Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovar-ian clear cell adenocarcinoma OVISE and gastric carcinoma MKN-45, highly produc
Trang 1matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1
(IGFBP-rP1/angiomodulin/mac25)
Sanjida Ahmed1,2, Xinlian Jin1, Motoki Yagi1,2, Chie Yasuda1, Yuichiro Sato1,2, Shouichi Higashi1, Chen-Yong Lin3, Robert B Dickson3and Kaoru Miyazaki1,2
1 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan
2 Graduate School of Integrated Sciences, Yokohama City University, Japan
3 Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
Insulin-like growth factor (IGF) binding proteins
(IGFBPs) regulate cellular proliferation by modulating
the actions of insulin and IGFs [1,2] Recent studies
have revealed a group of IGFBP-related proteins (IGFBP-rPs), which have low affinity for IGFs⁄ insulin and low structural homology to IGFBPs [3]
Keywords
angiomodulin; insulin-like growth factor;
insulin-like growth factor binding
protein-related protein-1; matriptase; proteolytic
processing
Correspondence
K Miyazaki, Division of Cell Biology, Kihara
Institute for Biological Research, Yokohama
City University, 641–12 Maioka-cho,
Totsuka-ku, Yokohama 244–0813, Japan
Fax: +81 458201901
Tel: +81 458201905
E-mail: miyazaki@yokohama-cu.ac.jp
(Received 25 August 2005, revised
15 November 2005, accepted 8 December
2005)
doi:10.1111/j.1742-4658.2005.05094.x
Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF⁄ insulin-dependent or ininsulin-dependent manner It also shows tumor-suppressive activity
in vivo We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine protein-ase, changing its biological activities In this study, we attempted to iden-tify the IGFBP-rP1-processing enzyme Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovar-ian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1 Serine proteinase inhibitors effect-ively blocked the IGFBP-rP1 cleavage in the OVISE cell culture The con-ditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity The mem-brane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1)
by immunoblotting When the membrane fraction was separated by SDS⁄ PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase
A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1 Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface
Abbreviations
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; AGM, angiomodulin; FBS, fetal bovine serum; HAI-1, hepatocyte growth factor activator inhibitor-1; HGF, hepatocyte growth factor; HLE, hepatocellular carcinoma; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; IGFPB-rP1, IGFBP-related protein-1; MMP, matrix metalloproteinase; MT-SP1, membrane-type serine proteinase matriptase; PSF, prostacyclin-stimulating factor; TAF, tumor-derived cell adhesion factor; siRNA, small interfering RNA; tPA, tissue plasminogen activator uPA, urokinase-type plasminogen activator.
Trang 2Angiomodulin (AGM) was initially purified as a
tumor-derived cell adhesion factor (TAF) from human
bladder carcinoma cells [4,5] Its cDNA was cloned
from human leptomeningial cells as mac25 [6] and from
human fibroblasts as prostacyclin-stimulating factor
(PSF) [7] Since AGM has a relatively low structural
homology to IGFBPs, the name of IGFBP-related
pro-tein-1 (IGFBP-rP1) has recently been proposed for
AGM⁄ mac25 ⁄ PSF AGM ⁄ IGFBP-rP1 exerts a weak
cell adhesion activity through heparan sulfate
proteog-lycans on the cell surface [4,5,8] and stimulates cell
growth in culture medium containing insulin or IGFs
[9,10] The IGFBP-rP1 mRNA is expressed in a wide
range of normal tissues including the heart, spleen,
ovary, small intestine and colon [11]
Immunohisto-chemical analysis has shown that IGFBP-rP1 is highly
expressed in the blood vessels of various human cancer
tissues [5] and in invading tumor cells [12] On the other
hand, other studies have shown that IGFBP-rP1
exhib-its a tumor-suppressive activity when overexpressed in
cancer cells [13–15] Thus, exact biological functions of
IGFBP-rP1 remain to be clarified
Various extracellular proteinases regulate cellular
functions by degrading or processing protein
sub-strates including extracellular matrix proteins, growth
factors and cell surface proteins For example,
mat-rix metalloproteinases (MMPs), such as membrane
type-1 matrix metalloproteinase (MT1-MMP),
matri-lysin (MMP-7) and gelatinases A⁄ B (MMP-2 ⁄ 9), are
known to play important roles in the process of
tumor invasion and metastasis [16–18] Serine
pro-teinases such as plasminogen activators, plasmin and
trypsin also contribute to expression of malignant
phenotypes in tumor cells [19,20] Recently,
consider-able attention has been focused on the physiological
and pathological functions of a membrane-bound
serine proteinase, matriptase (MT-SP1) [21–23] It is
well known that IGFBPs often undergo proteolytic
processing in various kinds of biological fluids such
as blood, synovial fluid and interstitial fluid, as well
as culture media [24,25] Several types of proteinases,
such as pregnancy-associated plasma proteins [26,27],
prostate specific antigen [28] and MMP-3 [29], have
been reported to cleave IGFBPs The proteolysis of
IGFBPs is thought to modulate the actions of IGFs
towards cells [29]
We recently found that IGFBP-rP1 is converted
from a single-chain form to a two-chain form by the
action of a trypsin-like serine proteinase [10] The
pro-teolytic processing of IGFBP-rP1 greatly reduced its
insulin⁄ IGF-dependent growth promoting activity but
enhanced its syndecan-1-mediated cell adhesion activity
[10] We report here that the membrane-bound serine
proteinase matriptase is responsible for the processing
of IGFBP-rP1
Results
Expression and processing of IGFBP-rP1
in various cell lines
We have reported that IGFBP-rP1 is proteolytically converted to a two-chain form during purification [10] The cleavage of IGFBP-rP1 leads to complete loss
of insulin⁄ IGF-1-dependent cell growth-stimulatory activity due to its loss of insulin⁄ IGF-binding ability
To examine whether the specific cleavage of IGFBP-rP1 also occurs in cultured cell systems, we tested expression and processing of IGFBP-rP1 in eight human cancer cell lines and one immortalized epithe-lial cell line (HEK293) When the conditioned media were analyzed by immunoblotting after reducing SDS⁄ PAGE, seven of the nine cell lines tested were found to secrete IGFBP-rP1 protein (Fig 1) Among them, OVISE ovarian adenocarcinoma cells and MKN-45 gastric adenocarcinoma cells secreted signifi-cant levels of the 25-kDa, cleaved form of IGFBP-rP1
On nonreducing SDS⁄ PAGE, IGFBP-rP1 in the two-conditioned media was separated to a single band of
33 kDa (data not shown), indicating that the cleaved IGFBP-rP1 was a two-chain form consisting of a
25 kDa chain and an 8 kDa chain [10] These results suggested that OVISE and MKN-45 cells expressed a high level of proteinase(s) responsible for the process-ing of IGFBP-rP1
To determine the class of the IGFBP-rP1-cleaving proteinase, OVISE cells were cultured in the presence
of various proteinase inhibitors and the processing of IGFBP-rP1 was analyzed As shown in Fig 2, serine proteinase inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and aprotinin, signifi-cantly inhibited the production of the cleaved form of IGFBP-rP1 (25 kDa), whereas a cysteine proteinase inhibitor (leupeptin), an aspartic proteinase inhibitor (pepstatin) and a metalloproteinase inhibitor (N-(R)- (2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-l-3-(2¢-naphthyl)alaninyl-l-alanine 2-aminoethyl amide; TAPI-1) did not affect the processing A commercially available proteinase inhibitor mixture containing AEBSF, aprotinin and some other inhibitors inhibited the processing to the same extent as AEBSF alone
As trypsin-type serine proteinases, but neither chymotrypsin-type nor elastase-type, are susceptible to the AEBSF inhibition, a trypsin-type serine proteinase was thought to be responsible for the IGFBP-rP1 cleavage
Trang 3Analysis of serine proteinases secreted
by various cell lines
Trypsin-type serine proteinases present in the
condi-tioned media of the nine cell lines were analyzed by
gelatin zymography (Fig 3A) To eliminate the activit-ies of metalloproteinases, the renatured gel was first treated with 1 mm EDTA and then incubated in the presence of 10 mm CaCl2, as described in Experimental procedures Of the nine cell lines tested, OVISE,
Fig 1 Analysis of noncleaved and cleaved
forms of IGFBP-rP1 in conditioned media of
eight cancer cell lines and one immortalized
cell line (HEK293) Concentrated conditioned
media were prepared from the cultures of
the nine indicated human cell lines, and
each sample containing the same amount of
protein (5 lg) was subjected to SDS⁄ PAGE
under reducing conditions on a 14% gel,
followed by immunoblotting with the
anti-TAF ⁄ IGFBP-rP1 antibody (upper panel) Bars
indicate the noncleaved form (33 kDa) and
the cleaved form (25 kDa) The cleaved form
is detected highly in OVISE and MKN-45 cell
lines and slightly in HLE and HEK293 cell
lines As a loading control, the same
vol-umes of conditioned media as those for the
immunoblotting were subjected to SDS ⁄
PAGE followed by protein staining with
Coomassie Brilliant Blue R-250 (lower
panel) Bars indicate the molecular size in
kDa Other experimental conditions and the
types of the cell lines used are described in
‘Experimental procedures’.
Fig 2 Effects of proteinase inhibitors on
processing of IGFBP-rP1 in culture of OVISE
cells OVISE ovarian carcinoma cells were
incubated in a serum-free culture medium
supplemented without (None) or with one
of the following proteinase inhibitors:
AE-BSF (100 l M ), aprotinin (75 n M ), leupeptin
(10 l M ), pepstatin A (1 l M ), TAPI-1 (2 l M ),
and a mixture (100 l M AEBSF, 75 n M
aprotinin, 5 l M bestatin, 1.5 l M E-64, 2 l M
leupeptin, and 1 l M pepstatin A) After
incubation for 2 days, IGFBP-rP1 in each
conditioned medium was analyzed by
immunoblotting, as described in Fig 1 The
upper panel shows immunoblots for
matriptase Bars indicate the noncleaved
form (33 kDa) and the cleaved form
(25 kDa) of IGFBP-rP1 The lower panel
shows protein staining patterns of
conditioned media Bars indicate the
molecular size in kDa.
Trang 4MKN-45 and DLD-1 cells commonly expressed a
major gelatinolytic activity at approximately 75 kDa
and additional weak activities at 95 kDa, 105 kDa and
lower molecular weight positions The 75 kDa activity
was faintly detected in HSC-4 and HEK293 cells
To date, several families of type II transmembrane
serine proteinases have been identified [30] Among
them, MT-SP1 is known to be expressed in many types
of epithelial cell lines and carcinoma cell lines, and to
be proteolytically released from the cell membranes
[21,31,32] The molecular size of a major form of
sol-uble matriptase is approximately 75 kDa To
deter-mine whether or not the 75 kDa proteinase in Fig 3A
was matriptase, the conditioned media of the nine cell
lines were subjected to immunoblotting with an
anti-matriptase antibody (M32) As shown in Fig 3B, the
conditioned media of OVISE, MKN-45 and DLD-1
cells clearly showed doublet bands at approximately
75 kDa and two minor bands at 95 and 110 kDa The
conditioned media of HSC-4 and HEK293 cells also
showed weak immunoreactive bands at the same
posi-tions These results attributed the gelatinolytic activit-ies at 75, 95 and 105 kDa in Fig 3A to matriptase The 75 kDa doublet has been reported to result from glycosylation [33] The glycosylation is thought to increase the stability of this protein against trypsin and other proteinases [34]
To further characterize the gelatinolytic activities in the OVISE cell conditioned medium, we examined effect of a serine proteinase inhibitor, AEBSF, on the proteinase activities (Fig 4) The sample was treated with 2 mm AEBSF before and⁄ or after SDS ⁄ PAGE and subsequent renaturation of separated proteins The pretreatment of the sample with the inhibitor par-tially blocked only the 75 kDa activity, whereas the inhibitor treatment after the protein renaturation strongly blocked the activities at 75, 95 and 110 kDa This indicated that only a part of the 75 kDa enzyme existed in an inhibitor-free active form in the concen-trated conditioned medium A weak activity of 40 kDa was not inhibited by the inhibitor, suggesting that it might be a metalloproteinase It has been reported that
110 95 75
EJ-1 ECV
-304 HT1080 OV
IS E MKN-45 HLE HSC-4 DLD-1 HEK293
110 95 75
EJ-1 ECV
-304
HT1080 OV
IS E MKN-45 HLE HSC-4 DLD-1 HEK293
A
B
Fig 3 Analysis of serine proteinases secre-ted by nine human cell lines Conditioned media were prepared from the cultures of the indicated, nine human cell lines The concentrated conditioned media containing the same amount of protein (5 lg) were subjected to gelatin zymography (A) and immunoblotting with the antimatriptase antibody M32 (B) as described in ‘Experi-mental procedures’ In the gelatin zymogra-phy, metalloproteinase activities were eliminated by incubating the renatured gel with 1 m M EDTA for 30 min (A) Gelatin zymograms of the conditioned media from the nine cell lines The conditioned media of OVISE and MKN-45, which showed high IGFBP-rP1 cleavage in Fig 1, showed a major activity at 75 kDa and minor activities
at 95 and 110 kDa, which were indicated by bars (B) Immunoblots for matriptase The
75 kDa major band seems to correspond mainly to a single-chain, latent matriptase, while the 95 and 110 kDa bands seem to correspond to two-chain, active forms complexed with HAI-1 [18,32].
Trang 5membrane-bound matriptase is shed into culture
med-ium in some different forms [21] The soluble
matrip-tase includes a 70 kDa single-chain, latent form as a
major component, a two-chain, active enzyme
com-plexed with two different sizes of hepatocyte growth
factor (HGF) activator inhibitor (HAI-1) (total
molecular sizes of 95 and 110 kDa), and a trace of an
inhibitor-free active enzyme [21,31,32] The 95 and
110 kDa matriptase⁄ HAI-1 complexes are expected
not to react with AEBSF, because their active serine
residues are bound to HAI-1 Based on these facts,
the 95- and 110-kDa bands in zymography (Figs 3A
and 4) and immunoblotting (Fig 3B) are most likely
to correspond to the matriptase⁄ HAI-1 complexes
The 95- and 110-kDa gelatinolytic activities seemed to
result from partial dissociation of an active matriptase
from its inhibitor HAI-1 after SDS⁄ PAGE Similarly,
the 75-kDa activity that was not inhibited by the
pre-treatment with AEBSF was thought to be an active
matriptase, which had been dissociated from HAI-1 by
the SDS treatment In addition, immunoblotting under
reducing conditions suggested that the majority of the
75-kDa immunoreactive band was a single-chain latent
matriptase (data not shown) [32]
The correlation between the IGFBP-rP1-cleaving activity (Fig 1) and the expression of matriptase in MKN-45 and OVISE cell lines suggested matriptase as
a candidate for the IGFBP-rP1-processing enzyme Moreover, it has been reported that matriptase prefers basic P1, P3 and P4 residues to cleave substrates [33] This consensus sequence is found in the cleavage site sequence of IGFBP-rP1, where Lys97, Lys95 and Arg94 residues are located at P4, P3 and P1 sites, respectively [10] These facts prompted us to examine whether or not matriptase is the IGFBP-rP1-processing enzyme However, when purified IGFBP-rP1 was incubated with the conditioned medium of OVISE or DLD-1 cells, the latter of which contained the highest amount of matriptase, its processing to a two-chain form was not observed (data not shown)
IGFBP-rP1-cleaving activity of membrane fraction from OVISE cells
As IGFBP-rP1 is cleaved in cultured OVISE cells, we next examined the possibility that cell-associated serine proteinase(s) might cleave IGFBP-rP1 A membrane fraction of OVISE cells was prepared as described under ‘Experimental procedures’ and incubated with purified IGFBP-rP1 Figure 5A (lanes 1 and 2) shows that the cleaved form of IGFBP-rP1 significantly increased during the incubation This cleavage was inhibited effectively by aprotinin, suggesting that a serine proteinase(s) catalyzed this processing (Fig 5A, lane 3) We further analyzed serine proteinase activit-ies in the membrane fraction of OVISE cells by gelatin zymography This analysis revealed the presence of a gelatinolytic enzyme, with an approximate molecular size of 80 kDa, in the membrane fraction (Fig 5B, lane 1) When analyzed on the same gel, the apparent molecular size of the gelatinolytic activity in the membrane was slightly higher than the 75 kDa activity found in the conditioned medium (data not shown) Immunoblotting with the antimatriptase antibody showed an immunoreactive band at the same position
as the gelatinolytic activity (Fig 5B, lane 2), suggest-ing that the 80-kDa serine proteinase was a mem-brane-bound matriptase
To verify that the 80 kDa proteinase has the IGFBP-rP1-cleaving activity, we separated the proteins
in the membrane fraction of OVISE cells by SDS⁄ PAGE After electrophoresis, the proteins on the gel were renatured and the gel was horizontally divided into eight equal parts Each piece of the gel was then incubated with purified IGFBP-rP1 and the cleavage
of the protein was analyzed as described under Experi-mental procedures As shown in Fig 6A, gel fractions
Fig 4 Effect of serine proteinase inhibitor AEBSF on gelatinolytic
activities of OVISE cell conditioned medium AEBSF was added to
make a final concentration of 2 m M into the OVISE cell conditioned
medium before SDS ⁄ PAGE (lanes 2 and 4) and ⁄ or into the reaction
buffer after the SDS ⁄ PAGE and subsequent renaturation (lanes 3
and 4) –, No addition, +, addition Ordinate indicates the molecular
size in kDa Arrowheads show the matriptase bands Other
experi-mental conditions are described in ‘Experiexperi-mental procedures’ and
in the legends to Fig 3A.
Trang 64–6 had the IGFBP-rP1-cleaving activity and fraction
6 showed the highest activity When the proteins in
the gel pieces were analyzed by immunoblotting, an
80 kDa matriptase was also detected in fraction 6
(Fig 6B), indicating that this membrane-bound serine proteinase is the prime candidate for the IGFBP-rP1-cleaving proteinase in OVISE cells
IGFBP-rP1-cleaving activity of purified matriptase The results of the SDS⁄ PAGE separation of the mem-brane-bound enzyme suggested that the soluble form
of matriptase might cleave IGFBP-rP1 if separated from inhibitors To test this possibility, the condi-tioned medium of DLD-1 cells, which contained the highest activity of matriptase, was separated by SDS⁄ PAGE and assayed for the IGFBP-rP1-cleaving
80
B
80
33 25
Fig 5 Analysis of IGFBP-rP1-cleaving enzyme present in
mem-brane fraction of OVISE cells A memmem-brane fraction was prepared
from OVISE cells as described in ‘Experimental procedures’ and
used for the following assays (A) IGFBP-rP1-cleaving activity
Puri-fied IGFBP-rP1 (500 ng) was incubated with the membrane fraction
(20 lg protein) in the presence (lane 3) or absence (lane 2) of
75 n M aprotinin (serine proteinase inhibitor) The incubated samples
were subjected to immunoblotting under reducing conditions with
the anti-TAF ⁄ IGFBP-rP1 antibody Lane 1, no incubation Bars
indi-cate the noncleaved form (33 kDa) and the cleaved form (25 kDa).
(B) Gelatin zymography (lane 1) and immunoblotting with the
anti-matriptase antibody M32 The membrane fraction containing 15 lg
protein was run on a gelatin-containing gel under nonreducing
con-ditions for the zymography (lane 1), and the same sample
contain-ing 10 lg protein was subjected to immunoblottcontain-ing with the
antimatriptase antibody M32 The bar indicates a gelatinolytic band
at approximately 80 kDa in lane 1 and a matriptase band at almost
the same position in lane 2 Other experimental conditions are
des-cribed in ‘Experimental procedures’.
Fig 6 Fractionation of IGFBP-rP1-cleaving enzyme and matriptase present in OVISE cell membrane by SDS ⁄ PAGE The membrane fraction (20 lg proteinÆlane)1) of OVISE cells was separated by non-reducing SDS ⁄ PAGE on two lanes of a 7.5% gel After the gel was washed with 1% Triton X-100 for protein renaturation, each of the two lanes was divided into eight fractions and used for one of the two following assays (A) IGFBP-rP1-cleaving activity Each gel frac-tion was incubated with IGFBP-rP1 (500 ng) at 4 C for 24 h fol-lowed by 37 C for 24 h The incubated samples were subjected to immunoblotting under reducing conditions with the anti-TAF ⁄ IGFBP-rP1 antibody (None), IGFBP-rP1 incubated without gel frac-tion Bars indicate the noncleaved form (33 kDa) and the cleaved form (25 kDa) Fraction 6 shows the highest activity (B) Fraction-ation of matriptase Gel fractions from another lane were used to detect matriptase Each gel fraction was extracted with the SDS sample buffer and applied to SDS ⁄ PAGE under nonreducing condi-tions on a 10% gel and immunoblotted with the antimatriptase anti-body M32 An arrow indicates an immunoreactive band of matriptase MF, the membrane fraction before separation Other experimental conditions are described in ‘Experimental procedures’.
Trang 7activity The IGFBP-rP1-cleaving activity was detected
at fractions corresponding to approximately 75 and
95 kDa, which also showed immunoreactive bands to
the antimatriptase antibody in immunoblotting (data
not shown) A similar result was also obtained when
the conditioned medium of OVISE cells was separated
by SDS⁄ PAGE (data not shown) These results
strongly suggested that soluble forms of matriptase are
able to cleave IGFBP-rP1 in the absence of HAI-1 or
other inhibitors
To further confirm this possibility, we purified an
inhibitor-free, soluble form of matriptase from the
conditioned medium of DLD-1 cells, as described in
‘Experimental procedures’ The purified matriptase
preparation contained the 75-kDa matriptase and a
few minor proteins, as analyzed by SDS⁄ PAGE
(Fig 7A) Gelatin zymography under nonreducing
conditions confirmed that the 75 kDa matriptase had a
proteolytic activity (Fig 7B) Immunoblotting analysis
under nonreducing conditions showed a single
immuno-reactive band for matriptase at 75 kDa, but under
reducing conditions it was split into a major 75-kDa
band of the single-chain, latent enzyme and a minor
50 kDa band of the two-chain, active enzyme (Fig 7C) [10] Based on the relative band intensity, the percent-age of the active enzyme to the total matriptase was estimated to be approximately 30%
We previously reported that trypsin cleaves IGFBP-rP1 at the same site as an endogenous processing enzyme [10] The IGFBP-rP1-cleaving activities of matriptase and trypsin were compared at varied con-centrations (Fig 8) Fifty nanograms of the 33 kDa IGFBP-rP1 was almost completely cleaved to the
25 kDa form by 5 ng of the total matriptase, which contained the active enzyme as a minor component
On the other hand, 10 ng of trypsin converted a major part of the 33 kDa IGFBP-rP1 to the 25 kDa form, but an increased amount (50 ng) of trypsin nonspecifi-cally degraded both forms of IGFBP-rP1 These results indicated that the two-chain, active matriptase has a much higher IGFBP-rP1-cleaving activity than trypsin Furthermore, we tried to identify the cleavage site of IGFBP-rP1 by the purified matriptase The 25 kDa form of IGFBP-rP1 obtained by the treatment with matriptase was applied to an automated protein sequencer The N-terminal amino acid sequence of the
25 kDa band of IGFBP-rP1 was determined to be
A98GAAAGGPG106, suggesting that this protein had been cleaved between K(Lys)97 and A(Ala)98 This cleavage site was identical to that previously deter-mined for a naturally cleaved IGFBP-rP1
All these results demonstrate that the soluble form
of active matriptase cleaves IGFBP-rP1 but HAI-1
or some other inhibitors block its activity in culture medium
Effects of matriptase siRNAs on IGFBP-rP1 cleavage in OVISE cells
To show that matriptase is an endogenous IGFBP-rP1-processing enzyme in OVISE cells, we designed three siRNAs for matriptase and examined their effects
on the IGFBP-rP1 cleavage As negative controls, OVISE cells were treated with a scrambled RNA or with the lipofectamine reagent alone Although the scrambled RNA had some cytotoxic effect, there was
no significant difference in the profiles of secreted pro-teins among the control and siRNA-treated cultures (Fig 9A) Immunoblotting with the antimatriptase antibody showed that all of the three siRNAs decreased the amount of soluble matriptase to less than 20% of the control levels (Fig 9B) When the IGFBP-rP1 secreted into culture medium by OVISE cells was analyzed by immunoblotting, contrasting patterns of the secreted IGFBP-rP1 were obtained
Fig 7 Electrophoretic analyses of purified matriptase Soluble
mat-riptase was purified from the conditioned medium of DLD-1 cells
as described in the text The purified matriptase (approximately
200 ng of total proteins) was subjected to the following analyses.
(A) SDS ⁄ PAGE under nonreducing conditions followed by silver
staining Arrowhead indicates the matriptase band Bars indicate
the molecular size in kDa (B) Gelatin zymography Arrowhead
indi-cates the gelatinolytic activity by matriptase (C) Immunoblotting
with the antimatriptase antibody M32 under nonreducing conditions
(lane 1) and reducing conditions (lane 2) In lane 2, the 75-kDa band
corresponds to the single-chain, latent enzyme, while the 50 kDa
band corresponds to the heavy chain of the two-chain, active
enzyme Other experimental conditions are described in
‘Experi-mental procedures’.
Trang 8between the control and siRNA-treated cultures
(Fig 9C) In the two control cultures, the 33-kDa,
uncleaved IGFBP-rP1 was faintly detected compared
with the 25-kDa, cleaved form, whereas in the
siRNA-treated cultures, the uncleaved IGFBP-rP1 was a
major component This indicated that the cleavage of
IGFBP-rP1 was effectively reduced by the siRNA
treatment These results confirmed that matriptase acts
as an IGFBP-rP1-processing enzyme in the culture of OVISE cells
Discussion
In this study, we first identified the type II membrane-bound serine proteinase matriptase as a processing enzyme of IGFBP-rP1⁄ AGM Matriptase was initially
Fig 8 Cleavage of IGFBP-rP1 by purified matriptase and trypsin (A) Matriptase IGFBP-rP1 (50 ng) was incubated with the indicated amounts of purified matriptase at 37 C for 5 h in 10 lL of a reaction mixture containing 20 m M Tris ⁄ HCl (pH 7.5), 0.1 M NaCl and 10 m M
CaCl2 The original concentration of matriptase was determined for the total 75-kDa protein based on the band intensity relative to that of bovine serum albumin as standard (Fig 7A) (B) Trypsin IGFBP-rP1 was incubated with the indicated amounts of TPCK-trypsin under the same conditions except for the absence of CaCl 2 in the reaction mixture The proteolytic cleavage of IGFBP-rP1 in (A) and (B) was analyzed
by immunoblotting as described in Fig 1 Bars indicate the 33 kDa, uncleaved form and the 25 kDa, cleaved form of IGFBP-rP1.
Fig 9 Effects of matriptase siRNAs on IGFBP-rP1 processing in culture of OVISE cells OVISE cells at 50–60% confluence in 60 mm culture dishes were transfected with 200 pmol of each of three siRNAs (si973, si1513 and si2578) using Lipofectamine 2000 reagent As negative controls, the cells were treated with a scrambled RNA (sc) or with the lipofectamine reagent alone (Cont.) These cultures were incubated in serum-containing medium overnight, and then in serum-free medium for 2 days The resultant conditioned media were collected and con-centrated The concentrated samples containing 10 lg protein were subjected to reducing SDS ⁄ PAGE followed by the Coomassie Brilliant Blue staining (A), nonreducing immunoblotting to detect matriptase (B), and reducing immunoblotting to detect IGFBP-rP1 (C) Bars indicate the molecular sizes of marker proteins in (A), the 110-, 95- and 75-kDa bands of matriptase in (B), and the 33-kDa, uncleaved form and the 25-kDa, cleaved form of IGFBP-rP1 in (C) The scrambled RNA-treated culture (2nd lane) was low in the total band intensity of matriptase (B) and IGFBP-rP1 (C) as compared with the control or siRNA-treated cultures This seemed due to the cytotoxic effect of the scrambled RNA Other experimental conditions are described in Experimental procedures.
Trang 9found as a trypsin-like serine proteinase secreted by
human breast cancer cells [35] and later purified as a
complex with its natural inhibitor HAI-1 from human
milk [21] Matriptase is expressed in normal epithelial
tissues such as the skin, stomach, colon, kidney, breast,
ovary and pancreas, but not in mesenchyma [36,37] A
recent study with matriptase-deficient mice has shown
that matriptase plays critical roles in the epidermal
barrier function, hair follicle development and thymic
homeostasis [38] Matriptase is also expressed in cancer
tissues of the breast, ovary, uterus and colon and also
by some mammary and ovarian carcinoma cell lines
in vitro[36] Since matriptase is able to degrade
extra-cellular matrix proteins and to activate
urokinase-type plasminogen activator (uPA) [33,39], hepatocyte
growth factor (HGF) [39] and protease-activated
receptor-2 (PAR-2) [33], it is expected to play some
roles in the growth, invasion and metastasis of human
carcinoma cells
We previously reported that IGFBP-rP1 is cleaved
to a two-chain form by a trypsin-like serine proteinase
[10] In the present study, we found that OVISE
ovar-ian carcinoma cells cleaved endogenous IGFBP-rP1,
and their membrane fraction cleaved exogenous
IGFBP-rP1 in a cell-free solution The
IGFBP-rP1-cleaving activity in the OVISE cell membrane
comi-grated with membrane-bound matriptase on SDS⁄
PAGE Furthermore, the treatment of OVISE cells
with matriptase siRNAs efficiently blocked both
mat-riptase expression and the cleavage of IGFBP-rP1 It
is also noted that the cleavage sequence of IGFBP-rP1
is consistent with the most preferable sequence in
sub-strate proteins of matriptase [33] Indeed, a soluble
form of matriptase purified in an inhibitor-free form
efficiently cleaved IGFBP-rP1 at the same site as that
found in a naturally cleaved IGFBP-rP1 All these
facts indicate that the membrane-bound matriptase is
a natural processing enzyme of IGFBP-rP1 On the
other hand, soluble forms of matriptase, which are
released from cell membranes by proteolysis, were
detected in the conditioned media of at least 5 cell
lines out of 9 cell lines tested The processing of
endo-genous IGFBP-rP1 was correlated with the amount of
soluble matriptase in the conditioned media Although
the purified soluble matriptase could cleave
IGFBP-rP1, the conditioned media of OVISE and DLD-1 cells
did not show the IGFBP-rP1-processing activity unless
they were separated by SDS⁄ PAGE Our recent
analy-sis has detected soluble matriptase in 19 of 24 human
carcinoma cell lines tested, which included carcinomas
of the breast, lung, stomach and colon [32] The
sol-uble matriptase mostly existed in a single-chain, latent
form as a major component and two-chain forms
complexed with its inhibitor HAI-1, in agreement with the past reports [18,30] Therefore, soluble matriptase released from cell membrane is expected to have a very low, if any, proteolytic activity The IGFBP-rP1-clea-ving activities of the SDS⁄ PAGE fractions and the matriptase purified from DLD-1 conditioned medium indicate that the soluble form of activated matriptase can cleave IGFBP-rP1, but its activity is masked by HAI-1 in culture medium It was recently reported that the matriptase zymogen might be auto-activated by interacting with HAI-1 on the cell surface [40] The proteolytic action of matriptase, including the process-ing of IGFBP-rP1, seems to be restricted to the cell surface and its close vicinity
Although the present study demonstrates that mat-riptase cleaves IGFBP-rP1, our data do not exclude the possibility that other proteinases, especially serine proteinases, also cleave IGFBP-rP1 We previously reported that many human cancer cell lines secrete an active or latent form of trypsin and a 75-kDa serine proteinase [20], the latter of which was identified as matriptase in a recent study [32] We have also repor-ted that OVISE cells secrete uPA, but neither trypsin nor tissue plasminogen activator (tPA) [41] In addi-tion, it was previously found that trypsin cleaves IGFBP-rP1 to the same two-chain form as that found
in conditioned media [10] In the present study, we examined whether or not uPA and tPA cleave IGFBP-rP1 in test tubes, but the IGFBP-IGFBP-rP1 cleavage was seen with neither uPA nor tPA (data not shown) There-fore, matriptase seems to be a major IGFBP-rP1-pro-cessing enzyme, at least in OVISE cells, and possibly
in MKN-45 cells Recent studies have revealed the presence of four families of type II transmembrane ser-ine proteinase [30] The matriptase subfamily consti-tutes three members (matriptase, matriptase 2 and matriptase 3) It is conceivable that some of these mem-brane-bound serine proteinases or their soluble forms are also involved in the processing of IGFBP-rP1
It is well known that some IGFBPs undergo proteo-lytic cleavage [24,25] In biological fluids, IGFBPs bind IGFs with high affinity, protecting the growth factors from proteolytic degradation The proteolytic cleavage
of IGFBPs is thought to contribute to the release of IGFs from IGF⁄ IGFBP complexes to interact with IGF receptors on the cell surface This mechanism may not be directly applicable to the case of IGFBP-rP1, because IGFBP-rP1 has a far lower affinity for IGFs than IGFBPs The high affinity binding of IGFs
to IGFBPs limits the interaction of the growth factors with the cell surface receptors Therefore, IGFBPs gen-erally inhibit IGF-stimulated cell growth in vitro [25]
In contrast, IGFBP-rP1 stimulates cell growth in the
Trang 10presence of insulin or IGF-1 in vitro, presumably due
to its low affinity for the factors [9,10] On the other
hand, some studies have shown that IGFBP-rP1
exhib-its a tumor-suppressive activity when overexpressed in
cancer cells [13–15] The apparent discrepancy between
the in vitro and in vivo studies is not clearly explained
IGFBP-rP1 also shows IGF⁄ insulin-independent
activ-ities It has affinity for heparin, type IV collagen and
syndecan-1 [5,8,10] These activities seem to be
respon-sible for the cell adhesion activity of IGFBP-rP1
in vitroand its dense deposition on the basement
mem-brane of blood vessels in tumor tissues [5] These
IGF⁄ insulin-dependent and independent activities of
IGFBP-rP1 are notably altered by its proteolytic
clea-vage [10] For example, IGFBP-rP1 loses its IGF⁄
insulin-binding activity and IGF⁄ insulin-dependent
growth-stimulating activity but acquires high cell
adhe-sion activity by proteolytic cleavage It seems
conceiv-able that matriptase regulates tumor growth by
modulating biological activities of IGFBP-rP1, as well
as other growth-regulating proteins, in vivo
In this study, we identified a new substrate of
mat-riptase Matriptase may exert a broad range of
func-tions in regulating cellular growth, apoptosis and
differentiation by degrading or processing a variety of
extracellular proteins
Experimental procedures
Materials
The sources of materials used are as follows: aprotinin,
leu-peptin, pepstatin A, AEBSF and proteinase inhibitor mixture
that contains AEBSF, aprotinin, bestatin, E-64, leupeptin
and pepstatin A from Wako Pure Chemical Industries
(Osaka, Japan); gelatin from Difco (Detroit, MI, USA);
N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-l-3-(2¢-naphthyl)alaninyl-l-alanine 2-aminoethyl amide
(TAPI-1) from Peptide Institute (Osaka, Japan) IGFBP-rP1
was purified from the conditioned medium of the human
bladder carcinoma cell line EJ-1, as described previously [10]
An anti-TAF⁄ IGFBP-rP1 monoclonal antibody (#88) [5]
and an antimatriptase monoclonal antibody (M32) [21] were
raised against purified IGFBP-rP1 and purified matriptase,
respectively All other chemicals were of analytical grade or
the highest quality commercially available
Cell cultures and preparation of conditioned
medium
Types of human cancer cell lines used are as follows:
HSC-4, tongue squamous cell carcinoma; HT1080, fibrosarcoma;
EJ-1 and ECV-304, bladder carcinomas; DLD-1, colon
adenocarcinoma; OVISE, ovarian clear cell adenocarcino-ma; MKN-45, adenosquamous carcinoma of the stomach; HLE, hepatocellular carcinoma The source and properties
of OVISE cells were described before [41] The human embryonic kidney cell line HEK293 (ATCC CRL-1573) was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) The other cell lines were obtained from Japanese Cancer Resources Bank (JCRB) in National Institute of Biomedical Innovation (Osaka, Japan) To prepare conditioned medium, each cell line was grown to semiconfluence in 90-mm culture dishes contain-ing a 1 : 1 mixture of Dulbecco’s modified Eagles medium and Ham’s F12 medium (Gibco; Grand Island, NY, USA), DME⁄ F12, supplemented with 10% fetal calf serum (FCS) The cells were rinsed three times with serum-free DME⁄ F12, and the culture was further continued in the presence or absence of various proteinase inhibitors in serum-free DME⁄ F12 After incubation for 2 days, the resultant conditioned medium was collected, clarified by centrifugation and dialyzed against distilled water at 4C The dialyzed sample was then lyophilized and dissolved in
a 100th volume of 10 mm Tris⁄ HCl (pH 7.5) to the original conditioned medium
Preparation of membrane fractions
OVISE cells were grown to confluence in the serum-contain-ing medium, rinsed three times with ice cold NaCl⁄ Piand then scraped in the presence of 20 mm Hepes (pH 7.5) con-taining 250 mm sucrose at 4C The cell suspension was then homogenized with a Dounce homogenizer The homogenate was centrifuged at 1500 g for 7 min to remove the nuclei The postnuclear supernatant was further centrifuged at
50 000 g for 30 min The resultant pellet was then dissolved
in 20 mm Tris⁄ HCl (pH 7.5) containing 0.5 m KCl, 0.15 m NaCl and 1% Triton X-100, and the insoluble substances were removed by centrifugation at 14 000 g for 20 min The supernatant was used as a crude membrane fraction
SDS–polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting
SDS⁄ PAGE was performed on 7.5, 10, or 14% polyacryl-amide gel slabs (85 mm wide, 1 mm thick, and 70 mm long) under reducing or nonreducing conditions Separated pro-teins were stained with silver Immunoblotting was per-formed as described previously [42] Briefly, proteins on gels were transferred onto nitrocellulose membranes (Schlei-cher & Schuell, Keene, NH, USA) The membrane was blocked with skimmed milk and successively treated with
an anti-TAF⁄ IGFBP-rP1 monoclonal antibody (#88) or an antimatriptase monoclonal antibody (M32) as the first anti-body, the second antibody (biotinylated antimouse IgG, Vector Laboratory, Burlingham, CA, USA), and alkaline