From the
Two major tenascin-C (TN-C) isoforms are generated by the
alternative splicing of the pre-mRNA. The large isoform contains seven
extra type three repeats that, by contrast, are omitted in the small
TN-C isoform. The large TN-C isoform is mainly expressed at the onset
of cellular processes that entail active cell migration, proliferation,
or tissue remodeling such as occur in neoplasia, wound healing, and
during development. Thus, the large TN-C isoform seems to be a specific
component of the provisional extracellular matrix. Here we have studied
the degradation of the large and small TN-C isoforms by matrix
metalloproteinases (MMPs) 2, 3, 7, and 9. Among these proteolytic
enzymes only MMP-7 can degrade the small TN-C isoform removing the
NH
Human tenascin-C (TN-C)
Here, we have compared the
susceptibility of small and large TN-C isoforms to degradation by
MMP-2, MMP-3, MMP-7, and MMP-9. Results show that the large TN-C
isoform is much more sensitive to MMP proteolytic activity.
Large TN-C Isoform Digestion
MMP-2, MMP-3, MMP-7, and MMP-9 were used to digest the large TN-C
isoform under the conditions described under ``Materials and
Methods.'' The products obtained were analyzed by SDS-PAGE under
reducing conditions and are shown in Fig. 1 A. The
digests were also analyzed by immunoblotting using 10 mAbs specific for
different epitopes of TN-C (see Fig. 3) and the
NH
All of the
results of large TN-C-resistant fragment analysis by immunoblotting are
summarized in and in Fig. 3, which also shows the
localization within the TN-C molecules of the epitopes recognized by
the various mAbs and the amino-terminal sequences of the fragments. Small TN-C Isoform Digestion
Digestion of the small TN-C isoform was carried out using the same
enzymes and under the same conditions as for large TN-C. The results
have shown that small TN-C is much more resistant than the large
isoform. MMP-2, MMP-3 (small and large form) and MMP-9 do not cleave
small TN-C. Only MMP-7 cleaves 100% of the molecules, giving two
fragments detectable in SDS-PAGE (Fig. 1 A). One has an
apparent molecular mass of 170 kDa and the second of about 16 kDa
(S-170 and S-16), respectively. Analysis with specific mAbs showed that
the S-16 kDa is the NH
Degradation of the extracellular matrix (ECM) is a crucial
step of tumor invasion and progression
(33) . Although many
proteases can cleave ECM molecules, MMPs are believed to be the main
physiologically relevant mediators of matrix degradation
(33, 40, 41) . The human MMP family consists of
at least 11 highly homologous zinc endopeptidases which are active at
neutral pH and collectively cleave most, if not all, constituents of
the ECM. More recently, it has been elegantly demonstrated that a mixed
substrate of TN-C and FN collaborate in the up-regulation of
collagenase (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9) gene
expression by rabbit fibroblasts
(42) , this phenomenon being
dependent on an epitope in the carboxyl-terminal fibronectin-like type
III repeats. Thus, transient alteration of the composition of the ECM
by TN-C presence up-regulates the expression of genes involved in cell
migration, tissue remodeling and invasion in regions of tissues
undergoing morphological modifications.
TN-C expression has been
documented in a wide variety of solid tumor tissues
(1, 17, 43, 44) . Moreover, much
evidence has shown an increased expression of the TN-C isoform with a
higher molecular mass with the onset of neoplastic transformation both
in vitro and in vivo (12, 13, 14, 15) . Furthermore, we
have recently shown that proliferating cells produce only the large
TN-C isoform, while resting cells mainly produce the small isoform
(18) .
Analysis of TN-C sensitivity to proteolytic
degradation may provide information about the mechanisms involved in
ECM degradation. Little information is available about this topic.
Friedlander et al. (45) digested TN-C with
chymotrypsin and separated the resistant fragments by gel filtration.
Taylor et al. (46) described TN-C digestion by
We have studied the degradation patterns
of small and large human TN-C isoforms obtained with different MMPs, a
group of enzymes which are known to be more highly expressed in
neoplastic and remodeling tissues than in the corresponding normal
adult tissues
(33) . We have been able to precisely localize the
cleavage sites through the analysis of resistant fragments with mAbs
whose epitope localization within the TN-C molecule was already known.
Results were confirmed by determination of the NH
Degradation by MMPs of TN-C purified from human melanoma cells has
recently been reported by Imai et al. (48) . They found
that TN-C produced by melanoma cells (essentially the large isoform) is
sensitive to MMP-1, 3 and 7 degradation while it is resistant to MMP-2
and MMP-9. The data on MMP-2 digestion do not entirely agree with ours,
even though degradation by this enzyme is only partial in our hands as
well. However, these authors neither report information about the
localization within the TN-C molecule of the cleavage sites nor compare
the sensitivity of the two major TN-C isoforms to these enzymes.
The
greater sensitivity of the large TN-C isoform to MMP degradation,
together with the higher expression of this isoform observed in
neoplastic and remodeling tissues
(25, 49) , may support
the hypothesis that the large TN-C isoform is a specific component of
the provisional extracellular matrix and may contribute to the
generation of a more suitable environment for cellular proliferation
and migration. Furthermore, degradation by MMPs of TN-C could modify
its biological activities. In fact, proteolytic cleavage may represent
a tool to either abolish or unmask specific TN-C functional sites.
We thank Antonella Gessaga for skilful secretarial
assistance and Mr. Thomas Wiley for manuscript revision. We are
indebted to Prof. L. Santi for his support and encouragement.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal knob. The large TN-C isoform shows the same
MMP-7-sensitive site adjacent to the NH
-terminal sequence,
but is further degraded in the splicing area where three
fibronectin-like type III repeats are completely digested. Moreover,
the large TN-C isoform is degraded by MMP-2 and MMP-3 which completely
digest a single type III repeat inside the splicing area. By contrast,
the large TN-C isoform is resistant to MMP-9 digestion. The results
show that the presence of the spliced sequence introduces new
protease-sensitive sites in the large TN-C isoform.
(
)
is an
extracellular matrix glycoprotein composed of six similar subunits
joined at their NH
-terminal by disulfide bonds
(1, 2, 3, 4, 5, 6, 7) .
During development, TN-C displays a time and space-dependent tissue
distribution with morphogenetically significant boundaries. Each human
TN-C subunit includes three types of structural modules: 14
epidermal growth factor-like repeats, 15 fibronectin (FN) type three
homology repeats, and a COOH-terminal knob made up of a sequence with
homology to the globular domain of the
and
chains of human
fibrinogen (see Fig. 3). TN-C is coded for by a single gene and
its expression is regulated by a single promoter
(8, 9) . Structurally and functionally different human
TN-C isoforms are generated by alternative splicing of the TN-C
transcript, seven type three repeats being included or omitted in the
mRNA (see Fig. 3)
(10, 11) generating two major
TN-C isoforms with molecular masses of about 300 and 200 kDa,
respectively. Analysis of TN-C isoform ratio has shown that, in human
malignantly transformed cultured cells as well as in breast carcinoma
and in some cases of lung carcinoma, the higher molecular mass isoform
is preferentially expressed
(12, 13, 14, 15, 16, 17) .
More recently it has been shown that mitogenic stimulation of cultured
fibroblasts induces a modification of the ratio between the two TN-C
isoforms, the expression of the larger isoform being increased to a
much higher extent
(18) . Differences in biological functions
and sites of expression between TN-C isoforms have been reported
(19, 20, 21, 22, 23, 24, 25) .
Murphy-Ullrich et al. (26) have demonstrated that
focal adhesion integrity is down-regulated by the alternative spliced
domain of human TN-C. Chiquet-Ehrismann et al. (27) have shown that the chicken TN-C isoform lacking the
alternatively spliced domain binds more strongly to FN than the isoform
in which this domain is included, and Kaplony et al. (28) reported that expression of high molecular mass TN-C
correlates with corneal cell migration. Moreover, recent reports have
shown that the two TN-C isoforms bind to the cell surface through
different receptors; in fact, the large TN-C isoform strongly interacts
with annexin II in glioma and endothelial cells
(29) while the
neuronal cell adhesion molecule contactin/F11 mainly binds the small
TN-C isoform
(30, 31) .
Figure 3:
Model
of the domain structure of the large isoform subunit of human TN-C and
MMP resistant fragments. The ovals and the squares represent the
EGF-like and FN-like repeats, respectively. The globular
NH-terminal and fibrinogen-like COOH-terminal domains are
also represented. The FN-like repeats A1 to D, whose expression is
regulated by alternative splicing of pre-mRNA, are shaded. The
upper part of the figure reports the mAbs used and the brackets show
the shortest sequence in which each epitope was localized. The lower
part represents the resistant fragments obtained by large TN-C
digestion with MMP-7 and MMP-2, respectively, the mAbs reacting with
the various fragments, as well as the amino-terminal sequences of the
resistant fragments.
Since extracellular matrix
degradation is a crucial step in tumor invasion, a differential
susceptibility of the different TN-C isoforms to the proteolytic
enzymes released by neoplastic cells may take part in this mechanism.
In particular, matrix metalloproteinases (MMPs) are known to be
produced by several tumor tissues and are involved in proteolytic
degradation of tumor extracellular matrix. In fact, it has been
reported that various murine and human tumor-derived cell lines
transcribe, synthesize and secrete MMP enzymes
(32, 33) . These enzymes are also synthesized and
secreted by normal cells under conditions that may be associated with
physiologic tissue remodeling.
Cells and Monoclonal Antibodies
Baby hamster
kidney (BHK) cells transfected with TN-C cDNA constructs
(11) in the pNUT expression vector were a gift of Dr H. P.
Erickson
(34) . Two clones, HxB.L and HxB.S produce the large
and the small TN-C splice variants, respectively (large TN-C and small
TN-C). Cells were grown in Dulbecco's modified Eagle's
medium (HyClone Europe Ltd. Cramlington, UK) containing 10% fetal calf
serum obtained from Boehringer (Mannheim, Germany). Preparation and
characterization of seven mAbs used has already been reported
(35) . Three new mAbs, BC-8, BC-9, and BC-10, not previously
described, have been characterized in this study using the procedure
previously described
(35) based on analysis of mAb reaction in
immunoblotting with different TN-C--galactosidase fusion proteins.
Purification and Activation of Recombinant
MMPs
Recombinant MMP-2, MMP-3, MMP-7, and MMP-9 were purified
from conditioned culture medium of transfected NSO mouse myeloma cells
essentially as previously described
(36, 37, 38) . The purified proenzymes were
activated by treatment with 1 m
M aminophenyl-mercuric acetate
for 1 h at 25 °C (MMP-2) or 37 °C (MMP-3, -7, and -9).
Purification and Digestion of Large and Small TN-C
Isoforms by MMP-2, -3, -7, and -9
Small and large TN-C splicing
variants were purified from conditioned culture medium of
BHK-transfected cell lines using an immunoadsorbent prepared with the
mAb BC-4 as already reported
(12) . Each isoform was more than
95% pure as judged by SDS-polyacrylamide gel electrophoresis (PAGE) and
the amount of TN-C was evaluated using the absorption coefficient
A(1 mg/ml, 1 cm) of 0.97
(1) . 300
µg of the two TN-C isoforms were incubated with either 5 µg of
active MMP-7, 13 µg of active MMP-2, or 15 µg of active MMP-3
or MMP-9 in 100 m
M Tris/HCl, pH 7.5, 100 m
M NaCl, 5
m
M CaCl
for 24 h at 37 °C. The digestion
products were analyzed by SDS-PAGE under reducing conditions followed
by Coomassie Blue staining or immunoblotting carried out as previously
reported
(39) . Amino-terminal sequencing of the fragments was
performed by TIB MolBiol (Genoa, Italy). Fragments to be analyzed were
run on SDS-PAGE under reducing conditions and blotted on an Immobilon-P
membrane (Millipore, Bedford, MA). The bands were revealed by staining
with Ponceau S (Gelman Sciences, Ann Arbor, MI) and isolated by cutting
the membrane.
-terminal sequences of the major fragments were
determined. Three different patterns were observed.
Figure 1:
Analysis on SDS-PAGE of the large and
small TN-C digested by MMP-7 and MMP-2. A, large TN-C
undigested ( lanes 1 and 3) and digested by MMP-7
( lane 2) and MMP-2 ( lane 4). In the MMP-7 digest,
three major resistant fragments were detectable: according to the
molecular mass L-170, L-65, and L-16. In the
MMP-2 digest two resistant fragments were detectable: L-190 and L-120. The values on the left are the
molecular masses (in kilodaltons) of the standards. B,
undigested small TN-C isoform ( lanes 1 and 3) and
digested by MMP-7 ( lane 2) or by MMP-2 ( lane 4) were
run in 4-18% SDS-PAGE in reducing conditions and stained with
Coomassie Blue. Two major resistant fragments were identified in the
MMP-7 digest ( S-170 and S-16), while degradation was
not observed using MMP-2. The values on the left are the molecular
masses (in kilodaltons) of the standards.
Pattern 1
MMP-7 degraded 100% of large TN-C molecules
and three major fragments with molecular masses of 170, 65, and 16 kDa,
were generated (fragments L-170, L-65, and
L-16) (Fig. 1). Analysis of these bands by
immunoblotting with specific mAbs is shown in Fig. 2and
. According to molecular mass and reaction with the
different mAbs, the L-170 band was identified as a fragment including
the EGF-like repeats and the FN like repeats 1 to A2. From the
molecular mass and mAb reactivity we deduced that the L-65 fragment
contains the fibrinogen-like COOH-terminal knob plus the FN like
repeats 6 to 8. The mAb BC-7, specific for an epitope located in the
amino-terminal portion of the molecule, reacted with the L-16 band.
Since no other band with a higher molecular mass was stained by this
mAb, the results show that the cleavage of the amino-terminal sequence
occurred in 100% of the large TN-C molecules.
Figure 2:
Analysis using different mAbs of large
TN-C digested by MMP-7. To the left, an SDS-PAGE in which undigested
( lane U) and digested with MMP-7 ( lane D) large TN-C
isoform were run under the conditions described under ``Materials
and Methods.'' To the right, immunoblottings of samples
as in lane D stained with the mAbs indicated above the
lanes.
Three mAbs reacting
with epitopes located in the splicing areas, -A3,
-B,
-D, were negative both with fragment L-170 and with fragment L-65
(), indicating that the MMP-7 completely degraded the
FN-like repeats A3 to D included in the splicing area under these
conditions. The model of the fragment generated by MMP-7 is depicted in
Fig. 3
. Amino-terminal sequences of the fragments L-170 and L-65
confirmed this degradation pattern (Fig. 3).
Pattern 2
MMP-2 and MMP-3 cleaved about 60% of the
large TN-C isoform into two major fragments of about 190 and 120 kDa
(fragments L-190 and L-120), respectively. The polypeptide pattern of
the large TN-C digested by MMP-2 was analyzed on a 4-18% SDS-PAGE
gradient using the conditions described under ``Materials and
Methods'' and is shown in Fig. 1 A. Analysis of
these fragments with specific mAbs () has demonstrated that
the L-190 includes the amino-terminal end of the molecule, the EGF-like
and the FN-like repeats 1 to A2. The 120-kDa fragment includes the
FN-like repeats A4 to D and 6 to 8 as shown by mAb reaction and,
according to the molecular mass, the fibrinogen like COOH-terminal
sequence. The absence of reaction of mAb -A3 with either L-190 or
L-120 fragments suggests that its epitope in the A3 repeat is
completely digested by these MMPs. The model of the fragment generated
by MMP-2 is depicted in Fig. 3. Amino-terminal sequences of the
fragment L-120 confirmed the pattern of degradation (Fig. 3).
Pattern 3
MMP-9 did not show detectable
degradation of the large TN-C isoform (data not shown).
-terminal knob, while the S-170
fragment includes all the remaining part of the molecule from the
beginning of the EGF-like repeats to the fibrinogen like COOH-terminal
sequence. The model of the fragment generated by MMP-7 is depicted in
Fig. 4
. Amino-terminal sequence of the fragment S-170 confirmed
this pattern of degradation (Fig. 4). The results are summarized
in .
Figure 4:
Model of the domain structure of the small
isoform subunit of human TN-C and MMP-7 resistant fragments. Symbols
are as in Fig. 3 and show the small TN-C isoform in which all the
repeats undergoing alternative splicing are omitted. Fragments obtained
in MMP-7 digestion are shown, as are NH-terminal sequence
and mAbs reacting with the various
fragments.
-chymotrypsin and trypsin. However, the exact location of cleavage
sites in the TN-C structure were not determined in these reports. More
recently, Chiquet et al. (47) analyzed resistant
fragments obtained by digestion with pepsin and Pronase of chicken TN-C
preparations enriched in small or large splicing variants. These
authors have localized the resistant fragment in the TN-C molecule
using specific mAbs and have shown that the large isoform is cleaved
inside the spliced region.
-terminal
sequences of the major fragments and have shown that the presence of
the spliced sequence strikingly modifies TN-C sensitivity to these
enzymes. In fact, while large TN-C can be cleaved by MMP-7, -2, and -3
inside this sequence, small TN-C contains only a sensitive site close
to the NH
-terminal knob that is cleaved by MMP-7.
Table: 1696613199p4in
ND, not
determined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.