
were examined by an experienced pathologist (A.L.) in adrenal tissues.
RNA extraction and Northern blotting
A Northern blot assay with 2 ␮g polyadenylated [poly(A)⫹] RNA
from normal adult endocrine tissues was purchased from CLONTECH
Laboratories, Inc. Total RNA was extracted from frozen adrenal tissues
by the CsCl/guanidine isothiocyanate method (37). Samples of poly(A)⫹
or total RNA (10 ␮g) were loaded on a 1% agarose-2.2 mol/liter formaldehyde gel, subjected to electrophoresis, and transferred onto nylon
membranes. The membranes were hybridized as previously described
with the 1.9-kb EcoRI novH probe (20, 24) labeled by random hexamer
priming (Amersham Pharmacia Biotech) using [32P]deoxy-CTP. The
signal for novH was normalized using the intensity of signal for ␤-actin
or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; CLONTECH
Laboratories, Inc.). For Northern blot comparisons, total RNA extracted
from HeLa cells (15 ␮g) was used as a reference sample.
Densitometry
Western immunoblots were analyzed by scanning with a GS700 imaging densitometer and processing with the Molecular Analyst data
system (Bio-Rad Laboratories, Inc.).
Northern blots were analyzed with a Storm PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical analysis
Data are expressed as the mean ⫾ sem. The two groups of tumors
were compared by Mann-Whitney’s U test for unpaired data using
Statistica software (Stat-Soft Inc., Tulsa, OK). This software was also
used to apply a statistical test for percentage to enable the comparison
of N-glycosylation in these groups. P ⬍ 0.05 was considered significant.
Results
The adrenal cortex is a major site of novH expression in
nontumoral adrenal gland and in human embryos
A Northern blot was performed using RNA extracted from
different human adult endocrine tissues. As shown in Fig.
1A, after a short exposure, novH RNA was only detected in
the adrenal cortex and medulla. However, a very weak novH
expression could be detected in pancreas, thyroid, and stomach after a longer exposure (not shown). Immunohistochemical experiments were then carried out to localize the sites of
NOVH expression in fetal and nontumoral postnatal adrenal
gland, using the human K19M polyclonal antibody (24). After 12 wk gestation, the adrenal cortex consists principally of
two distinct zones, the definitive and fetal zones (see Ref. 38
for a review), both of which contained NOVH expression
(Fig. 1B). At this stage, NOVH expression is more strongly
expressed in the definitive zone, which is composed of a
narrow band of tightly packed basophilic cells (Fig. 1B, c).
After 20 wk gestation, NOVH was still detectable in both the
definitive and fetal zones (Fig. 1B, d and e), except in small
islands of cells present in a restricted area of the innermost
fetal zone that had a barely detectable level of NOVH (Fig.
1B, e and f). Immunostaining subsequently performed on
several sections containing this same restricted zone, using
anti-PS100 antibody (Fig. 1B, g and h), which is specific for
peripheral neuronal Schwann cells, and the antichromogranin A antibody (Fig. 1B, i and j), which is specific for the
chromaffin cells that colonize the medullary part of the adrenal gland, revealed that the same restricted zone contained
cells of neuroblastic and neuroendocrine origins.
Neuroendocrine cells also contained very low levels of
NOVH in a human postnatal adrenal gland taken from a
patient with Cushing’s disease, whereas high levels of
NOVH were detected in adrenocortical clear cells (Fig. 1C, c
and d). Thus, during embryogenesis and in nontumoral adrenal gland, NOVH is mostly expressed in adrenocortical
cells. The apparent discrepancy between the results of the
Northern blot and those of immunohistochemistry is probably due to contaminating adrenal cortex tissue in the medullary sample. This was confirmed by the detection in both
adrenal cortex and medullary samples of a 1.7-kb mRNA
encoded by the 3␤-hydroxysteroid dehydrogenase (probe
provided by Dr. Van Luu-The) specifically expressed in the
adrenal cortex (data not shown).
Normal, benign, and malignant adrenocortical tissues have
different NOVH protein profiles
NOVH expression was also studied in both normal and
tumoral adrenocortical extracts (Fig. 2A). The K19M antibody detected a major 46-kDa band and a faint smear
extending from 46 –52 kDa in all normal adult adrenocortical tissues tested. In benign and suspect tumors from
group 1 (Fig. 2A), various amounts of the 46-kDa form of
NOVH were detected, but densitometric analyses of the
NOVH smear extending from 46 –52 kDa in 6 of the 11
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The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940
Martinerie et al. • Expression of novH in Adrenocortical Tumors
FIG. 1. A, novH mRNA levels in adult endocrine tissues. Northern blot analysis of novH in human adult endocrine tissues. Blot [2 ␮g poly(A)⫹
RNA] was successively hybridized with novH and ␤-actin probes and processed for autoradiography as described in Subjects and Methods. A
1-h exposure is presented. B, Expression of NOVH in sections through 12- and 20-wk-old human fetal adrenal gland. Hematoxylin-eosin-saffron
Martinerie et al. • Expression of novH in Adrenocortical Tumors
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940 3933
establish a correlation between the presence of this
NOVH-related form and the stage of the tumor.
It is noteworthy that although classified as benign, tumor
4, in which the IGF-II gene is overexpressed, contained no
detectable NOVH.
We found variable amounts of NOVH in tumors from
groups 1 and 2, with overlap with the normal range. However, the total immunoreactivity of the different forms of
NOVH in tumors from group 2 was significantly less than
that in group 1 (Fig. 2B; P ⬍ 0.004).
The limited number of normal adrenal tissues available in
this study did not allow us to measure significant statistical
variations in NOVH levels between normal adrenal gland
and tumors from group 1 or 2; however, the general trend is
for NOVH levels to be higher in group 1 tumors and lower
in group 2 tumors than in normal tissues. Thus, these data
strongly suggest that NOVH protein expression is qualitatively and quantitatively altered during adrenocortical
tumorigenesis.
Differences in the levels of N-glycosylation of NOVH protein
FIG. 1. Continued
tumors tested was significantly (P ⬍ 0.05) more intense
(mean ⫾ sd, 2.1 ⫾ 1.2) than that in normal tissues (0.4 ⫾
0.2). An additional NOVH-related 31- to 32-kDa doublet
was also detected in samples 1, 2, 3, 9, and 11, with different intensities. Lower molecular mass forms of NOVH,
possibly generated by cleavage of the full-length protein,
have been observed in Wilms’ tumors (24) and in the
conditioned medium of insect cells infected with a recombinant baculovirus expressing NOVH (39).
In a large proportion (14 of 18) of the tumors from group
2, little or no NOVH expression (Fig. 2A) was detected. In
the malignant tumors in which NOVH expression could be
detected, two different patterns were observed: 1) an intense smear (no. 21, 25, 28, and 30) as observed in benign
tumors with different intensities of the 46-kDa band; the
average intensity of the smear detected in these four tumors (1.3 ⫾ 0.3) was also significantly (P ⬍ 0.05) higher
than that in normal adrenocortical tissues, but did not
differ significantly from that in group 1 (2.1 ⫾ 1.2); or 2)
the 46-kDa form present at various levels with no smear
(no. 15, 17, 20, 22, 23, and 29). The 31- to 32-kDa doublet
was detected in various amounts in several malignant
tumors (no. 18, 19, 22, 25, 28, and 29), but we could not
NOVH protein contains two potential sites of N-glycosylation, at positions 97 (NQTG) and 280 (NCTS), and the
treatment of novH-transfected MDCK cells with tunicamycin reduces the apparent molecular mass of NOVH in
these cells to 39 kDa (24). This suggested that the 46- to
52-kDa smear of NOVH observed in several tumors results
from different degrees of N-glycosylation. We investigated this possibility by subjecting total protein extract (40
␮g) derived from one benign (tumor 1) and two malignant
tumors (tumors 21 and 28) to N-glycosidase F treatment.
The apparent molecular mass of NOVH under reducing
conditions after N-glycosidase F treatment decreased from
46 to 44 kDa in tumors 1, 21, and 28 (Fig. 3). The forms of
NOVH with the lowest molecular mass detected in samples 1 and 28 were also reduced from 31–32 kDa to the
same molecular mass (24 kDa). Therefore, these results
show that during adrenocortical tumorigenesis, changes
in NOVH N-glycosylation can be detected as early as the
benign stage. Serum-free conditioned medium from insect
cells infected with a recombinant baculovirus expressing
NOVH (SF9/82) was subjected to the same conditions and
used as a control of N-glycosidase F treatment. The apparent molecular mass of these NOVH recombinant forms
before N-glycosidase F treatment was lower (44 and 27
kDa) than that in adrenal tissues, indicative of different
levels of posttranslational modifications. N-Glycosidase F
(HES) staining of prenatal human adrenals from 12-wk-old (a) and 20-wk-old (b) fetuses. The gland consists predominantly of an inner
eosinophilic fetal zone capped by a rim of smaller cells that comprise the definitive cortex. Immunohistochemistry of 12-wk-old (c) and 20-wk-old
(d–f) stages performed with the K19M antibody. Insets in c and d show controls in which the primary antibody was omitted. Note a restricted
zone in e and different cells of this zone in f that are not labeled with K19M (black arrowheads). Immunohistochemistry was performed at 20
wk of embryonic development with anti-PS100 antibody, a specific marker for Schwann cells (g and h). Note in h that only a few cells of this
restricted area of the adrenal gland (g) are labeled, suggesting that the labeled cells (nb) probably correspond to immature neuroblasts. Labeling
of chromaffin cells with antichromogranin A antibody (i and j). Note in i that chromaffin cells are only present in a restricted zone of the adrenal
gland. Neuroendocrine cells (nen) are indicated by arrowheads. Magnification, ⫻400 for all except e, g, and i, ⫻80. C, Expression of NOVH in
adrenal gland from a patient with Cushing’s disease. HES staining showing two different types of cells (a): adrenocortical clear cells (adc) and
chromaffin cells of the medulla (nen). Immunohistochemistry was performed with the K19M antibody (b and c represent different areas of the
same section); the inset in b shows the control, in which the primary antibody was omitted. Immunohistochemistry was performed with
antichromogranin A antibody on a serial section (d). Magnification, ⫻400.
3934
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940
Martinerie et al. • Expression of novH in Adrenocortical Tumors
FIG. 2. A, NOVH protein expression in normal adrenal cortex and in tumors of the adrenal cortex. Western blot analysis (40 ␮g protein) for
NOVH in normal adrenal cortex (Nl ad; a– c), in tumors from group 1 (no. 1–12) and group 2 (no. 13–30) using K19M antibody at a 1:500 dilution.
Tumor 10 is not presented. The various forms of NOVH are indicated by asterisks. Protein integrity in each sample tested was checked after
transfer with Ponceau Red staining. B, Quantitative analysis of total NOVH protein in normal adrenal cortex and in tumors of the adrenal cortex.
The immunoblots were scanned, and the sum of the various forms of NOVH was compared with the amount of NOVH in sample 5, which was
tested in each experiment, used for normalization, and expressed as 1 in arbitrary units. The mean NOVH protein level for each group is indicated
by a horizontal dash. AU, Arbitrary units.
treatment also reduced NOVH recombinant sizes to 42 and
24 kDa. In our experimental conditions, however, the apparent molecular masses of the various forms of NOVH
were not reduced to the predicted 39 and 19 kDa, indicating that either the N-glycosidase F treatment was not
complete or NOVH undergoes other posttranslational
modifications in these samples.
No histological differences are detected in malignant tumors
with different NOVH profiles
As different NOVH-sized proteins due to glycosylation
variations were observed in malignant adrenocortical tumors, we investigated whether a relationship could be established between these profiles and the histology of these
tumors. For this experiment, we studied benign tumor 9 and
malignant tumors 19, 21, and 28 with a very low level of
NOVH expression or with different electrophoretic profiles,
as detected by Western blotting. Histological examination of
tumors 19, 21, and 28 revealed no striking differences. Tumors (no. 19, 21, and 28; Fig. 4B, a, d, and g) consisted mostly
of compact cells grouped in large sheets or trabeculae separated by a fine fibrovascular stroma. Large areas of necrosis
were also present. Few mitoses were observed in nuclei with
abnormalities.
Immunohistochemical experiments were then carried out
to determine the sites of NOVH expression in both benign
and malignant adrenocortical tumors. In benign tumor 1 (Fig.
4A, b– d), NOVH protein was detected both in the part of the
adrenocortical gland that was normal and in the adenomatous tissue. The level of NOVH expression seemed to be
higher in the adrenal compartment next to the tumoral tissue
than in the adenomatous tissue (Fig. 4A, b). In contrast, little
or no NOVH expression was detected in the capsule composed of connective tissue. At higher magnification, the clear
cells of the adrenal cortex in normal (Fig. 4A, c) and adenomatous (Fig. 4A, d) tissue expressed NOVH. In malignant
tumors, consistent with Western blot analysis, NOVH was
detected from weak in tumor 19 to high levels in tumors 21
and 28, essentially in compact tumoral cells (Fig. 4B, b, e, and
h). No NOVH expression was detected in necrotic areas. In
Fig. 4B (c, f, and i), a section is shown at higher magnification
to demonstrate that NOVH detection was not uniform in all
tumoral cells. Within the fibrovascular stroma, some fibroblasts were also positive (Fig. 4B, j). As previously observed
Martinerie et al. • Expression of novH in Adrenocortical Tumors
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940 3935
a homozygous deletion of this region was responsible for the
lack of novH expression in this tumor.
Discussion
FIG. 3. N-Glycosidase F treatment of recombinant and native NOVH
protein produced by insect SF9 cells and adrenocortical tumors. Western blot analysis was performed with the K19M antibody at a 1:500
dilution for SF9/82-conditioned medium (20 ␮L) and protein extracts
(40 ␮g) from adrenocortical tumors (benign, no. 1; malignant, no. 21
and 28) untreated (⫺) or treated (⫹) with N-glycosidase F (5 U). The
various forms of NOVH are indicated by asterisks. The slight difference between patterns for untreated samples (⫺) 1, 21, and 28 in Fig.
3 and for samples 1, 21, and 28 in Fig. 2A is due to the overnight
incubation at 37 C in N-glycosidase F buffer of the control samples 1,
21, and 28 (⫺). This was confirmed by running the three samples, not
incubated, ⫺, and ⫹, on the same gel (data not shown).
in other tissues (24), NOVH protein was also present in
differentiated structures within the tumor, such as endothelial cells, and in the surrounding smooth muscle of some
blood vessels (Fig. 4B, k).
Transcriptional regulation of novH also occurs in
adrenocortical tumors
We investigated whether the differences in the amounts of
NOVH proteins detected in adrenocortical tumors resulted
from differences in levels of novH mRNA by performing
Northern blot analyses. Various amounts of the 2.5-kb novHspecific mRNA species were detected in different tumors
(Fig. 5A), with significantly more (P ⬍ 0.001) in tumors from
benign group 1 tumors than in those from malignant group
2 tumors (Fig. 5B). There was a correlation between novH
protein and RNA levels in the various samples tested (Fig.
5C; P ⬍ 0.001). However, the ratio of protein to RNA was 6
to more than 100 times higher in several group 2 tumors such
as 15, 17, 20, and 23, than in group 1 tumors 1, 2, and 3,
indicating that the level of novH mRNA translation or
NOVH protein stability may be higher in some group 2
tumors. Similar results were obtained when levels of novH
mRNA were normalized relative to GAPDH (Fig. 5, B and C)
or to the ribosomal protein S26 mRNA (40) (data not shown).
Neither novH mRNA nor NOVH protein was detected in
some samples, such as that for tumor 26. We therefore carried
out Southern blot analysis (data not shown) with control
(leukocytes) and tumor (adrenocortical) DNA from the same
patient (no. 26). After AvaII digestion, electrophoresis, and
blotting onto membranes, both DNA samples were hybridized with a novH-specific probe. Restriction fragments of
identical size (2.2, 1.6, 0.7, and 0.5 kb) were detected in both
samples, indicating that neither a major rearrangement nor
Several studies (10, 31, 41) have shown that nov expression
is widely distributed in normal adult tissues. Levels of nov
mRNA have been reported to be high in the brains of adult
humans, rats, and chickens, but to differ considerably between species in other organs such as the lung or heart (10,
31, 41). Spatio-temporal regulation of nov expression has also
been described in the chicken, because nov mRNA was detected in embryonic, but not adult, heart, muscle, and kidney,
whereas in the lung, nov expression was only detected in
adult tissue (10). nov expression is tightly regulated during
development of the central nervous system (32) and throughout chondrogenesis (Laurent, M., personal communication),
suggesting that nov may be involved in the development and
differentiation of these tissues.
In this paper we report for the first time that in humans,
novH is more strongly expressed in the adrenal gland than
in several other adult endocrine tissues. nov was strongly
expressed in the adrenal gland cortex derived from the celomic epithelium, and lower levels of its expression were
detectable in cells of the medulla of the neural crest origin.
NOVH, which is a secreted protein (24), was present in both
the definitive and fetal zones of the adrenal cortex in 12- and
20-wk-old fetuses and may therefore play an autocrine/paracrine role in the development and/or differentiation of this
tissue.
As novH expression was altered in Wilms’ tumors (20, 24),
which have some physiopathological and molecular alterations in common with adrenocortical tumors (2), we investigated whether novH was involved in adrenocortical tumorigenesis by studying its expression in 30 adrenocortical
tumors of different types. The major finding of our study is
that novH expression is reduced with malignant progression
and that different glycosylation patterns can be detected as
early as the benign stage.
Both overexpression and down-regulation of nov have
been reported in tumors depending on the tumor studied
and the histological composition of the tumor. In Wilms’
tumors, high levels of nov expression have been associated
with heterotypic tumoral differentiation (e.g. muscle and cartilage) (24). The low levels of novH expression observed in
malignant adrenocortical tumors are consistent with the dedifferentiation of tumoral cells and with novH having a
potential inhibitory role in the growth of certain types of cells
(10). However, immunohistochemical analyses showed that
in both benign and malignant tumors, NOVH was not uniformly present in all cells, indicating that novH expression
may be dependent on the cell cycle or the stage of differentiation of the cells.
Other members of the CCN family also have different
patterns of expression in tumors. The expression of elm1/
wisp1 is inversely correlated with the incidence of metastasis
and the growth of melanoma cells (16), but is overexpressed
in colon tumors. r-Cop1/wisp2 expression is up-regulated in
Wnt-1-transformed C57MG cells, but down-regulated in
transformed fibroblasts in rats and mice (17). Its level of
3936
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940
Martinerie et al. • Expression of novH in Adrenocortical Tumors
FIG. 4. A, Expression of NOVH protein in sections from a group 1 adrenocortical tumor (no. 9). HES staining (a) showing that remnant of the
adrenal cortex in contact with the adenoma. nac, Normal adrenal cortex; fs, fibrovascular stroma; ad, adenoma; adc, adenoma cells. Immunohistochemistry was performed with the K19M antibody (b– d). Higher magnifications of b are presented in c (for nac) and d (for ad). Insets
in c (nac) show controls in which the primary antibody (K19M) was omitted (left) or preincubated with K19M-specific peptide (right). Note in
d, a nonuniform distribution of NOVH in adrenocortical clear cells of the adenoma (adc). The inset in d (adc) shows the control in which the
primary antibody (K19M) was omitted. Magnifications: ⫻300 (a), ⫻200 (b), and ⫻800 (c and d). B, Expression of NOVH protein in sections from
group 2 adrenocortical tumors (no. 19, 21, and 28). Three adrenocortical tumors [no. 19 (a– c), 21 (d–f), and 28 (g– k)] exhibiting different NOVH
profiles were studied. HES staining was used (a, d, and g). Immunohistochemistry was performed with the K19M antibody (b, c, e, f, h, i, j,
and k). Insets in b, e, and h show controls in which the primary antibody was omitted. cpc, Tumoral compact cells; nt, necrotic tissue; fb,
fibroblasts; fs, fibrovascular stroma. Note in k that NOVH protein is also present in smooth muscle (sm) and in the endothelial cells (ec) of vessels.
Magnifications: ⫻400 (a, b, d, e, g, and h), ⫻800 (c, f, i, and k), ⫻2160 (j).
expression is significantly lower in colon tumors than in
normal colon mucosa (15). In breast tumors (15, 42) and in
pancreatic tumors (43), the expression of elm1/wisp1,
r-Cop1/wisp2, and ctgf has been detected essentially in the
stroma cells surrounding tumor cells, with little or no expression in tumor cells. NOVH was detected in adrenocortical tumors, with different amounts in the two compartments. Therefore, although changes in the expression of CCN
members are detected in tumors, suggesting a role in tumor
growth, no unifying hypothesis has yet been established.
In this study we found that N-glycosylation of the NOVH
protein in several benign and malignant adrenocortical tumors differed greatly from that in normal tissue. This modification seems to be specific to NOVH, because no significant
alteration of the N-glycosylation profile of other proteins
such as IGFBP-3 was detected in the same samples (5) (our
data not shown). An increased N-glycosylation of the NOVH
protein was significantly (P ⬍ 0.05) more frequent in benign
(54.5%) than in malignant (22.2%) tumors. In 58% of the
benign tumors tested the 46-kDa form of NOVH was also
present at higher or similar levels than in normal tissues,
whereas in only 11% of the malignant tumors could the level
of the 46-kDa band be compared with benign or normal
tissues. In a large majority of the malignant samples tested
(77%) an important decrease in this form was observed. In
several benign and malignant tumors, additional glycosylated 31- to 32-kDa forms of NOVH were observed. No
correlation was found between the amounts of these forms
and modifications to the N-glycosylation of the 46-kDa form
in the various samples studied. This indicates that changes
in N-glycosylation do not affect the amount of the lower
molecular mass forms of NOVH probably generated by proteolytic cleavage. Changes in NOVH glycosylation may,
however, affect the ability of NOVH to interact with other
proteins. It has been shown that the extent of N-glycosylation
can modulate the cell-binding activity of IGFBP-3 (44). Aberrant N-glycosylation of NOVH could also affect NOVH
stability, as suggested by our previous results (24). Along this
line, it has been reported that aberrant N-glycosylation of von
Willebrand factor type C (45) leads to an increase in clearance
from plasma and accounts for a low von Willebrand factor
type C phenotype. Further studies are required to elucidate
Martinerie et al. • Expression of novH in Adrenocortical Tumors
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940 3937
FIG. 4. Continued
3938
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940
Martinerie et al. • Expression of novH in Adrenocortical Tumors
FIG. 5. A, novH mRNA levels in normal adrenal cortex and in tumors from adrenocortical tissues. Northern blot analysis of novH in
representative samples (10 ␮g total RNA) of normal adrenal gland (Nl ad; b and c) and of tumors from group 1 (no. 1–11) and group 2 (no. 13–25).
Blots were successively hybridized with novH and GAPDH probes and processed for autoradiography as described in Subjects and Methods.
B, Densitometric analysis of novH mRNA levels in normal adrenal cortex and tumors from adrenocortical tissues. The groups are defined in
Table 1. For each of the 28 samples tested, the amount of novH RNA was normalized relative to GAPDH, compared with the amount of normalized
novH RNA in HeLa cells (15 ␮g) that were used as a control, and expressed as 1 in arbitrary units. The mean novH RNA level for each group
is indicated by a horizontal dash. C, Correlation between novH RNA and protein levels in various adrenocortical samples, expressed in arbitrary
units (AU).
the role of N-glycosylation in the biochemical properties of
NOVH.
Variable amounts of NOVH were observed within group
1 and 2 tumors. However, these variations could not be
strictly correlated to any of the clinical or molecular data
indicated in Table 1 in particular. They are more likely to be
due to a combination of several different elements characterizing the tumors, including the hormonal pattern, size of
the tumor, presurgery treatment, and variable amounts of
IGF-II.
A growing body of evidence suggests that the IGF system
is involved in tumor proliferation (reviewed in Ref. 46) and
particularly in the development of adrenocortical tumors
(2– 6, 47). In this study no overexpression of the IGF-II gene
was detected in tumors from group 1, except for tumor 4,
which did not express novH, whereas 16 of the 18 malignant
tumors overexpressed the IGF-II gene. Twelve of these 16
tumors displayed a strong down-regulation of novH expression. Therefore, although no strict inverse correlation can be
drawn, these results suggest that IGF-II and novH may be
regulated in opposite ways by a common transcription factor. In some Wilms’ tumors, we have previously reported
that levels of novH mRNA were inversely correlated to levels
of wt1 mRNA (20) and that wt1 indirectly regulated novH
expression ex vivo (25). wt1 also acts as a negative transcriptional regulator for IGF-II (30), and IGF-II gene expression is
also altered in Wilms’ tumors (26 –28). wt1 can up-regulate
or down-regulate gene expression depending on the inter-
acting proteins and gene promoters (48 –51). It has recently
been shown that wt1 expression is required for development
of the adrenal cortex (52), but nothing is known about its
possible involvement in IGF-II and novH regulation in this
tissue. It is also possible that IGF-II and/or novH each regulate the other’s expression.
In conclusion, the significant differences in the levels of
novH protein and RNA between benign and malignant tumors may be of importance. For many genes such as IGFII,
IGFBP2 (5), p57Kip2, G1 cyclins, and G1 CDKs (7), altered
expression has only been observed in malignant tumors. In
contrast, differences in novH expression between benign
tumors and normal tissue can be detected, suggesting that
novH could participate in the early stages of tumorigenesis.
Relatively little is known to date about the function of
novH. In several cell systems nov has been reported (53, 54)
to be associated with cell quiescence. Together with previous
observations that overexpression of nov in chicken embryo
fibroblasts led to an inhibition of growth (10), this suggests
that nov is a negative regulator of growth. However, different cells may respond differently to NOV, because recombinant NOV stimulates 3T3 cell proliferation (41), but has no
effect on the proliferation of vascular smooth muscular cells
(VSMC) (54).
Several lines of evidence suggest that nov is more likely to
be involved in cell adhesion. The multidomain structure of
NOV and the other CCN members suggests that they bind
to components of the extracellular matrix, including heparin-
Martinerie et al. • Expression of novH in Adrenocortical Tumors
The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3929 –3940 3939
like oligomers (14). The finding that fibulin 1C, an extracellular matrix-associated protein (55, 56), interacts with the
NOVH protein (39) provides a clue for the possible participation of NOVH in signaling pathways involving the extracellular matrix. More recently, it has been shown that the
recombinant NOV protein can promote cell adhesion ex vivo
and that changes in nov expression occur in response to
injury of the arterial walls (54). It has been proposed that
reduced nov expression is involved in releasing VSMC for
migration and proliferation and that nov can be reexpressed
during the late stages of repair, when migration and proliferation slow down. It is thus tempting to speculate that the
enhanced expression of nov in adrenocortical tumors participates in the benign phenotype by increasing adhesion of
cells, and that decreased levels of NOV in malignant tumors
are involved in cell invasiveness. Alternatively, novH could
act as a tumor suppressor in adrenocortical tumors. Further
investigation of the biological properties of NOVH in adrenocortical cells will enable us to determine whether changes
in the expression of novH play a key role in the development
of these tumors.
Note Added in Proof
While this manuscript was submitted, tight regulation of nov expression during mouse development in skeletal and visceral muscles
and in nervous system was described (Natarajan D, Andermarcher E,
Schofield PN, Boulter C 2000 Mouse Nov gene is expressed in hypaxial
musculature and cranial structures derived from neural crest cells and
placodes. Dev Dyn 219:417– 425).
Acknowledgments
We thank Prof. B. Perbal for helpful discussions and Dr. X. Bertagna
and Dr. H. Kleinman for critical reading of this manuscript. We are
grateful to A. M. Henere for skillful assistance with the immunohistochemistry experiments, to S. Kyurkchiev for purifying the K19M antibody, and to J. M. Ricort for performing Western ligand blotting for
IGFBP-3 detection in adrenocortical samples. We thank Dr. M. Tantau
for provision of fetal material. We also thank Mr. J. Grellier for assistance
with photography.
Received November 17, 2000. Accepted April 4, 2001.
Address all correspondence and requests for reprints to: Dr. C. Martinerie, INSERM, U-515, Hôpital Saint-Antoine, 184 rue du Faubourg
Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: martiner@
st-antoine.inserm.fr.
This work was supported by Assistance Publique des Hopitaux de
Paris (Contrat de Recherche Clinique 97133), University Paris VI, Faculté
Saint-Antoine (UPRES EA 1531), Association de Recherche contre
le Cancer (no. 1364), Centre National de la Recherche Scientifique,
INSERM (U-515), and Programmes Hospitaliers de Recherches Cliniques Grant AOM95201 for the Comete Network.
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