From the Departments of Bone and Cartilage Biology,
¶ Gene Expression Sciences, and
Protein Biochemistry,
SmithKline Beecham Pharmaceuticals,
King of Prussia, Pennsylvania 19406
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have identified and cloned a novel connective
tissue growth factor-like (CTGF-L) cDNA from primary human
osteoblast cells encoding a 250-amino acid single chain polypeptide.
Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was
obtained from a murine lung cDNA library. CTGF-L protein bears
significant identity (~60%) to the CCN (CTGF, Cef10/Cyr61, Nov)
family of proteins. CTGF-L is composed of three distinct domains, an
insulin-like growth factor binding domain, a von Willebrand Factor type
C motif, and a thrombospondin type I repeat. However, unlike CTGF,
CTGF-L lacks the C-terminal domain implicated in dimerization and
heparin binding. CTGF-L mRNA (~1.3 kilobases) is expressed in
primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a
~26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high
expression in osteoblasts forming bone, discrete alkaline phosphatase
positive bone marrow cells, and chondrocytes. Specific binding of
125I-labeled insulin-like growth factors to CTGF-L was
demonstrated by ligand Western blotting and cross-linking experiments.
Recombinant human CTGF-L promotes the adhesion of osteoblast cells and
inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest
that CTGF-L may play an important role in modulating bone turnover.
Connective tissue growth factor
(CTGF)1 was originally
identified as a major chemotactic and mitogenic factor from endothelial cells (1). CTGF is distinct from, but immunologically related to,
platelet-derived growth factor and competes for binding to the
platelet-derived growth factor receptor (1). In fibroblasts, transforming growth factor Members of CCN family are cysteine-rich proteins that are organized
into four distinct motifs (9). The first motif contains an insulin-like
growth factor (IGF) binding domain (GCGCCXXC) common to all
seven known IGF-binding proteins (IGFBPs). The second domain
contains a von Willebrand factor type C (VWC) module that is suspected
to be involved in oligomerization. The third is a thrombospondin type I
repeat (TSP1) that is thought to play a role in cell attachment and
binding to matrix proteins and sulfated glycoconjugates (9). The
fourth, a C-terminal (CT) domain, has been implicated in heparin
binding and dimerization (10). The CT domain (~10 kDa) of CTGF is
present in biological fluid and is sufficient for some biological
activities (10).
Osteoblasts are specialized mesenchymal cells that are responsible for
synthesizing and secreting the complex mixture of collagenous and
noncollagenous proteins that make up bone matrix. These cells are also
responsible for subsequent mineralization of this matrix (11). During
the process of bone formation and remodeling, there is an integrated
process of osteoclast-mediated bone resorption and osteoblast-derived
bone formation (11, 12). The process of bone formation and remodeling
is under tight regulation by numerous factors, including endocrine
hormones, cytokines, growth factors, adhesion molecules, and
extracellular matrix components.
In the present report, we describe the identification, cloning,
expression, and functional characterization of a novel CTGF-like (CTGF-L) cDNA from primary human osteoblast cells. CTGF-L contains the first three domains present in CCN family members but lacks the
fourth CT domain. We show that CTGF-L is expressed at high levels in
human bone tissue. Recombinant hCTGF-L protein binds to IGFs and
promotes adhesion of osteoblast cells. In addition, rhCTGF-L inhibits
the binding of fibrinogen to integrin receptors and inhibits
osteocalcin production from rat osteoblast-like cells.
Cells and Cell Culture--
Primary osteoblasts were grown from
explants of human trabecular bone fragments from knee joints taken at
surgery (kindly provided by the Rothman Institute, Pennsylvania
Hospital, Philadelphia, PA). The osteoblasts were cultured in Eagle's
modified minimum essential medium supplemented with 10% fetal calf
serum (Hyclone, Logan, UT), 2 mM L-glutamine,
and antibiotics for 2-3 weeks as described previously (13, 14). Cells
from up to three passages were used for all experiments. Primary human
fibroblasts, human osteosarcoma MG 63 and SaOS-2, HeLa, and human
mesenglial cells were obtained from ATCC (Manassas, VA). Human stromal
TF274 cells have been described previously (15).
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from primary human osteoblasts and different cell lines using
Trizol reagent (Life Technologies, Inc.) according to the
manufacturer's recommendation. RNA was fractionated by electrophoresis
on 1.2% agarose-formaldehyde gels, transferred to Genscreen plus
membranes, and cross-linked using an UV Stratalinker-180 (Stratagene,
La Jolla, CA). The blots were probed with 32P-labeled
CTGF-L cDNA and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) according to standard procedures (16). A
commercial human multiple tissue Northern blot
(CLONTECH, Palo Alto, CA) containing 2 µg of
poly(A)+ RNA from various tissues was processed according
to the manufacturer's instructions.
Cloning of CTGF-L cDNAs and Expression of Recombinant
Protein--
Human CTGF-L cDNA was identified by expressed
sequenced tag (EST) analysis (17) of a cDNA library derived from
primary human osteoblasts. After sequencing, the clone HOEBG39 was
found to contain the entire open reading frame of hCTGF-L. An EST
(AA754979) from a murine lung library, with high homology to the human
CTGF-L cDNA, was identified from the GenBankTM data
base. This EST contained the 3'-untranslated region and encoded about
35 amino acids from the C terminus of CTGF-L protein. Based on the most
5' sequence of this EST, primers were designed, and the full-length
cDNA was isolated using the 5' rapid amplification of cDNA ends
from a marathon cDNA library prepared from murine lung
(CLONTECH). For recombinant protein expression, the
coding region of hCTGF-L was subcloned into CDN vector, where the
expression of hCTGF-L was driven by the cytomegalovirus promoter (18). Two epitope tags, an N-terminal human immunodeficiency virus GP120 V3
(SKSIRIQRGPGR) and His6, were inserted after the signal
sequence, and an enterokinase cleavage site was engineered between the
epitope tags and CTGF-L protein. The plasmid was transfected into
Chinese hamster ovary cells by electroporation, and cells expressing
hCTGF-L were bulk-selected in nuceloside-free medium. Conditioned
medium from a large-scale culture of Chinese hamster ovary-hCTGF-L
cells was used to purify rhCTGF-L using a TALON metal affinity column (CLONTECH). Poor expression of soluble secreted
CTGF-L was observed possibly due to the high number of cysteine
residues (11% of total protein), and hence only small amounts of
purified recombinant protein were obtained. The expression and
authenticity of rhCTGF-L was confirmed by immunoblotting with
anti-CTGF-L antibodies and N-terminal sequencing of purified
protein. Anti-CTGF-L antibodies were prepared by injecting
KLH-conjugated peptides derived from hCTGF-L
(135SEDVRLPSWDCPHPRRV151 and
234SRPCPPSRGRSPQNS248) into rabbits according
to standard procedures.
Tissue Processing, in Situ Hybridization, and
Immunocytochemistry--
Human tissues were obtained through the
Anatomical Gift Foundation of Arizona (Phoenix, AZ). Human osteophytes
were dissected from osteoarthritic femoral heads. Osteoclastoma tissue
was prepared from freshly dissected giant cell tumors. All tissues were
processed for cryosectioning as described (19). The hCTGF-L cDNA
cloned into pBluescript was linearized and transcribed using T3- and T7-promoter sites to generate the antisense and sense strand riboprobes using the Promega (Madison, WI) In Vitro transcription kit
with [35S]thio-CTP (Amersham Pharmacia Biotech).
Riboprobes with a specific activity in excess of 108
cpm/µg were used for in situ hybridization as described
previously (20, 21). For immunocytochemistry, cryostat sections (8 µm) were placed onto 3-aminopropyltriethoxy silane-coated glass
slides, fixed in 10% formalin, and washed in phosphate-buffered saline (PBS). Antigen retrieval was performed by heating the sections at
95 °C for 20 min in a Coplin jar containing appropriately diluted target retrieval buffer (Dako, Carpenteria, CA). Endogenous peroxidase activity was blocked by incubating the sections for 10 min in 0.03%
hydrogen peroxide and sodium azide. The slides were washed in PBS and
blocked with PBS containing 10% goat and 1% human AB serum. The
sections were reacted with anti-CTGF-L or preimmune serum (1:250 in
PBS), washed and reacted with horseradish peroxidase-conjugated secondary antibody, and visualized by labeled streptavidine-biotin 2 peroxidase kit and the liquid diaminobenzidine substrate/chromogen system, according to the manufacturer's protocols (Dako). A brown stain indicated positive reactivity.
Immunoprecipitation of CTGF-L from Fibroblast and Osteoblast
Cells--
For metabolic labeling, exponentially growing primary human
osteoblasts and fibroblasts were incubated for 4 h in
cysteine-free medium containing 5% dialyzed fetal bovine serum with
100-150 µCi/ml of 35S-labeled cysteine (~1000 Ci/mmol,
ICN Biomedicals, Costa Mesa, CA). For immunoprecipitation, 5.0 µl of
preimmune or anti-CTGF-L immune-serum was mixed with precleared
35S-labeled conditioned medium and 20 µl of protein
A-agarose (Life Technologies, Inc.) and incubated overnight at 4 °C.
Immune complex beads were collected by centrifugation and washed three
times with PBST (PBS containing 0.1% Tween 20) buffer. The beads were solubilized in sample buffer and resolved through SDS-PAGE, fixed, dried, and processed for fluorography and autoradiography.
Ligand Western Blotting and Cross-linking--
Recombinant
hCTGF-L (10-300 pmol) was separated by SDS-PAGE under nonreducing
conditions. The protein was transferred to nitrocellulose membrane,
blocked with 5% nonfat dry milk in PBST, and probed sequentially with
1 × 106 cpm of 125I-labeled IGF-I or -II
(~2000 Ci/mmol, Amersham Pharmacia Biotech) or anti-CTGF-L antibody
(1:1000) in PBST containing 0.1% BSA for 18 h. The membrane was
then washed 4 times for 15 min each with PBST and either dried and
exposed to autoradiographic film for ligand blots or developed with ECL
for Western blot (Amersham Pharmacia Biotech). For cross-linking, 300 pmol of rhCTGF-L was mixed with 1 × 105 cpm of
125I-labeled IGF-I or -II for 2 h at 4 °C. Water
soluble homobifunctional cross-linker BS3 (Sigma) was added
to the reaction mixture at a final concentration of 5 mM
for 1 h. The reaction was stopped by addition of SDS-PAGE sample
buffer, and the products were separated by 12.5% SDS-PAGE. In some
cases, the cross-linking was performed in the presence of excess
unlabeled IGF-I or IGF-II (Life Technologies, Inc.).
Cell Adhesion Studies--
Corning 96-well enzyme-linked
immunosorbent assay plates (Corning, NY) were precoated overnight at
4 °C with various concentrations of rhCTGF-L, 0.1 ml of human
vitronectin (0.2 µg/ml in PBS), or BSA (3 mg/ml). The plates were
washed once with PBS and blocked with 3% BSA in PBS for 1 h at
room temperature. Cells were trypsinized and resuspended in RPMI medium
and supplemented with 20 mM Hepes, pH 7.4, and 0.1% BSA at
a density of 5 × 105 cells/ml, and 0.1 ml of cell
suspension was added to each well. Following 1 h of incubation at
37 °C, the cells were fixed by the addition of 25 µl of a 10%
formaldehyde solution, pH 7.4, at room temperature for 10 min. The
plates were washed three times with 0.2 ml of PBS, and the adherent
cells were stained with 0.1 ml of 0.5% toluidine blue for 20 min at
room temperature. Excess stain was removed by extensive washing with
deionized water. The toluidine blue incorporated into cells was eluted
by the addition of 0.1 ml of 50% ethanol containing 50 mM
HCl and quantitated by measuring absorbance at 630 nm on a microtiter
plate reader (Titertek Multiskan MC, Sterling, VA).
Integrin Binding--
Vitronectin receptor,
Osteocalcin Measurement--
ROS 17/2.8 cells were seeded in
24-well plates at a concentration of 3 × 104/0.5
ml/well in assay medium (Ham's F-12 medium supplemented with 1% fetal
calf serum, 2 mM L-glutamine, and antibiotics)
and incubated for 3-4 h. Fresh medium, with or without 10 nM 1,25(OH)2D3 and various
concentrations of CTGF-L, was then added in duplicate, and the cultures
were incubated for an additional 48 h. Osteocalcin in the culture
supernatants was measured by radioimmunoassay using reagents purchased
from Biomedical Technologies (Stoughton, MA).
Identification and Cloning of CTGF-L cDNA--
By analyzing a
human osteoblast cDNA library using expressed sequence tag
analysis, we identified an EST (HOEBG39) that contained an IGF binding
domain (GCGCCXXC). Complete sequencing of HOEBG39 revealed
that it contained an open reading frame encoding a polypeptide of 250 amino acids. Based on sequence analysis, the HOEBG39-encoded protein
exhibited significant identity to the CCN family of proteins of which
CTGF and Cyr61 are the most fully characterized members. HOEBG39
encoded protein is 30-60% identical to CCN proteins (Fig. 1A) and is most closely
related to CTGF (~60% identity). Therefore, we termed it CTGF-L
protein. The first 23 amino acids of CTGF-L encode a putative signal
sequence followed by three of the four distinct domains that are found
in the CCN family of proteins (9). An IGF binding domain is present
from residue Gln24 to Leu93. A VWC repeat is
found from residue Ser98 to Gly164. A third
TSP1 domain is contained within residues Cys194 to
Cys237 (Fig. 1, A and B). A fourth CT
domain (~100 residues) that has been implicated in the heparin
binding and mitogenic activity of CTGF is missing from CTGF-L. An
alignment of CTGF-L to various members of the CCN family is shown in
Fig. 1A.
An EST data base search with hCTGF-L sequence identified a murine EST
(AA754979) that exhibited significant identity (~74% at nucleotide
level) to hCTGF-L. We cloned the full-length mCTGF-L cDNA from a
murine lung cDNA library using marathon 5' rapid amplification of
cDNA ends as described under "Materials and Methods." The
protein encoded by mCTGF-L cDNA is ~70% identical to hCTGF-L
protein (Fig. 1A). Similar to hCTGF-L, the mCTGF-L also
encodes a protein of 251 amino acids and contains all but the fourth CT
domain (Fig. 1, A and B). Phylogentic analysis
with all available members of the CCN family grouped both human and
murine CTGF-L together in a separate group (Fig. 1C). It
appears that elm is the oldest gene from which both CTGF-L
and other CCN members originated (Fig. 1C). Given the
similar arrangement, composition, and length of various domains between
the murine and human CTGF-L protein (Fig. 1, A and
B), it is clear that the murine cDNA that we isolated is
the murine orthologue of human CTGF-L.
Expression of CTGF-L mRNA and Protein--
Northern blots
containing RNAs from various human tissues and bone-derived cells were
hybridized to a 32P-labeled CTGF-L cDNA probe. A
~1.3-kilobase CTGF-L mRNA was highly expressed in primary human
osteoblasts, fibroblasts, ovary and testes (Fig.
2, A, lanes 1 and
9, and B, lanes 12 and 13). A lower level of expression of CTGF-L was also observed in heart, lung, skeletal muscle, prostate, and colon (Fig. 2B, lanes 1, 4, 6, 11, and 15). However, CTGF-L was not expressed in human
osteosarcoma SaOS-2 or MG 63 cells, stromal TF274 cells, osteoclastoma
tissue, or HeLa or mesenglial cells (Fig. 2). The expression of CTGF-L in primary osteoblast or fibroblast cultures appears to be constitutive as treatment with a variety of osteotropic agents, including
parathyroid hormone, transforming growth factor-
In situ hybridization indicated strong expression of CTGF-L
mRNA in bone-forming osteoblasts on calcified cartilage spicules (primary spongiosa) in fetal bone (Fig.
3, A and B) and
human osteophytic tissue (Table I).
Discrete alkaline phosphatase positive cells in bone marrow of
osteophyte and discrete macrophage-like cells from giant cell tumor
also exhibited strong expression of CTGF-L mRNA. Weaker expression
was noted at sites of secondary remodeling (secondary spongiosa),
chondrocytes, and osteoclasts. These data are summarized in Table
I.
Polyclonal antibodies were generated to peptides derived from CTGF-L
and used to immunolocalize CTGF-L protein in bone. Intense CTGF-L
staining was associated with osteoblasts lining trabecular and
periosteal bone surfaces from human fetal bone tissue (Fig. 3,
C and D). We also examined the expression of
CTGF-L protein in primary human osteoblasts and fibroblasts that show
high expression of CTGF-L mRNA. In both cell types, a ~26-kDa
protein was specifically immunoprecipitated by anti-CTGF-L antibodies
from 35S-labeled conditioned medium (Fig.
4, A, lanes 2 and
4) and cell lysate (not shown). The apparent molecular mass
(~26 kDa) of immunoprecipitated protein is consistent with the
expected size of the protein encoded by the open reading frame of
CTGF-L mRNA after cleavage of the signal peptide. A similar sized
protein was also immunoprecipitated from HeLa and TF274 cells that were
transfected with an expression vector encoding hCTGF-L (data not
shown).
Recombinant hCTGF-L was expressed as an N terminus human
immunodeficiency virus GP120 V3 and His6 epitope tag in
Chinese hamster ovary cells and was purified by TALON metal affinity
chromatography as described under "Materials and Methods." The
purified tagged protein of ~30 kDa (expected size due to the tag) was
detected by Coomassie Blue staining (Fig. 4B, lane 1) and
immunostaining (Fig. 4B, lane 2).
Binding of CTGF-L to IGF-I and IGF-II--
The presence of an IGF
binding domain within CTGF-L prompted us to investigate the binding of
CTGF-L to IGFs using ligand Western blotting and cross-linking.
Increasing amounts of rhCTGF-L were separated by nonreducing SDS-PAGE
and transferred onto a nitrocellulose membrane. The blot was probed
sequentially with 125I-labeled IGF-I,
125I-labeled IGF-II, and anti-CTGF-L antibody. As little as
10 pmol of CTGF-L bound to labeled IGFs (Fig.
5A). rhCTGF-L showed a
dose-dependent binding to 125I-labeled IGFs,
with IGF-II exhibiting relatively higher binding than IGF-I.
Immunostaining confirmed the increasing amounts of rhCTGF-L loaded in
each lane (Fig. 5A, bottom panel). As a control, an
unrelated His-tagged protein, p38 MAP kinase, did not bind either IGF-I
or IGF-II (Fig. 5A, lane 5). In a cross-linking experiment using a homobifunctional cross-linker BS3, both
125I-labeled IGF-I and -II bound to rhCTGF-L (Fig.
5B, lane 1). The size of cross-linked product (~37-38
kDa) is consistent with the cross-linking of rhCTGF-L (~30 kDa) to
IGFs (~7-8 kDa). We also performed a competitive cross-linking
experiment in which the cross-linking was competed with increasing
amounts of unlabeled IGF-I (Fig. 5B, lanes 2-5) or IGF-II
(lanes 6-9). The signal obtained by cross-linking of
rhCTGF-L to 125I-labeled IGF-II was much higher than with
125I-labeled IGF I. In addition, only 30 ng of IGF-II was
needed to compete rhCTGF-L cross-linking to IGF-I (Fig. 5B,
lane 7, upper panel), whereas even 300 ng of IGF-I could not
compete the cross-linking of rhCTGF-L to IGF-II (lane 5, lower
panel). These data suggest that both IGF I and IGF II bind to
rhCTGF-L and that IGF-II has at least 10-fold higher affinity for
CTGF-L compared with IGF-I. It should be noted that the specific
activity of both 125I-labeled IGF-I and IGF-II was
comparable (~2000 Ci/mmol).
Adhesion of Osteoblast Cells to CTGF-L--
The TSP1 domain has
been implicated in the promotion of cell attachment (9). Therefore, we
tested whether rhCTGF-L was able to promote the adhesion of
osteoblastic cells. Primary human osteoblast, osteosarcoma MG63, and
rat osteoblast-like osteosarcoma Ros 17/2.8 cells attached to
immobilized rhCTGF-L in a dose-dependent manner (Fig.
6A). Although maximal adhesion
was observed with Ros 17/2.8 cells, the extent of cell adhesion to
rhCTGF-L for all cells was comparable to that observed with
vitronectin. Half-maximal adhesion to rhCTGF-L was observed at a
coating concentration of ~300 ng/ml (~10 nM), whereas
no cell adhesion was observed using a protein preparation in which
CTGF-L was depleted using anti-CTGF-L antibodies or to an unrelated
His6-tagged protein, p38 (Fig. 6B).
Binding of rhCTGF-L to Purified Integrin Receptors--
The
attachment of cells to CTGF-L-coated plates and a previous report that
Cyr61, another CCN family member, binds
Effect of rhCTGF-L on Osteocalcin Production--
To determine
whether rhCTGF-L could modulate osteoblast function, we examined its
effect on osteocalcin production in rat osteoblast-like osteosarcoma,
ROS 17/2.8 cells. Osteocalcin is a marker of mineralizing osteoblasts,
and its expression is used routinely as a measure of osteoblast
function. ROS 17/2.8 cells endogenously secrete low levels of
osteocalcin, which can be up-regulated by treatment with
1,25(OH)2D3. As shown in Fig.
8, rhCTGF-L inhibited both basal and
1,25(OH)2D3-stimulated osteocalcin production
in a dose dependent manner with an IC50 of ~300 ng/ml
(~10 nM). Treatment with a CTGF-L depleted protein
preparation or with an unrelated His6-tagged protein, p38,
had no effect on osteocalcin production (data not shown). The
inhibition of osteocalcin expression was at the mRNA level because
rhCTGF-L also inhibited the osteocalcin mRNA induction by
1,25(OH)2D3 both in Ros 17/2.8 and in primary human osteoblasts (data not shown).
During the past few years, several proteins belonging to the
expanding CCN family have been described (9). Most members of this
family are immediate-early genes that are induced by treatment with
serum or growth factors (1, 3, 5, 24, 25). All members of this family
contain four distinct domains, including the IGF binding domain, the
VWC domain, the TSP1 domain, and the CT domain.
We have identified human (and murine) CTGF-L as a novel member of the
CCN family. There are two striking features that distinguish CTGF-L
from the other CCN family members. First, CTGF-L lacks the fourth CT
domain found in all other family members. Second, CTGF-L is not induced
by serum or growth factors. Although an alternatively spliced form may
exist, we have been unable to identify a CTGF-L variant that encodes a
protein containing the CT domain in libraries derived from cells and
tissues expressing CTGF-L.
In addition, immunoprecipitation of CTGF-L by anti-CTGF-L antibodies
detected only a single ~26-kDa protein in both osteoblasts and
fibroblasts. We did not detect a protein of higher molecular mass in
the anti-CTGF-L immunoprecipitates that might contain the fourth CT
domain. Furthermore, murine CTGF-L cDNA encodes an open reading
frame that terminates at the same amino acid residue as human CTGF-L.
CTGF-L without the CT domain, therefore, is the functional protein
being produced and secreted by osteoblast and fibroblast cells.
Another feature that distinguishes CTGF-L from all other members of the
CCN family is the inability of serum or various growth factors to
induce its expression. The expression of CTGF-L in primary osteoblast
or fibroblast cultures appears to be constitutive, and to date, we have
not been able to modulate its expression by treatment with a variety of
osteotropic agents, including parathyroid hormone, transforming growth
factor- Human tissues expressing the highest levels of CTGF-L mRNA are
bone, ovary, and testes. We have shown that both CTGF-L mRNA and
protein are expressed at high levels in osteoblasts forming bone in the
periosteum and primary spongiosa of human fetal and osteophytic bone.
These bone formation areas are zones of appositional and longitudinal
bone growth. Lower levels of expression were observed in osteoblasts
lining the relatively slower remodeling trabeculae of the secondary
spongiosa. Thus, CTGF-L mRNA was highly expressed by osteoblasts at
sites of high bone turnover (Table I) associated with net gain in bone
mass. This highly selective expression is suggestive of a specific role
for CTGF-L in the control of bone formation.
We have examined the activity of rhCTGF-L in several assays to
elucidate potential functions for various domains of this protein, including cell adhesion, IGFs and integrin binding, and osteocalcin production. IGFs have growth promoting activity and play an important role in bone formation and remodeling (26). Several IGF-binding proteins regulate the activity and availability of IGFs in bone and
other tissues (26). Although we have unequivocally shown that rhCTGF-L
binds to both IGF-I and IGF-II, to date, no significant change in IGF
action has been observed in the presence of CTGF-L. This suggests that
CTGF-L may serve not to modulate IGFs activity, but rather to increase
the local concentrations of IGFs. Although cross-linking and ligand
Western blotting studies did not allow a precise determination of
binding affinities, it appears that the relative affinity of rhCTGF-L
for IGF-II is at least 10-fold higher than for IGF-I. In this regard,
it has been reported that human osteoblasts produce and respond better
to IGF-II, whereas rodent osteoblasts produce and are more responsive
to IGF-I (26). In a recent report, rhCTGF was also shown to bind both
IGF-I and IGF-II (27) but with much lower affinity than the binding of IGFBP3 to IGFs. Unlike IGFBPs, no reports on modulation of IGFs activity by CCN family of proteins have been described.
Whereas the binding of CTGF-L to IGFs is most likely mediated via the
IGF binding domain similar to the IGFBPs, cell adhesion is most likely
mediated via the TSP1 domain, perhaps through binding to integrin
receptors. Indeed, we have demonstrated that rhCTGF-L directly inhibits
fibrinogen binding to purified integrin receptors. Fibrinogen, like
many extracellular matrix proteins, contains an RGD motif that is
crucial for integrin binding. However, no RGD sequence is present in
CTGF-L or Cyr61, which has also been shown to bind
Although the VWC domain has been implicated in oligomerization, we have
not observed either dimers or any other higher order species, nor have
oligomers been reported for other CCN proteins. CTGF-L lacks the CT
domain, which alone is sufficient for heparin binding and mitogenic
activity of CTGF (10). This suggests an interesting possibility that
CTGF-L may antagonize the effect of CT domain in one or more CCN
proteins, as osteoblasts are capable of expressing various CCN members,
including CTGF,2 Cyr61 (24),
and CTGF-L.
Due to the multidomain structure of CTGF-L, antisera generated against
peptides derived from the VWC domain and the C terminus of CTGF-L were
nonneutralizing and, therefore, were not useful for blocking
experiments. Alternatively, we used a similarly tagged but unrelated
protein, p38, or a preparation in which rhCTGF-L was depleted with
anti-CTGF-L antibodies. Neither protein preparations had any effect in
any of the assays tested, suggesting that the functional results
obtained were due to CTGF-L protein. Similarly, we have also confirmed
various activities reported here using a rhCTGF-L preparation in which
the epitope tags were removed by enterokinase digestion, suggesting
that the presence of epitope tags did not have any effect on the
rhCTGF-L protein activity (data not shown).
The multidomain structure of the CCN family of proteins, of which Cyr61
and CTGF are the best characterized members, suggests that they may
have multiple functions (9). CTGF protein has mitogenic and chemotactic
activity and may have a role in wound repair and/or fibrosis (1, 10,
31-33). Cyr61 has been shown to be an extracellular matrix-associated
molecule that has mitogenic activity when used in conjunction with
basic fibroblast growth factor and can also promote adhesion and
migration of fibroblasts and vascular endothelial cells (34, 35).
Recently, it has been determined that Cyr61 mediates the adhesion of
cells via While this report was in preparation, two laboratories reported the
cloning of murine and rat CTGF-L. WISP-2, the murine orthologue of
hCTGF-L, was cloned by subtractive hybridization as a Wnt-1 inducible
gene, the expression of which was reduced in some tumors (38). The rat
orthologue of CTGF-L, rCOP-1, was cloned by differential display
analysis as a gene of which the expression was lost as a result of cell
transformation (39). An identical sequence has also been submitted to
the GenBankTM data base as a CTGF-related protein, CT 58, which was found in a yeast two-hybrid screen from epithelial cell
library using mucin, Muc1, as a bait (GenBankTM accession
number AF074604). Mucin is a family of highly glycosylated secreted
proteins that are aberrantly expressed in epithelial tumors, including
breast carcinomas (40). Although these studies suggest a role for
CTGF-L in tumorigenesis possibly as a tumor suppressive gene, they are
yet to be confirmed using purified protein. Given the range of
activities associated with this class of proteins, the exact
function(s) of CTGF-L is not readily predictable.
We have shown that rhCTGF-L is active at nanomolar concentrations in
assays measuring the inhibition of osteocalcin production, the
promotion of cell adhesion, and integrin binding, suggesting that these
could be among the major functions of this protein. We do not yet know
whether CTGF-L is deposited in bone matrix, where it might facilitate
osteoblast adherence leading to osteoid formation. By virtue of its
ability to promote cell adhesion and binding to IGFs, CTGF-L may
increase the local concentration of IGFs to augment osteoblast
activity. In this regard, it is tempting to speculate that CTGF-L may
also function to maintain or extend the osteoblast matrix maturation
phase by inhibiting the production of osteocalcin, a standard marker of
mineralizing osteoblasts. The high expression of CTGF-L in bone and
suggested tumor suppressive activity of some CCN protein suggests that
it may also play a role in preventing tumor metastasis to bone and
other tissues. The identification of CTGF-L adds to the diversity of
the CCN family and suggests their potential role in bone metabolism.
Further studies with CTGF-L will help elucidate its role in bone and
other tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
induces the production of CTGF mRNA and protein, which in turn induces type I collagen gene expression (2).
Other members of this family include Fisp12 (3), which is the murine
orthologue of human CTGF; human and murine Cyr61 (4, 5); the chicken
orthologue of Cyr61, Cef10 (6); and human and Xenopus Nov
(7, 8). These genes, with the exception of nov, are
immediate-early genes that are induced by serum, growth factors, or
certain oncogenes and are collectively referred to as the CCN (CTGF,
cef10/cyr61, nov) family (9). The nov
gene was identified as a gene that was induced as a result of proviral rearrangement due to insertion of nephroblastosis associated virus (7).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v
3 (0.12 µg), and fibrinogen receptor,
IIb
3 (1 µg), purified from human
placenta and blood, respectively (22) were added to 96-well microtiter
plates and incubated overnight at 4 °C. At the time of experiment,
the protein solutions were aspirated and the wells were incubated in
0.1 ml of Buffer A (50 mM Tris, 100 mM NaCl, 1 mM MgCl2, 1 mM MnCl2,
pH 7.4) containing 3% BSA for 1 h at room temperature to block
the nonspecific binding. After aspirating the blocking solution,
various concentrations of CTGF-L was added to the wells followed by the
addition of 8 nM biotinylated fibrinogen in 100 µl of
Buffer A containing 0.1% BSA. The plates were incubated for 1 h
at room temperature and washed twice with 100 µl of binding buffer.
Anti-biotin antibody conjugated to alkaline phosphatase (1:2000
dilution, Sigma) was then added for 10 min followed by two washes with
binding buffer containing 0.1% Tween 20. The reaction was quantitated
with alkaline phosphate substrate kit (Bio-Rad) by measuring absorbance
at 405 nm using a microtiter plate reader.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
[in a new window]
Fig. 1.
Alignment of CTGF-L with various members of
the CCN family of proteins. A, protein sequence
alignment was performed using the MEGALIGN program of Lasergene
(DNASTAR Inc., Madison, WI) according to the clustal algorithm. The
amino acids identical to CTGF are hidden and indicated by
dots, whereas dashes indicate gaps. The
broken, dashed, solid and dotted lines with
arrows indicate the IGF binding domain, the VWC domain, the
TSP1 domain, and the CT domain, respectively. B, schematic
representation of different domains, including signal sequence
(SS) of CCN proteins. IGF BD, IGF binding domain.
The amino acid number flanking each domain of hCTGF-L is also shown.
Note that the CT domain is missing in CTGF-L. C,
phylogenetic relationship between various members of the CCN
family.
,
1,25(OH)2D3, and estrogen, failed to modulate
its expression (data not shown).
View larger version (30K):
[in a new window]
Fig. 2.
Expression of human CTGF-L mRNA in
various human cells and tissue preparations. A, total
RNA from primary human osteoblasts (lane 1), human
osteosarcoma SaOS2 (lane 2), human osteoclastoma tissue
(lane 3), human osteosarcoma HOS (lane 4), human
osteosarcoma MG63 (lane 5), human stromal TF274 (lane
6), HeLa (lane 7), human mesenglial (lane
8), and synovial fibroblasts (lane 9) was hybridized
with 32P-labeled hCTGF-L and GAPDH cDNA probe. The
location of 28 S and 18 S ribosomal RNA, CTGF-L, and GAPDH are
indicated. B, a multiple tissue Northern blot containing
poly(A)+ RNA from heart (lane 1), brain
(lane 2), placenta (lane 3), lung (lane
4), liver (lane 5), skeletal muscle (lane
6), kidney (lane 7), pancreas (lane 8),
spleen (lane 9), thymus (lane 10), prostate
(lane 11), testes (lane 12), ovary (lane
13), small intestine (lane 14), colon (lane
15), and peripheral blood leukocytes (lane 16) was
hybridized with 32P-labeled CTGF-L and GAPDH cDNA
probe. The locations of RNA markers (in kilobases), CTGF-L, and GAPDH
are indicated.
View larger version (142K):
[in a new window]
Fig. 3.
CTGF-L mRNA and protein expression during
human endochondral ossification. A, a section of human
fetal femoral growth plate was hybridized with antisense CTGF-L
riboprobe. Osteoblasts (Obs) (arrows) forming
osteoid on calcified cartilage spicules demonstrated intense mRNA
expression; adjacent formation sites showed reduced expression
(arrowheads). Magnification, × 50; counter-stained with
methylene blue. B, high power view of the area shown in
A demonstrates CTGF-L mRNA expression in osteoblasts
(Obs) (arrows) and osteoprogenitor cells
(Opg) within the marrow (magnification, × 100).
C, a section of fetal bone stained with the anti-CTGF-L
polyclonal antibody. Osteoblasts lining the trabecular
(arrows) and periosteal (arrowheads) bone surface
express high levels of CTGF-L (magnification, × 25). D,
high power view of the area shown in C shows expression of
CTGF-L in osteoblasts (Obs) (arrows) and discrete
cells within the marrow (arrowheads) (magnification, × 100).
Expression of CTGF-L mRNA in human bone by in situ hybridization
View larger version (34K):
[in a new window]
Fig. 4.
Expression of endogenous and recombinant
CTGF-L protein. A, primary human fibroblasts
(Fib.) and osteoblasts (Obs.) were labeled with
[35S]cysteine, and the conditioned medium was
immunoprecipitated with preimmune (PI) or immune
(IM) anti-CTGF-L antibody. The location of ~26-kDa CTGF-L
is indicated. B, recombinant CTGF-L was expressed as N
terminus GP120/His6-tagged protein in Chinese hamster ovary
cells and purified by TALON metal affinity chromatography. Molecular
weight markers (in thousands) and the location of purified rhCTGF-L as
detected by Coomassie Blue staining (lane 1) and Western
blot (lane 2) are shown.
View larger version (39K):
[in a new window]
Fig. 5.
Binding of rhCTGF-L to IGFs.
A, various amounts of CTGF-L (10-300 pmol) and p38 (300 pmol) were separated by SDS-PAGE under nonreducing conditions and
probed sequentially with 125I-labeled IGF-I or IGF-II or
with anti-CTGF-L antibody. Between experiments, the blot was stripped
and extensively washed. The final immunoblot was developed with ECL.
The locations of the ~30-kDa marker and CTGF-L as detected with each
probe are indicated. B, rhCTGF-L was cross-linked to
125I-labeled IGF-I (top) or
125I-labeled IGF-II (bottom) with a
homobifunctional cross-linking agent, BS3. The
cross-linking was competed with increasing amounts of unlabeled IGF-I
(lanes 2-5) or IGF-II (lanes 6-9) as
indicated.
View larger version (21K):
[in a new window]
Fig. 6.
Promotion of cell adhesion by rhCTGF-L.
A, 96-well microtiter plates were coated with or 3 mg/ml
BSA, 200 ng/ml vitronectin (Vn), or various amounts of
rhCTGF-L as indicated. After blocking with BSA, MG 63 (black
columns) primary human osteoblasts (gray columns), and
Ros 17/2.8 (white columns) cells were allowed to adhere for
30 min, washed extensively, fixed, and stained to quantitate the
adhesion. B, adhesion of MG 63 (black columns)
and Ros 17/2.8 (white columns) cells to microtiter plates
coated with equal amounts (1 µg/ml) of CTGF-L, antibody depleted
CTGF-L (depleted) or unrelated protein, p38. The data are
representative of three independent experiments. Values shown are the
means ± S. E. of triplicate determinations.
v
3 (23) prompted us to examine the effect
of CTGF-L on the binding of purified integrins to matrix protein. The
purified integrin receptors,
v
3 and
IIb
3, demonstrated high affinity binding
to biotinylated fibrinogen. As shown in Fig.
7, rhCTGF-L inhibited the binding of
fibrinogen to both integrins in a dose-dependent manner.
The IC50 for
v
3 (Fig. 7,
filled triangles) was ~100 nM, whereas weaker
inhibition was observed for
IIb
3
(IC50 ~1 µM) (open circles), suggesting some degree of selectivity.
View larger version (13K):
[in a new window]
Fig. 7.
Inhibition of fibrinogen binding to purified
integrins by rhCTGF-L. Biotinylated fibrinogen (8 nM)
was added to microtiter wells coated with integrin receptors in the
presence or absence of various concentration of CTGF-L and incubated
for 1 h at room temperature to measure the effect of CTGF-L on the
binding of biotinylated fibrinogen to v
3
(filled triangles) and
IIb
3
(open circles). Inhibition of fibrinogen binding by CTGF-L
was quantitated by staining with alkaline phosphatase conjugated
anti-biotin antibody and by measuring absorbance at 405 nM.
Total binding (control = 100%) was determined in absence of
CTGF-L. The inhibition is expressed as a percentage of control. The
data are representative of three independent experiments. Values shown
are the means ± S. E. of triplicate determinations.
View larger version (18K):
[in a new window]
Fig. 8.
Inhibition of osteocalcin production by
rhCTGF-L. Ros cells were treated with different concentrations of
rhCTGF-L in the absence (open circles) or presence
(filled triangles) of 10 nM
1,25(OH)2D3. Osteocalcin present in 48-h
culture supernatants was measured by radioimmunoassay. The data are
representative of three independent experiments. Values shown are the
means ± S. E. of triplicate determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 1,25(OH)2D3, and estrogen. An
additional feature is the absence of CTGF-L mRNA expression in
various tumor cell lines, including osteosarcoma, and it appears that
its expression is restricted to primary cells, such as osteoblasts and fibrolasts.
v
3 integrin (23). Therefore, CTGF-L
binding must occur through an RGD-independent mechanism. Consistent
with this notion, the adhesion of osteoblasts to vitronectin, but not to CTGF-L, was inhibited by the RGD-containing snake venom protein echistatin (data not shown). Therefore, CTGF-L may inhibit
matrix/integrin interaction by binding close to the RGD recognition
site due to steric hindrance or by inducing a conformational change.
Similarly, the effect of CTGF-L on osteocalcin release may occur
through the TSP1 domain and integrin binding because it is known that integrin binding to extracellular matrix proteins can result in signal
transduction leading to changes in cell proliferation, gene expression,
and cellular differentiation (28). It is possible that these changes
may be modulated directly or indirectly via interactions of CTGF-L with
matrix molecules and integrin receptors. Indeed, the TSP1 domain has
been implicated in binding to both soluble and matrix molecules and to
sulfated glycoconjugates (9, 29). It was recently reported that binding
of integrin antagonist echistatin inhibits IGF-I signaling in vascular
smooth muscle cells (30). This led to the proposal that ligand-induced
activation of
v
3 integrin receptor
results in its interaction with the IGF-I signal transduction pathway.
It is possible that CTGF-L by its ability to bind both integrin and
IGFs may modulate the activity of IGFs. However, we did not observe any
significant modulation of IGF activity by CTGF-L, at least in
osteoblastic cells (data not shown). It is also possible that CTGF-L
may bind to some as yet unidentified receptor.
v
3 integrin binding and has
been suggested to promote tumor growth and angiogenesis (36, 37). Elm,
another family member, is expressed at low levels in metastatic cancer
and suppresses metastasis (25).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Michael Lark, Larry Suva, and Peter Young for critical reading of the manuscript; Dr. Rothman of the Rothman institute, Pennsylvania Hospital, for providing bone samples; Dr. Kyung Johanson for purified integrin receptors; Heather McClung for technical assistance; DNA synthesis group for oligonucleotide synthesis and DNA sequencing; and Wendy Crowell for artwork.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF083500 and AF126063.
§ To whom correspondence should be addressed: SmithKline Beecham, Bone and Cartilage Biology, UW2109, 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-7245; Fax: 610-270-5598; E-mail: Sanjay_Kumar{at}sbphrd.com.
2 S. Kumar, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CTGF, connective tissue growth factor; CTGF-L, CTGF-like; rhCTGF-L, recombinant human CTGF-L; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; TSP1, thrombospondin type I domain; VWC, von Willebrand factor type C domain; CT, caboxyl terminal; TGF, transforming growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; EST, expressed sequenced tag; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|