Endocrine Reviews 20 (2): 189-206
Copyright © 1999 by The Endocrine Society
The Connective Tissue Growth Factor/Cysteine- Rich 61/Nephroblastoma Overexpressed (CCN) Family1
David R. Brigstock
Departments of Surgery and Medical Biochemistry, The Ohio State
University, Columbus, Ohio 43210; and Department of Surgery,
Childrens Hospital, Columbus, Ohio 43205
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Abstract
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- I. Introduction
- II. The Modular Structure of the Connective Tissue Growth
Factor/Cysteine-Rich 61/Nephroblastoma Overexpressed (CCN)
Family Members
- III. Connective Tissue Growth Factor (CTGF)
- A. Discovery
- B. Structure of the CTGF gene and protein
- C. CTGF mRNA production
- D. CTGF production, secretion, and processing
- E. CTGF-heparin interactions
- F. Biological properties of CTGF
- G. Mechanism of action of CTGF
- IV. Cyr61
- A. Discovery of cyr61
- B. Structure of the cyr61 gene and protein
- C. Cyr61 mRNA production
- D. Cyr61 production and secretion
- E. Biological properties of cyr61
- F. Mechanism of action of cyr61
- V. Nov
- A. Discovery and structure of nov
- B. Nov mRNA production
- C. Biological properties of nov
- VI. Elm1/WISP-1
- VII. Heparin-Induced CCN-Like Protein (HICP)/rCop-1/CTGF-3/WISP-2
- VIII. WISP-3
- IX. Other CCN-Like Molecules
- X. Regulation of Cellular Functions by the CCN Family
- A. Cell cycle control
- B. Cell adhesion and migration
- C. Extracellular matrix (ECM) production
- XI. Biological Processes Involving the CCN Family
- A. Development and differentiation
- B. Female reproductive tract function
- C. Angiogenesis
- D. Wound repair
- E. Fibrotic disorders
- F. Inflammation
- G. Tumor growth
- XII. Perspectives and Future Directions
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I. Introduction
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THE LAST 56 yr have seen the emergence of a new gene
family that currently comprises connective tissue growth factor (CTGF;
also termed fisp-12), cysteine-rich 61 (cyr61), nephroblastoma
overexpressed (nov), expressed low in metastasis 1 (elm1; also termed
WISP-1), heparin-inducible CTGF/cyr61/nov (CCN)-like protein (HICP;
also termed rCop-1, CTGF-3 or WISP-2), and WISP-3 (Fig. 1
). Family members have been
characterized from human, mouse, rat, pig, cow, chicken, quail, and
frog and are predicted to have arisen from a common ancestral gene more
than 40 million years ago (Fig. 2
).
Although these proteins were initially classified as immediate early
gene products or growth factors, this concept has had to be modified in
light of more detailed studies of their activities as well as the
discovery of unique family members that exhibit quite different
biological properties. Since CTGF, cyr61, and nov were the prototype
members of this family, this article adopts the term "CCN family"
as introduced by Bork in 1993 (1). Implicit in this usage is that
reference to the CCN family applies to all paralogs, and that CTGF,
cyr61, and nov are not necessarily representative of the full range of
gene or protein structures and biological properties.

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Figure 1. Amino acid sequence alignment of human CCN
paralogs. Sequences were aligned using the J. Hein method.
Shading represents residues that are identical to the
consensus sequence.
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The primary translational products of most CCN family members contain
343381 residues and generate secreted proteins of 3540 kDa that
contain 38 conserved cysteine residues that are organized into four
distinct structural modules. Exceptions are rCop-1 and its orthologs,
which lack the fourth structural module and contain only 28 conserved
cysteine residues, and WISP-3, which lacks 4 of the cysteine residues
that are usually present in module 2. The biological properties of CTGF
and cyr61 include stimulation of cell proliferation, chemotaxis,
adhesion, and extracellular matrix (ECM) formation. CTGF and cyr61, but
not nov, elm1, or HICP/rCop-1, are encoded by growth factor-inducible
immediate early genes whereas nov, elm1, and HICP/rCop-1 are expressed
in cells demonstrating growth arrest or quiescence. The CCN family
appears to be involved in normal processes such as implantation,
placentation, embryogenesis, differentiation, and development as well
as processes related to tissue pathology, including wound healing and
fibrotic disorders.
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II. The Modular Structure of Connective Tissue Growth
Factor/Cysteine Rich 61/Nephroblastoma Overexpressed (CCN)
Family Members
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Through evolution, exons with specific biological functions
("modules") have been shuffled, forming genes that encode mosaic
proteins which exhibit new biological properties (2, 3, 4). CTGF, cyr61,
and nov were shown to contain four distinct structural modules that
exhibit homology to conserved regions in a variety of extracellular
mosaic proteins (1). All four modules are represented in WISP-1 and
WISP-3, and three of the modules are present in rCop-1 and its
orthologs (Fig. 3
). Each module is
involved in protein binding and contains conserved cysteine,
hydrophobic, and polar residues. Except for module 2 in WISP-3, the
modules in the various CCN proteins are 3898% conserved with the
corresponding module in human CTGF (hCTGF) (Table 1
). While complex intrachain disulfide
bridging is likely, this is predicted to occur within, rather than
between, the modules (1).

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Figure 3. Modular structure of individual CCN family
members. Proteins shown with black boxes are the
predicted primary translational products. Proteins shown with
open boxes are biologically active derivatives arising
by proteolysis (CTGF) or viral DNA integration (Nov). Question
marks indicate that the C termini of N-terminally truncated
CTGF proteins have not been experimentally determined.
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Table 1. Amino acid sequence homologies (%) of individual
modules and complete sequences of CCN proteins as compared to those
of hCTGF
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The proposed modular configuration of CCN family members has become a
provoking model for those engaged in determining their biological
functions. Introns occur between the modules in the CTGF, cyr61, and
nov genes (5, 6, 7, 8), a feature that is typical in genes of many other
modular proteins (9). The presence of the modules is further supported
by the susceptibility of CTGF to proteolysis at sites between, rather
than within, the modules (10, 11) and by functional properties such as
the binding of CTGF to insulin-like growth factors (IGF) or putative
cell surface receptors (10, 11, 12, 13, 14), and the promotion of cell adhesion by
CTGF or cyr61 (15, 16). However, it is unclear whether the biological
properties of CCN proteins reflect the individual properties of each
module or the overall combination of the modules and other sequences
within each protein.
Module 1 is approximately 32% identical with the N-terminal
cysteine-rich regions of the six "classic" IGF-binding proteins,
IGFBP-1 to -6 (1), and contains a motif (GCGCCXXC) that is involved in
binding IGF (17). 125I-labeled IGF-I or -II specifically
binds recombinant hCTGF (rhCTGF) (12), although with much lower
affinity than classic IGFBPs and more comparable to mac25, a
low-affinity IGFBP, also termed IGFBP-7 or IGFBP-related protein
(IGFBP-rP)-1 (18). Nov was also reported to bind IGFs (19), but this
result has not proven reproducible (20). Recently, the terms IGFBP-8,
-9, and -10 were introduced as synonyms for CTGF, nov, and cyr61,
respectively (12), although it has since been proposed to use the names
IGFBP-rP-2, -3, and -4 until their relationship to the classic IGFBPs
has been determined (21). Indeed, a case against classifying the CCN
family as IGFBPs has recently been made (22). Whatever the terminology,
it remains unclear whether the interaction of IGFs with CCN family
members occurs physiologically, whether sequences within module 1 are
actually responsible for the observed binding of IGF, and what
consequences this interaction has on the respective half-life,
bioavailability, or activity of each of the molecules involved.
However, there is general agreement that direct, IGF-independent
effects of CTGF on the cell cycle are likely to be more significant
than its low affinity binding of IGF (12, 22).
Module 2 comprises a Von Willebrand type C domain (VWC) that occurs in
Von Willebrand factor as well as various mucins, thrombospondins, and
collagens (1). Many proteins that contain VWC modules participate in
oligomerization, which may be preceded by a dimerization event (23).
Since module 4 of CTGF is a putative dimerization domain, module 2 may
mediate the formation of complexes from CTGF dimers. Unlike all other
CCN proteins, module 2 in WISP-3 contains only 6 of the 10 cysteines,
the functional significance of which has yet to be established (24).
Module 3 is a thrombospondin type 1 (TSP1) that contains the local
motif WSXCSXXCG (1) and appears to be a cell attachment motif that
binds sulfated glycoconjugates (1, 25, 26, 27). While cyr61 and CTGF
promote cell adhesion (15, 16), the role of module 3 in this process
remains unexplored.
Module 4 is a C-terminal (CT) module that also occurs in the C termini
of a variety of unrelated extracellular mosaic proteins (1). Six of the
10 cysteine residues in the CT module appear to adopt the cystine knot
motif that also occurs in nerve growth factor (NGF), transforming
growth factor-ß (TGF-ß), and platelet-derived growth factor (PDGF)
(1). This complex structure comprises two 2-stranded ß-sheets that
lie face-to-face and are linked by three interlocking disulfide bridges
and has defined a new superfamily of growth regulators (28, 29) to
which members of the CCN family (except orthologs of rCop-1, which lack
module 4; see below) may also belong. Since some of the receptor
binding properties of NGF, TGF-ß, and PDGF reside in variable regions
within the cystine knot (28), the CT module likely contains both
dimerization and receptor-binding domains (1). Although dimerization of
CCN proteins has not been reported, bioactive forms of 10-kDa porcine
CTGF (pCTGF) comprise the C-terminal 102 or 103 residues of the primary
translational product (Fig. 3
) (10) and thus support the proposed role
of module 4 in binding cell surface receptors (1). Module 4 is absent
from rCop and its orthologs (Figs. 1
and 3
), suggesting that they are
functionally distinct from other CCN proteins.
 |
III. Connective Tissue Growth Factor (CTGF)
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A. Discovery
The first description of a CTGF ortholog occurred in 1988 when
cDNA encoding "fibroblast-inducible secreted protein-12"
(fisp-12) was isolated by differential screening of a cDNA
library from serum-stimulated NIH 3T3 cells (30). The gene structure
and predicted protein sequence of fisp-12 were subsequently
reported in 1991 (5). Using a similar screening strategy, Brunner
et al. (31) independently isolated the same cDNA, termed
ßIG-M2, from TGF-ß2-stimulated mouse AKR-2B cells.
Fisp-12/ßIG-M2 comprises 348 amino acids and contains 39
Cys residues, one of which occurs in the 25-residue N-terminal signal
peptide (Fig. 1
) (5, 31). The human ortholog of CTGF was discovered in
1991 due to the cross-reactivity of a PDGF antiserum with 38-kDa hCTGF
secreted by cultured human vein endothelial cells (HUVECs) (13). The
corresponding cDNA was isolated by screening a HUVEC cDNA expression
library with anti-PDGF and shown to encode a 349-amino acid protein
that is 91% homologous to fisp-12 (13) (Fig. 1
). The
protein was termed "CTGF" because it was both mitogenic and
chemotactic for fibroblast-like cells in vitro. CTGF cDNAs
have since been reported for pig, rat, cow, and frog (10, 32, 33, 34, 35).
B. Structure of the CTGF gene and protein
The fisp-12 gene comprises five exons and four introns
and spans 3.1 kb (Ref. 5 ; Table 2
). The
organization and structure of the hCTGF gene are very similar to that
of fisp-12 except that exon 1 encodes one additional amino
acid in the signal peptide (13, 36, 37). There is 80% sequence
identity between CTGF and fisp-12 in the 300 nucleotides
that lie immediately upstream of the mRNA cap site (36), and the
5'-regions of both genes contain a variety of conventional regulatory
elements as well as a unique TGF-ß response element (Fig. 4
). Fisp-12 maps to the A3-B1
region of murine chromosome 10 (5) and hCTGF maps to human chromosome
6q23.1 (38).

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Figure 4. Key regulatory elements in the gene promoters of
hCTGF, mouse cyr61, and human nov. Note that the TGF-ß response
element (TßRE) is unique to CTGF.
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The CTGF primary translational product comprises 349 (human, pig, cow),
348 (mouse), or 343 (Xenopus) residues (5, 10, 31) and is
more than 90% conserved in mammals (Fig. 1
and Table 1
). Most of the
nonhomology occurs over the first 43 residues where the identity is
only 6065%. The secreted proteins from all five species are
predicted to comprise 323 residues and to contain 38 fully conserved
cysteine residues, which are evenly spread throughout the molecule
except for a cysteine free-region between Asp167 and
Asn198 in hCTGF. As assessed by SDS-PAGE, the molecular
mass of CTGF is 3638 kDa (5, 13, 14, 32, 39, 40).
Heterogeneity in the mass of native and recombinant forms of hCTGF is
due to variations in its degree of glycosylation (14, 40). hCTGF
contains predicted sites for N-linked glycosylation at
Asp28 and Asp225 (13) and is susceptible to
Endoglycosidase F, which decreases its mass by 28 kDa (12, 40). Neither fisp-12 nor pCTGF appears to be glycosylated
(5, 31, 32), suggesting that the glycan groups in hCTGF are either not
functionally relevant or confer additional properties on the human
ortholog.
C. CTGF mRNA production
Among the cell types that produce CTGF mRNA are fibroblasts (5, 14, 31, 35, 39, 41, 42), endothelial cells (13, 35, 43), vascular
smooth muscle cells (VSMC) (35, 44), epithelial cells (45, 46, 47),
chondroctyes (48), and glioblastoma cells (37).
Fisp-12/CTGF transcripts have been detected in
multiple tissues of at least four mammalian species (5, 12, 32, 35, 47, 49). Most studies have reported a single CTGF transcript of 2.4 kb,
although glioblastoma cells also contain 3.5-kb and 7.0-kb transcripts
(37). The 2.4-kb transcript is present in unstimulated fibroblasts (35, 39) but is rapidly induced after treatment with TGF-ß or serum and is
superinduced in stimulated fibroblasts in the absence of de
novo protein synthesis (5, 31, 41). These studies collectively
established that CTGF/fisp-12 are encoded by immediate early genes,
although their kinetics of induction are more rapid and sustained than
those of other immediate early genes (41). TGF-ß also increases CTGF
mRNA levels in cultured human or mouse lung mesenchymal cells (34) and
in human chondrocytic cells (48), the latter of which also produce CTGF
mRNA in response to bone morphogenic protein-2, a member of the TGF-ß
family (50). Using an enzyme-linked immunosorbent assay (ELISA),
TGF-ß treatment of human fibroblasts was shown to result in increased
production of the CTGF protein (51). The molecular basis for the action
of TGF-ß on CTGF gene transcription has been atributed to nucleotides
-157 to -145 of the hCTGF promoter (36). This region is a unique
TGF-ß-inducible element (Fig. 4
) that is fully conserved in the
promoter of the fisp-12 gene (5) but is absent from the
other parologs of the gene family (Fig. 4
) as well as from other
immediate early genes. In addition, inhibitors of protein kinase A, but
not of tyrosine kinases or protein kinase C, block TGF-ß-stimulated
CTGF transcription (52). Collectively, the TGF-ß response element and
the involvement of cAMP in CTGF mRNA production represent major
mechanistic differences in CTGF gene transcription as compared with
other CCN family members.
Although considerable attention has been directed toward the TGF-ß
inducibility of CTGF expression and the potential pathophysiological
consequences thereof (53), PDGF, epidermal growth factor (EGF), and
fibroblast growth factor (FGF) also stimulated hCTGF gene expression in
fibroblasts (41). As compared with the effects of TGF-ß on this cell
type, CTGF transcription is less robust in response to these growth
factors (41) and does not result in increased CTGF protein levels (51).
In contrast, pancreatic cancer cells of epithelial origin demonstrate
much stronger production of CTGF mRNA by EGF or TGF-
than TGF-ß,
although the lack of response to TGF-ß was due, at least partly, to
defects in TGF-ß signaling (54). Phenomenologically, the lack of
response to TGF-ß in certain pancreatic cells is similar to that of
skin epithelial cells, which (unlike fibroblasts) do not synthesize
CTGF in response to local TGF-ß administration in vivo
(14). Dexamethasone treatment of cultured 3T3 cells was recently shown
to stimulate CTGF expression while actually down-regulating TGF-ß
mRNA (55). Systemic administration of dexamethasone stimulated CTGF
expression in the heart, kidney, and skin. Basal and
dexamethasone-stimulated CTGF expression was strongly attenuated by
tumor necrosis factor-
(TNF-
) (55), which was independently shown
to suppress basal CTGF mRNA production in bovine aortic endothelial
cells (BAECs), fibroblasts, and VSMCs (35). Finally, increased CTGF
expression in renal epithelial cells in response to injury occurred
without an increase in TGF-ß expression (46). These results show, not
surprisingly, that mechanisms of CTGF gene regulation may be
cell specific and, in addition to the action of TGF-ß, involve a
variety of hormones, growth factors, and cytokines.
D. CTGF production, secretion, and processing
CTGF is present in HUVEC-conditioned medium (13) and undergoes
microsomal processing (5), consistent with the presence of a signal
peptide and its presumed secretion. CTGF mRNA is expressed in BAECs in
which it is present at higher levels during growth than confluence
(43). Similarly, expression of fisp-12 in cycling
fibroblasts is relatively high (15). CTGF produced by human foreskin
fibroblasts or mouse connective tissue cells under normal growth
conditions appears to be cell associated, long lived, and secreted
relatively inefficiently (39). On the other hand, fisp-12
was secreted efficiently from NIH 3T3 cells but was relatively unstable
in the medium (5). Kireeva et al. (15) showed that
fisp-12 was present in the cellular fraction, ECM, and
medium and that inhibition of lysosomal acid hydrolysis increased the
half-life of fisp-12 in the cellular fraction and stabilized
steady-state fisp-12 levels. These data suggest that
membrane-associated fisp-12 may form complexes with
receptors on the surface of fisp-12-producing cells that are
then internalized and degraded (15).
Various low mass (1020 kDa) forms of pCTGF were purified from uterine
secretory fluids and shown to commence between modules 2 and 3, at the
beginning of module 3, or between modules 3 and 4 (10, 11) (Fig. 3
).
Native 38-kDa pCTGF undergoes rapid proteolytic processing by uterine
fluids (11) and likely explains the presence of 38-kDa CTGF in uterine
tissues (32) but not in uterine fluids (10, 11). Native low-mass CTGFs
in uterine fluids are extremely stable, and their levels are strongly
correlated with those of CTGF-degrading proteases (11). Although
conditioned medium from serum-stimulated mouse fibroblasts did not
degrade fisp-12 (15), 10- to 12-kDa CTGF, but not 38-kDa
CTGF, was present in conditioned medium from cycling human or mouse
fibroblasts (39). Recently, Western blot analysis demonstrated 12- to
14-kDa C-terminal forms of CTGF in mouse uterine luminal flushings (47)
and 18- and 24-kDa CTGF isoforms in human serum and amniotic,
follicular, peritoneal, and cerebrospinal fluids (40). The presence of
CTGF in sera has been confirmed by ELISA (51). Whereas serum CTGF
levels in normal human subjects were less than 28 ng/ml, those of
patients with biliary atresia were up to 8-fold higher (51). These
results highlight potential diagnostic or prognostic applications of
measuring circulating or secreted CTGF levels.
Collectively, these data suggest that qualitative and quantitative
aspects of CTGF secretion may be regulated by numerous factors
including stage of cell cycle, growth factor pretreatment, cell type,
species and tissue of origin, and protease activity of the pericellular
environment. Moreover, the presence of CTGF in a variety of body fluids
suggests that they may be important reservoirs of CTGF in
vivo.
E. CTGF-heparin interactions
Heparin-affinity chromatography has been used to purify 38-kDa
rhCTGF (14) and 10- to 20-kDa pCTGF (10, 11), the latter of which are
eluted from heparin by 0.8 M NaCl. 35S-labeled
fisp-12 binds to heparin-agarose beads and is eluted by 0.4
M NaCl (15). Although module 3 is a binding motif for
sulfated glycoconjugates (1), residues 247260, 274286, and 305328
of hCTGF bind strongly to heparin (10, 56). Residues 247260 contain a
proposed heparin-binding consensus sequence (XBBXBX) that occurs in a
variety of heparin-binding proteins (57).
As with other heparin-binding growth factors, such as FGFs,
heparin-binding EGF-like growth factor, amphiregulin, keratinocyte
growth factor, and vascular endothelial growth factor (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70), the
biological activity of CTGF is modulated by heparin (10, 14). The
binding of fisp-12 to heparin has been proposed as the basis
for the association of fisp-12 with the ECM in cultured
cells (15). Heparin-like molecules on the cell surface or in the ECM
may constitute a high-capacity, low-affinity binding reservoir for CTGF
and thereby regulate its activity, bioavailability, or stability.
Indeed, the half-life of fisp-12 in ECM (1 h) is much less
than that of cyr61 (4 h) and appears to correlate with the relative
affinity of each paralog for heparin (15, 71).
F. Biological properties of CTGF
Native 38-kDa hCTGF is a mitogenic and chemotactic factor for NIH
3T3 cells in vitro (13). Ten-kilodalton forms of pCTGF are
mitogenic for Balb/c 3T3 cells, VSMCs, and endometrial stromal cells,
but not endothelial cells (10, 56). The mitogenic activity of 38-kDa
rhCTGF or 10-kDa pCTGF on fibroblasts is enhanced by EGF, PDGF, basic
FGF (bFGF), or IGF-I and is either enhanced or reduced by heparin
according to the CTGF-heparin ratio (10, 14). In monolayer cultures of
normal rat kidney (NRK) cells, 38-kDa rhCTGF stimulated DNA synthesis
and induced expression of type 1 collagen, fibronectin, and
5
integrin (14), as is characteristic of the effects of TGF-ß. These
various biological effects are elicted by CTGF concentrations of about
120 ng/ml. In vivo, injection of TGF-ß or CTGF into the
dermal/subcuticular area of the skin in neonatal mice produced nodules
comprising mainly connective tissue cells and ECM (14, 72), suggesting
that CTGF plays a role in TGF-ß-mediated formation of granulation
tissue. Recombinant hCTGF is also mitogenic for human lung fibroblasts
(73). However, recombinant 38 kDa fisp-12 did not directly
stimulate DNA synthesis in HUVECs or 3T3 cells at 0.33 µg/ml (15),
although other evidence suggests that endothelial cells do not respond
mitogenically to 10-kDa CTGF, which is nonetheless mitogenic for 3T3
cells and VSMCs (10). At 0.33 µg/ml, the same fisp-12
protein enhanced the mitogenic activity of 10 ng/ml bFGF on HUVECs
and NIH 3T3 cells and promoted attachment of HUVECs, NIH 3T3 cells,
AKR2B cells, and mink lung epithelial cells at 520 µg/ml (15).
These latter effects occurred at 100- to 1000-fold higher
concentrations than were needed for the stimulation of mitosis,
chemotaxis, or ECM production by CTGF in other studies (10, 13, 14).
Nonetheless, a role for CTGF in cell adhesion is further supported by
the localization of CTGF in ECM (15) and by the ability of antisense
CTGF to suppress growth and migration of BAECs in vitro
(43). While 38-kDa CTGF appears to be proteolytically cleaved into low
mass derivatives (11), the relationship (if any) between processing and
activity remains undefined.
Although TGF-ß induces CTGF expression and CTGF mimics some of the
effects of TGF-ß in fibroblasts, CTGF-independent pathways of TGF-ß
action have been identified in these and other cell types. For example,
TGF-ß is a potent inhibitor of the growth of mink lung epithelial
cells, whereas CTGF is not (14). In addition, while CTGF and TGF-ß
both stimulate DNA synthesis in monolayer cultures of NRK cells, only
TGF-ß is able to stimulate anchorage-independent growth (AIG) of the
same cells (14, 74). AIG induced by TGF-ß is antagonized by CTGF
antibodies or antisense CTGF and is restored by addition of CTGF to
TGF-ß-stimulated cells showing that CTGF is involved in the
stimulation by TGF-ß of this process (74). Since CTGF was induced by
TGF-ß in these cells, it was proposed that both CTGF-dependent and
-independent pathways are involved in the stimulation of AIG by TGF-ß
(74). The induction of CTGF mRNA and AIG by TGF-ß in NRK cells is
inhibited by elevated cAMP levels (52). This effect was reversed by
addition of CTGF, but not other growth factors, and was attributed to a
CTGF-specific restriction point in late G1 of
TGF-ß-activated cells (52).
G. Mechanism of action of CTGF
In addition to contributing to TGF-ß-mediated AIG (see above),
CTGF interacts synergistically with EGF, PDGF, IGF-I, or bFGF (10, 14, 56), suggesting that it activates distinct receptors and/or signaling
pathways to those used by other growth factors. Although 38-kDa hCTGF
was suggested to bind PDGF receptors (13, 75), this possibility was not
supported using a mitogenic form of 10-kDa murine CTGF (39).
Binding studies with 125I-labeled 38-kDa rhCTGF have
demonstrated the presence of specific high- and low-affinity binding
sites and a 280-kDa cross-linked CTGF complex (76). Although there was
no clear evidence for a signal-transducing receptor, these data are
nonetheless potentially very exciting. Alternatively, CTGF may function
primarily as a cell adhesion molecule that regulates cell function
either directly through its association with the ECM or indirectly by
synergizing with other growth factors (15).
 |
IV. Cyr61
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A. Discovery of cyr61
Cyr61 (originally termed 3CH61) was described in 1985 as an
immediate early gene in mouse Balb/c 3T3 cells that was induced by
serum or PDGF (77). It was named cyr61 when shown to encode a
cysteine-rich protein that contained 10% cysteine residues (78). The
same gene, named ßIG-M1, was also discovered by Brunner et
al. (31) in their studies of TGF-ß-inducible immediate early
genes in mouse AKR-2B cells. The gene for cef-10, the chicken ortholog
of cyr61, was described in 1989 as pp60v-src-inducible
immediate early gene in chicken embryo fibroblasts (CEFs) (79). cDNA
for the human ortholog of cyr61 (hcyr61) was isolated from a 6-week-old
human embryonic tissue cDNA library (80).
B. Structure of the cyr61 gene and protein
The murine cyr61 gene comprises five exons and four introns that
span 3.1 kb (Table 2
) and contains a serum-response element (SRE) (Fig. 4
) in the 5'-region that mediates serum- or PDGF-induced gene
transcription (6). Induction of cyr61 is similar to that of other
immediate early genes that contain SREs, consistent with its
coordinated expression with c-fos during G0 to
G1 (81), although its transcriptional suppression is
relatively inefficient (6). Although cyr61 expression is induced by
TGF-ß (31), its promoter lacks the novel TGF-ß response element
found in the promoter of CTGF (36) (Fig. 3
). Human cyr61 maps to p22.3
on chromosome 1 (80, 82). The mouse cyr61 gene encodes a 379-residue
protein, which, after signal peptide cleavage, yields a 355-residue
protein containing 38 conserved cysteine residues (31, 78) (Fig. 1
).
The cysteine-free portion of cyr61 is about twice the length of that in
other CCN paralogs (Fig. 1
).
C. Cyr61 mRNA production
A single cyr61 transcript of 2.4 kb has been reported in mouse
AKR2B cells, 3T3 fibroblasts, and PSA-1 teratocarcinoma cells (31, 77, 81, 83) and rat H197 embryonic neuronal hippocampal cells (84). A
1.8-kb cef-10 transcript is present in CEFs (79), and two cyr61
transcripts of 2.5 kb and 3.54 kb have been described in human
tissues (80, 82). Although cef-10 mRNA is expressed principally in the
adult lung (79), cyr61 is expressed in multiple adult tissues of the
mouse and human such as heart, uterus, skeletal muscle, and lung (49, 78, 80). Transcript levels in other tissues are variable and likely due
to differences in mRNA detection techniques as well as in the stage of
development or differentiation of the source material. For example,
human cyr61 mRNA expression is higher in the kidney of the fetus than
that of the adult (80). Cyr61 mRNA is present at high levels in mouse
embryos on days 9.514.5, whereas placental expression of cyr61 is
highest on days 17.518.5 (83).
Cyr61 gene expression is stimulated by diverse molecular signals, which
supports its role in a variety of biological processes. In mouse cells,
cyr61 mRNA is rapidly induced by serum, PDGF, FGF, TGF-ß, phorbol
ester, or cholera toxin (31, 77, 78, 81). Serum stimulation of Balb/c
3T3 cells resulted in a rapid increase and sustained high levels of
cyr61 mRNA, which were stabilized by cyclohexamide (78). Similarly,
human cyr61 mRNA is present in proliferating, but not quiescent, skin
fibroblasts (49). In CEFs, transcription of cef-10 is rapidly enhanced
by pp60v-src or serum (79). In human osteoblasts, cyr61
expression is enhanced by EGF, PDGF, and interleukins-1ß, -2, and -6,
as well as by 1
,25-dihydroxyvitamin D3, a mediator of
osteoblast differentiation and function (85). In the rat uterus, cyr61
is estrogen-responsive and strongly induced by tamoxifen (86). In rat
H197 cells, cyr61 is induced within 1 h by bFGF, a stimulator of
neuronal differentiation in this cell type (84). This process appears
to involve both mitogen-activated protein kinase-dependent and
-independent pathways (84).
D. Cyr61 production and secretion
The 41-kDa mouse cyr61 protein is present at very low amounts in
quiescent Balb/c 3T3 cells but reaches peak levels within 2 h of
serum stimulation (78). Cyr61 is differentially distributed in the ECM,
cellular fraction, or cell surface rather than the medium (71, 78). The
extracellular fate and half-life of cyr61 synthesized in response to
mitogenic stimulation were shown to be dependent upon the stage of the
cell cycle and presence of binding moieties in the ECM and on cell
surfaces such as heparin-like molecules from which cyr61 is eluted by
0.60.8 M NaCl (16, 49, 71).
E. Biological properties of cyr61
Recombinant murine cyr61 promotes dose-dependent attachment of
HUVECs or NIH 3T3 cells to plastic surfaces coated with 330 µg/ml
of the protein (16). Similar concentrations of cyr61 also stimulate
dose-dependent chemotaxis of NIH 3T3 cells (16) and human microvascular
endothelial cells (HMVEC) (87). These concentrations of cyr61 were not
mitogenic for NIH 3T3 cells or HUVECs, although they did potentiate the
mitogenic activity of bFGF for these cell types (16, 49). This latter
effect was abolished by cyr61 antiserum and attributed to the
displacement by cyr61 of bFGF from the ECM, thus increasing the
effective concentration of soluble bFGF (16, 49). In mouse limb bud
micromass cultures, cyr61 promoted expression of type II collagen,
incorporation of sulfate, and the production of cartilage nodules (88).
Moreover, cyr61 promoted the adhesion and aggregation of limb bud
mesenchymal cells. Cyr61 was also a weak stimulator of DNA synthesis
and proliferation of limb mesenchymal cells (88). Cyr61 was further
shown to promote chondrogenesis in cell cultures seeded at subthreshold
densities that do not normally undergo chondrogenic differentiation.
These various responses were elicted with 0.35 µg/ml cyr61. Limb
bud micromass cultures showed reduced levels of chondrogenesis when
incubated with neutralizing cyr61 antisera, suggesting that endogenous
cyr61 plays a normal physiological role in chrondrogenic
differentiation (88). Finally, cyr61 has been shown to induce
neovascularization in vivo (87), consistent with its
stimulation of directed migration of HMVECs in vitro (87).
Stable transfection of cyr61 cDNA into RF-1 gastric adenocarcinoma
cells had no effect on in vitro growth rate, yet produced
larger tumors in immunocompromized mouse hosts as compared with a
vector control, a phenomenon that was attributed to cyr61-stimulated
tumor vascularization (87).
F. Mechanism of action of cyr61
A mechanistic framework for the biological properties of cyr61 has
been provided by the finding that cyr61 binds to integrin
vß3, which represents the first (and only)
molecularly defined receptor for any member of the CCN family (89). The
interaction of
vß3 with cyr61 may account
for its promotion of chemotaxis and growth factor-mediated DNA
synthesis as well as cell adhesion since integrins are known to
modulate cell migration and growth factor signaling in other systems
(90). As well as demonstrating a direct interaction between the two
molecules in binding assays in vitro (89), cyr61-mediated
adhesion and migration of cultured endothelial cells were specifically
inhibited by the peptide RGDS and/or antiintegrin
vß3 (87, 89). In addition to its
integrin-binding property, cyr61 appears to be localized to its site of
synthesis by associating with the ECM, possibly by binding to
heparin-like molecules. This interaction may limit the extent of cyr61
diffusion so that its site of action is in close proximity to its site
of synthesis (15).
 |
V. Nov
|
|---|
A. Discovery and structure of nov
Nov (nephroblastoma overexpressed) was first recognized as an
overexpressed gene in nephroblastomas induced by
myeloblastosis-associated virus type 1 (MAV-1) in day-old chicks (7, 91). Subsequently, the human (novH), mouse nov (novM), quail (novQ),
and Xenopus (xnov) orthologs of chicken nov (novC) were
isolated by hybridization screening (8, 33, 38, 92). The nov gene
demonstrates a well conserved intron/exon structure that is similar to
that of other paralogs in the CCN family (Table 2
). In one chicken
nephroblastoma, MAV-1 envelope and long-terminal repeat sequences
integrated into the second exon of the novC gene at Cys63
(7); the first putative initiation codon downstream of this integration
site is Met99. The novH gene maps to chromosome 8q24.1 and
appears to contain at least three transcription initiation sites in the
promoter region (38). NovM maps to chromosome 15 between D15
Mit153 and D15 Mit 183 (92, 93).
Putative transciption factor-binding sites are present in the
5'-untranslated region of several nov genes (8, 33, 38, 92)
(Fig. 4
). However, SREs and the TGF-ß response element are absent,
suggesting that transcriptional regulation of nov is distinct from that
of CTGF or cyr61. The activity of the novH promoter is repressed in the
presence of the Wilms tumor suppressor gene WT-1 (94). While WT-1
binding sites are present in the novH promoter (Fig. 4
), WT-1-mediated
repression of novH transcription was experimentally attributed to other
domains in the promoter (94).
The primary translational products of nov orthologs are predicted to
comprise between 343 and 357 amino acids and to contain 38 conserved
cysteine residues (Fig. 1
). The signal peptide in novH appears to
comprise 27 residues (8). In CEFs, an immunoreactive 46-kDa novC
protein was localized to the ECM (93), although the underlying
mechanisms and functional significance remain unclear. NovH produced in
transfected Madin Darby canine kidney cells is a glycosylated protein
that is secreted about 1.5 h after synthesis and requires 0.5
M NaCl for elution from heparin (20). Secreted novH has a
half-life of more than 18 h, which is more than 10 times that of
intracellular novH (20). Levels of the endogenous 46-kDa novH protein
are repressed in 293 kidney cells that constitutively express WT1 (94).
B. Nov mRNA production
A single 2.2-kb novC transcript is usually detected in
MAV1-induced nephroblastomas (7). An exception was a tumor in which
MAV1 integrated within the novC gene itself, resulting in the
production of a 2.0-kb transcript (7). The 2.2-kb novC mRNA
transcript was detected at high levels in the brain and heart of
day 18 chick embryos, with weak signals in muscle and intestine and
nondetectable levels in liver, lung, and yolk sac. In the adult, 2.2-kb
novC mRNA was expressed at high levels in brain and lung, at low levels
in the spleen, and was nondetectable in heart, muscle, and liver (7).
Quiescent cultures of CEFs contain a 2.2-kb novC transcript that is
down-regulated during proliferation, mitogenic stimulation, or
activation of viral oncogenes (95). NovH is normally expressed as a
2.5-kb transcript in human glioma cell lines and Wilms tumors, although
an additional novH mRNA of 1.2 kb was observed in a single Wilms tumor
(8, 37). Unlike novC, this smaller transcript was not the result of
rearrangement of the novH gene (8).
There is little, if any, experimental evidence supporting a role for
nov as an immediate early gene. As explained above, there are no motifs
in the nov gene promoter that resemble either a SRE or a TGF-ß
response element. Serum stimulation of kidney-derived 293 cells or CEFs
failed to increase nov mRNA levels within 90 min, even though the genes
for CTGF and jun were rapidly activated (93). Moreover, in
normal CEFs, nov was expressed in quiescent cells but down-regulated in
proliferating cells (95). In contrast to cef-10, expression of
p60v-src in CEFs resulted in transcriptional
down-regulation of the novC gene (95). NovC expression was also
repressed by the viral oncogenes v-erbB, v-mil, and v-crk (95). In
addition, mRNA destabilization sequences in the 3'-region are absent
from most nov orthologs (except xnov), suggesting that nov RNA
degradation is regulated differently from that of CTGF, cyr61, and
other immediate early genes.
C. Biological properties of nov
Overexpression of full-length novC cDNA was shown to inhibit the
growth of CEFs grown in soft agar (7). In contrast, transformation of
CEFs occurred after overexpression of an N-terminally truncated form of
novC that initiated at Met99 and was thus comparable to
that expressed after integration of MAV-1 into the novC gene (Fig. 3
)
(7). This truncated form of nov also induced morphological
transformation of rat embryo fibroblasts (8). Within the CCN family,
the ability to function as a protooncogene is thus far unique to nov.
The dramatic biological differences between the normal and truncated
nov genes have led to suggestions that nov is a growth suppressor gene,
truncation of which results in oncogenic activation. These
interpretations are consistent with the low levels of nov mRNA in
mitogen-stimulated cells and the high levels of nov mRNA in resting
cells (93, 95). Although the truncated nov protein lacked the signal
peptide and the IGF-binding motif in module 1, it is not known how the
loss of one or both of these domains contributes (if at all) to its
transforming properties.
 |
VI. Elm1/WISP-1
|
|---|
Elm1 (expressed in low-metastatic type 1 cells) was initially
identified by mRNA differential display as being expressed in low, but
not in high, metastatic murine melanoma cells (96). The elm1 protein
was subsequently predicted to comprise 367 amino acids and to conform
to the CCN modular structure (97) (Figs. 1
and 3
; Table 1
). Elm1
transcripts of 1.8 and 5.0 kb were present in all mouse tissues
analyzed (97). The 5.0-kb elm1 transcript was induced within 3 h
of serum stimulation of quiescent Balb/c 3T3 cells, unlike cyr61 which
was induced within 30 min in the same cells (97). Elm1 maps to mouse
chromosome 15 between D15 Mit17 and D15 Mit3.
Southern blotting demonstrated that the elm1 gene is also present in
human, monkey, rat, mouse, dog, and cow (97). Transfection of elm1 into
highly tumorigenic and metastatic K-1735 M-2 cells resulted in a
decreased tumor growth in vivo and reduction in the
frequency of lung metastatic colonies (97). In vitro,
transfected cells that expressed relatively high amounts of elm1 grew
somewhat slower and to lower saturation density as compared with
control cells (97). Elm1 did not exhibit mitogenic activity for NIH3T3
cells, either alone or in combination with bFGF. The growth-suppressive
properties of elm1 have been tentatively linked to module 3 (97).
Recently, WISP-1 was identified as the human ortholog of elm1 and shown
to be a component of the Wnt-signaling pathway in transformed cells
(24). WISP-1 maps to human chromosome 8q24.18q24.3 and exhibits
tissue-specific patterns of expression (24).
 |
VII. Heparin-Induced CCN-Like Protein
(HICP)/rCop-1/CTGF-3/WISP-2
|
|---|
A novel 1.8-kb mRNA was identified by subtractive hybridization
screening of heparin-treated and nontreated rat VSMCs and shown to
encode a novel member of the CCN family that was termed HICP (98).
Independent studies resulted in the isolation of cDNA for the same
gene, termed rCop-1, when shown to be expressed in normal rat embryo
fibroblasts or mouse 3T3 cells, but not in their transformed
derivatives (99). The mouse ortholog, WISP-2, was recently identified
as a unique gene that was up-regulated in Wnt-1-transformed mouse
mammary epithelial cells in vitro (24). These molecules,
together with the human ortholog, CTGF-3 (100), demonstrate an overall
amino acid homology of
30% with hCTGF and conservation of all of
the cysteine residues in modules 13 (Fig. 1
). However, all of these
proteins are C-terminally truncated as compared with other CCN
paralogs, resulting in a total absence of module 4 (Figs. 1
and 3
).
While HICP/CTGF-3/WISP-2 transcripts were present in multiple tissues
(24, 98, 100), rCop-1 mRNA was not detected in any of the major organs
of the mouse embryo and adult rat (99). Although this discrepancy
requires further investigation, expression of rCop-1 has been linked to
aging and senescence (99). HICP expression patterns, similar to those
of growth arrest genes, occur at high levels in quiescent or
heparin-arrested VSMCs and at very low levels during
proliferation (98). Somewhat different expression patterns occur
for rCop-1 in cycling mouse 3T3 cells in which rCop-1 mRNA is absent
during quiescence or immediately after serum stimulation and appears
only during late S phase (99). Unlike normal cells, transfection of
rCop-1 into transformed cells suppressed their cell growth; this was
attributed to cell death rather than growth arrest (99). In transfected
cells, rCop-1 is localized primarily intracellularly and to the cell
surface, but not in conditioned medium or ECM. It has been speculated
(99) that the unique subcellular localization and expression pattern of
rCop-1/HICP is a reflection of its role in negative growth regulation,
the structural basis of which may lie in the absence of module 4.
Potential tumor-suppressive properties are further suggested by the
finding that WISP-2 is underexpressed in human colon tumors, unlike
WISP-1 and -3, which are overexpressed (24).
 |
VIII. WISP-3
|
|---|
Human WISP-3, a 354-residue protein containing 36 cysteine
residues, was identified by screening expressed sequence tag databases
with WISP-1 (24). The secreted WISP-3 protein is predicted to contain
34 of the 38 conserved cysteine residues found in most other CCN
proteins since it lacks 4 of the cysteines normally present in module
2. It is possible that this structural difference results in unique
properties and functions, although this aspect of WISP-3 biology has
not yet been explored (24).
 |
IX. Other CCN-Like Molecules
|
|---|
Twisted gastrulation (tsg) and short gastrulation (sog), which are
involved in dorsoventral patterning in Drosophila embryos
(101, 102), contain sequences that resemble, respectively, modules 1
and 2. As with CTGF and cyr61, tsg is eluted from heparin by 0.60.8
M NaCl, and the binding of tsg by somatic cells may involve
a heparin-like coreceptor (103). The cytoskeletal rearrangements that
take place in dorsal amnioserosa cells in the presence of tsg have been
likened to those in endothelial cells during CTGF-induced chemotaxis
(101). Sog, like CTGF, may be susceptible to proteolytic cleavage,
yielding diffusible bioactive fragments (102). The cDNA of "small
CCN-like growth factor" (SCGF) was recently isolated from an 9-week
human embryo library and shown to encode a 206-residue protein that had
limited structural similarity to the C-terminal region of CCN family
members, although cysteine residues occur at half the expected
frequency and are poorly conserved (104). Overall, tsg, sog, and SCGF
exhibit very weak alignment with the CCN family and are not genuine
paralogs.
 |
X. Regulation of Cellular Functions by the CCN Family
|
|---|
A. Cell cycle control
Members of the CCN family are products either of immediate early
genes (CTGF, cyr61) or of putative growth arrest/suppression genes
(nov, elm1, HICP). The expression of immediate early genes represents
the earliest genomic response to growth factors and is likely to
initiate the program leading to cell replication. Since they encode a
diverse array of regulatory molecules, the induction of immediate early
genes represents a central component in the proliferative response to
mitogenic stimuli (105). Since CTGF and cyr61 are transcriptionally
activated by TGF-ß, PDGF, EGF, FGF, TPA, and cholera toxin, it is
likely that the cellular responses to these diverse mitogenic stimuli
are controlled, at least partly, by CTGF and cyr61. On the other hand,
the expression of growth arrest-specific genes, DNA damage-inducible
genes, and the MyD growth-arrest genes are associated with the negative
regulation of cell growth (106). Growth arrest genes have been
implicated in cellular differentiation, embryonic development, and
apoptosis (107, 108, 109), and it is possible that these types of processes
are similarly regulated by nov, elm1, and rCop-1.
In addition, each CCN family member appears to further regulate the
cell cycle through their 1) direct mitogenic action (e.g.,
CTGF); 2) potentiation of the mitogenic activity of other growth
factors (e.g., CTGF, cyr61); 3) binding of IGFs
(e.g., CTGF); 4) regulation of ECM synthesis
(e.g., CTGF, cyr61); 5) regulation of cell attachment and
migration (e.g., CTGF, cyr61, elm1); 6) interactions with
the ECM (e.g., CTGF, cyr61, nov); 7) oncogenic properties
(e.g., nov); and 8) regulation of a cell cycle restriction
point (e.g., CTGF). This broad spectrum of biological
properties, many of which are interacting and interdependent,
demonstrate the complexity by which the cell cycle is likely regulated
by the CCN family.
B. Cell adhesion and migration
In vitro studies have shown that CTGF or cyr61 are
chemotactic and promote cell adhesion (13, 15, 16, 87). Elm1 is
expressed in low, but not high, metastatic cell lines and, when
transfected into high-metastatic cell lines, causes them to exhibit
slower rates of tumor growth and decreased incidence of metastasis
(97). Truncated nov stimulates AIG and colony formation in CEFs (7).
Collectively, these data demonstrate broad effects on cell adhesion and
locomotion, which likely reflects the ability of CCN family members to
regulate the composition of the ECM, the net balance between its
synthesis and degradation, as well as to bind directly to ECM
components such as integrins and heparan sulfate proteoglycans (see
below).
C. ECM production
Tissue formation and cell proliferation and differentiation are
dependent upon interactions between cells and the ECM. In processes
such as tissue remodeling and malignancy, ECM components are degraded
by proteases such as collagenase, plasmin, and matrix metalloproteases,
and the ability of cells to interact with the ECM is altered or
abolished. TGF-ß plays a central role in ECM production since it
stimulates synthesis of ECM proteins such as collagen, fibronectin,
laminin, elastin, glycosaminoglycans, and other glycoproteins as well
as synthesis of inhibitors of ECM degradation such as plasminogen
activator inhibitor and tissue inhibitors of metalloproteases (110). In
view of the TGF-ß-inducibility of CTGF and cyr61 gene expression,
these molecules may promote ECM deposition by stimulating synthesis of
ECM components and inhibiting their degradation. While detailed studies
of the regulation of ECM-degrading enzymes and their inhibitors by the
CCN family have yet to be reported, initial studies have shown that
38-kDa CTGF enhances fibronectin, type IV collagen, and integrin
production (14) and that cyr61 stimulates type II collagen production,
albeit in a specialized cell type (88). As discussed below, numerous
fibrotic disorders exhibit substantial ECM involvement and expression
of CTGF. In addition, there is a striking appearance of CTGF in uterine
stromal cells that are undergoing decidualization (47), a highly
regulated process involving increased mitosis and synthesis and
deposition of ECM molecules such as desmin, laminin, and fibronectin
(111). ECM production and remodeling is also a critical component of
development and differentiation during embryogenesis (112).
Collectively, these data suggest that CTGF may exert diverse and
important functions in normal physiology and pathological states via
its net induction of ECM protein deposition and content.
 |
XI. Biological Processes Involving the CCN Family
|
|---|
A. Development and differentiation
Immunohistochemical studies showed that CTGF is present in the
mouse as early as embryonic days 4.56.5, at which time it is most
abundant in the embryonic endoderm and mesoderm (47). At later stages
of gestation (days 1418), CTGF and cyr61 are both present in various
tissues and organs including the cardiovascular and pulmonary systems
as well as the skin and placenta (15). Secretory structures such as
kidney tubules and salivary, mucous, and sebaceous glands are positive
for CTGF, but not cyr61, whereas the nervous and skeletal systems are
positive for cyr61, but not CTGF (15). This differential distribution
may be indicative of unique roles for each factor. A role for nov
during Xenopus development is suggested by the production of
its mRNA in the oocyte at stages I, III, and IV and in the embryo at
all developmental stages between egg and tadpole (33). In chickens,
novC is expressed in kidney, heart, and muscle of the embryo but not of
the adult (7). Other organs (e.g., brain) demonstrate novC
expression in both embryonic and adult life, whereas expression of novC
in the lung occurs in the adult but not the embryo (7).
Evidence to date supports a role for cyr61 and CTGF in chondrogenesis.
The cyr61 protein is present in cartilage and bone in day 1318 mouse
embryos (15, 88), and cyr61 mRNA is present in a variety of developing
cartilaginous structures in day 8.514.5 embryos (83). A role for
cyr61 in normal growth and development of the cartilaginous skeleton is
further supported by the ability of cyr61 to stimulate collagen
synthesis, sulfate incorporation, and formation of cartilage nodules in
mouse limb bud micromass cultures, and adhesion, aggregation, and
growth of limb bud mesenchymal cells (88). In the case of CTGF, its
mRNA is present in hypertrophic chondroctyes from day 17 mouse embryos,
neonatal rabbit growth cartilage tissue and cultured cells, and human
chondrocytic cell lines, but not in human osteosarcoma or mouse
osteocytic cell lines (48). In cultured rabbit growth cartilage cells,
peak levels of CTGF occurred during the early hypertrophic stage. CTGF
mRNA levels in these cells were enhanced by treatment with TGF-ß or
bone morphogenic protein-2 (48). In addition to its potential role in
chondrogenesis, cyr61 may also be involved in hippocampal
differentiation, since it is induced in H197 cells by bFGF, which
stimulates their differentiation into nonproliferative neuronal cells
(84).
Several lines of evidence support a role for nov nephrogenesis. First,
novC expression occurs in normal avian kidney cells of the embryo and
neonate, yet is barely detectable in adult kidney cells (7). Second,
ureteric buds, which give rise to the kidney collecting ducts, fail to
demonstrate outgrowth in WT-1-null mice; as discussed above, novH
promoter activity is repressed by WT-1, and a reciprocal quantitative
gradient of novH and WT1 expression occurs during normal human
nephrogenesis (see Ref. 94). Third, novH is localized to
differentiating glomerular podocytes where it accumulates and may
regulate podocyte structure or function during both pre- and postnatal
life (20). Since the composition of the ECM changes during the earliest
stages of metanephric induction (113) and ECM components regulate
uteric bud morphogenesis, nov may also influence nephrogenesis by
binding to the ECM (93).
B. Female reproductive tract function
The actions of ovarian steroids are mediated, in part, by
polypeptide growth factors, which may also contribute to the
uterine-embryo signaling dialogue that initiates implantation and
stimulates embryonic development (114, 115, 116, 117, 118, 119, 120). Also, some growth factors
are secreted into the uterine lumen or localized at the implantation
site, where they may stimulate development of the embryo and placental
membranes (114, 121). CTGF is present in uterine fluids of the pig and
mouse, as well the uterus of the pig, mouse and human (10, 11, 13, 47, 56, 122) suggesting that it is involved in the regulation of uterine
function. In uterine fluids, the levels of low mass CTGFs and CTGF
proteases are highly correlated with each other and show cyclic
variations during the estrous cycle (11). Moreover, there are clear
differences in the levels of CTGF and CTGF protease(s) in uterine
fluids at equivalent stages of the estrous cycle and early pregnancy
suggesting that CTGF production or action is modified in the presence
of the embryo (11, 56). A relationship between CTGF expression and
TGF-ß action during the periimplantation period of the pig is
supported by the presence of both molecules at this time in uterine
and/or embryonic tissues (11, 123, 124, 125). In addition, a direct role of
CTGF on uterine cells is indicated by its stimulation of DNA synthesis
in pig stromal cells in vitro (56).
In mice and women, CTGF is localized primarily to uterine luminal and
glandular epithelial cells during the estrous cycle and during the
first few days of pregnancy (47, 122). On the day of implantation in
mice, epithelial staining for CTGF is strongly reduced and is followed
over the next 2 days by profound staining of decidualizing endometrial
stromal cells, suggesting that CTGF contributes to the decidualization
process or is produced as a result of it (47). Decidualization is a
highly regulated differentiation process involving increased vascular
permeability, DNA synthesis, and synthesis and deposition of ECM
molecules (111, 126, 127, 128). The distribution of uterine CTGF during
early pregnancy in the mouse does not entirely correlate with TGF-ß
and its receptors, suggesting that TGF-ß-independent synthesis of
CTGF may be operative (47). Alternative mechanisms may involve
glucocorticoids or estrogen, which stimulate CTGF and cyr61 gene
transcription, respectively (55, 86). The nonconcordance of CTGF and
TGF-ß may also be related to the initial production of TGF-ß in a
latent form (129). Whatever the explanation, these data support a role
for uterine CTGF at the time of implantation.
C. Angiogenesis
Angiogenesis, the formation of new capillaries from preexisting
blood vessels, occurs in processes such as tumor growth, diabetic
retinopathy, wound healing, and placental vascularization. It is a
complex process that involves degradation of the capillary basement
membrane, migration and proliferation of endothelial cells, and tube
formation; it is regulated by many factors (130), including cyr61,
which promotes directed migration of HMVECs via
vß3-integrin and induces
neovascularization in the rat cornea (87). Expression patterns of cyr61
support its contribution to angiogenesis in the embryo, placenta,
hypertrophic cartilage, wounds, and tumors (83, 87). Since cell
migration is the only component of angiogenesis to be directly
stimulated by cyr61, indirect effects of cyr61, such as release and
action of the angiogenic factor bFGF from ECM (16, 49), may contribute
to the overall process of neovascularization. These questions and the
role of other CCN family members in angiogenesis will require further
study.
D. Wound repair
Many studies have established that growth factors, including
TGF-ß, are involved in wound healing (see Ref. 131). Collectively,
CTGF and cyr61 exhibit numerous biological properties that are of
potential importance in the wound healing response, including
stimulation of cell proliferation, cell adhesion, chemotaxis,
angioge