In addition, TTSPs possess a stem region that may contain a diverse array Keywords activation of pro-hepatocyte growth factor; hepatocyte growth factor activator inhibitor type 1 HAI-1;
Trang 1inhibited by hepatocyte growth factor activator inhibitor type 1 and activates pro-hepatocyte growth factor
Tomio Hashimoto1, Minoru Kato2, Takeshi Shimomura2and Naomi Kitamura1
1 Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta,
Midori-ku, Yokohama, Japan
2 Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, Kamoshida-cho, Aoba-ku, Yokohama, Japan
Introduction
Type II transmembrane serine proteases (TTSPs) are
structurally defined by the presence of a short
N-termi-nal cytoplasmic domain, a transmembrane domain
located near the N-terminus, and a C-terminal extra-cellular serine protease domain In addition, TTSPs possess a stem region that may contain a diverse array
Keywords
activation of pro-hepatocyte growth factor;
hepatocyte growth factor activator inhibitor
type 1 (HAI-1); Kunitz-type inhibitor;
TMPRSS13; type II transmembrane serine
protease (TTSP)
Correspondence
N Kitamura, Department of Biological
Sciences, Graduate School of Bioscience
and Biotechnology, Tokyo Institute of
Technology, Nagatsuta, Midori-ku,
Yokohama 226-8501, Japan
Fax: +81 45 924 5771
Tel: +81 45 924 5701
E-mail: nkitamur@bio.titech.ac.jp
(Received 31 May 2010, revised 26 August
2010, accepted 24 September 2010)
doi:10.1111/j.1742-4658.2010.07894.x
Type II transmembrane serine proteases (TTSPs) are structurally defined
by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain The human TTSP family consists of 17 members Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigene-sis and viral infections The activities of TTSPs are regulated by endoge-nous protease inhibitors However, protease inhibitors of most TTSPs have not yet been identified In this study, we investigated the inhibitory effect
of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family We found that the protease activity of a member, TMPRSS13, was inhibited
by HAI-1 A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2) In addition, an in vitro protein binding assay showed that NK1 formed com-plexes with TMPRSS13, but NK1LK2 did not TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form
in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited
by NK1 The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2 These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1
Abbreviations
BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; HA, haemagglutinin; HAI-1, hepatocyte growth factor activator inhibitor type 1; HAI-2, hepatocyte growth factor activator inhibitor type 2; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; HPAI, highly pathogenic avian influenza; IC50,the concentration of inhibitor that inhibited the enzymatic activity by 50% compared with the uninhibited control; LDL, low-density lipoprotein; MSPL, mosaic serine protease large form; PBS, phosphate-buffered saline; TTSP, type II transmembrane serine protease.
Trang 2of protein domains [1,2] The human TTSP family
consists of 17 members, which are classified into four
subfamilies [2] TTSPs are synthesized as inactive
sin-gle-chain pro-enzymes, the proteolytic cleavage of
which is required for the enzymes to exert their activity
[2] Several members of the TTSP family have been
shown to have pivotal functions in development and
homeostasis [1,2] Moreover, recent studies revealed
that some members are involved in tumorigenesis
and viral infections [3] However, the physiological and
pathological functions of most members of the TTSP
family remain to be investigated
The activities of some members of the TTSP family
are regulated by endogenous protease inhibitors, which
include Kunitz-type inhibitors and serpins [2]
Hepato-cyte growth factor activator inhibitor type 1 (HAI-1),
a Kunitz-type serine protease inhibitor, is implicated in
the inhibition of two members of the TTSP family,
matriptase and hepsin HAI-1 was originally identified
as a potent inhibitor of hepatocyte growth factor
acti-vator (HGFA), a blood coagulation factor XII-like
serine protease that converts pro-hepatocyte growth
factor (pro-HGF) to the active form [4] HAI-1
was also isolated from human milk in a complex with
matriptase, and potentially inhibits the protease
activ-ity of matriptase [5] The physiological role of the
inhi-bition of matriptase by HAI-1 was determined by
analysing knockout mice The homozygous deletion of
HAI-1 resulted in embryonic lethality due to impaired
formation of the placental labyrinth layer [6,7],
whereas matriptase⁄ HAI-1 double-deficient mice
formed the placental labyrinth and developed to term,
indicating an essential role of the inhibition of
matrip-tase by HAI-1 during placental development in the
mouse embryo [8] Hepsin has an ability to convert
pro-HGF to the active form with an activity
com-parable with HGFA The HGF-converting activity is
inhibited by HAI-1 [9,10]
The protease inhibitors that regulate the activities of
most TTSPs have not been identified yet Because
HAI-1 is a potent inhibitor of matriptase and hepsin,
it might also inhibit the protease activities of other
TTSPs To test this possibility, we have searched for
TTSPs targeted by HAI-1, and found that the activity
of TMPRSS13 is potentially inhibited by HAI-1
TMPRSS13 is a splice variant of mosaic serine
pro-tease large form (MSPL), and belongs to the
hep-sin⁄ TMPRSS subfamily of the TTSP family MSPL
and TMPRSS13 were isolated by a PCR-based
screen-ing from a human lung cDNA library usscreen-ing degenerate
primers designed on the basis of the conserved
cata-lytic motif of known trypsin-type serine proteases
[11,12] The amino acid sequence of TMPRSS13 is
identical to that of MSPL except for an insertion of five amino acids in the N-terminal cytoplasmic region and the C-terminal end following the protease domain,
in which TMPRSS13 has eight amino acids and MSPL has a different 27 amino acids [12] MSPL and TMPRSS13 preferentially recognize cleavage sites con-sisting of paired basic amino acid residues [12] Recently, MSPL and TMPRSS13 have been shown to
be candidates for haemagglutinin (HA)-processing pro-teases of highly pathogenic avian influenza (HPAI) viruses Namely, a full-length recombinant HA of an HPAI virus was efficiently converted to mature HA subunits with membrane-fused giant cell formation in MSPL- or TMPRSS13-transfected cells, but not in untransfected cells Furthermore, infection and multi-plication of the HPAI virus were detected in the trans-fected cells [13] MSPL and TMPRSS13 are expressed
in a variety of tissues, and predominantly in lung, placenta, pancreas and prostate [12] Therefore, in addition to the function in HA processing, MSPL and TMPRSS13 may have physiological functions in these tissues that remain to be explored
Here, we characterize in detail the inhibitory effect
of HAI-1 on TMPRSS13 Moreover, we demonstrate
a possible physiological function of TMPRSS13, that
is its HGF-converting activity
Results
Search for TTSPs targeted by HAI-1
To search for targets of HAI-1, we constructed Escheri-chia coli expression vectors encoding protease domains with short pro-sequences of six members of the TTSP family These proteases have been shown to be co-expressed with HAI-1 in various tissues (database of BioExpress System, Gene Logic Inc., Gaithersburg,
MD, USA) Then, the putative activation cleavage sequences were replaced with the enterokinase recogni-tion sequence (DDDDK) for activation in vitro Escherichia colicells were transformed with the expres-sion vectors, and expressed proteins were purified from cell lysate The purified proteins were treated with enterokinase, and protease activity was measured using synthetic substrates suitable for each TTSP TMPRSS3 and TMPRSS4 expressed in this system did not show protease activity Thus, other TTSPs that did show activity were tested for the inhibitory activity of HAI-1 using the first Kunitz domain of HAI-1 (HAI-1–K1) Among these TTSPs, TMPRSS11A, HAT-like 4 and HAT-like 5 were not inhibited by HAI-1–K1 By contrast, the protease activity of TMPRSS13 was potentially inhibited by HAI-1–K1 We therefore
Trang 3characterized the inhibitory activity of HAI-1 against
TMPRSS13 in detail
Preparation and activation of a secreted form of
pro-TMPRSS13 expressed in mammalian cells
TMPRSS13 expressed in E coli showed weak protease
activity, probably because of incorrect protein folding
We therefore expressed pro-TMPRSS13 in mammalian
cells To obtain pro-TMPRSS13 from conditioned
medium of mammalian cells, we constructed an
expres-sion vector encoding a secreted form of this protein
that lacked the cytoplasmic and transmembrane
domains In addition, the putative activation cleavage
sequence (AMTGR325) was replaced with the
entero-kinase recognition sequence (DDDDK) for activation
in vitro, and the protein was tagged at the C-terminus
with myc-His for purification and immunoblot analysis
(Fig 1A) COS-7 cells were transiently transfected with
the expression vector The protein was purified from
the conditioned medium of the transfected cells The
immunoblot analysis of the purified protein using
an anti-c-Myc IgG showed a band of 63 kDa under
reducing and nonreducing conditions (Fig 1B,C), indi-cating that pro-TMPRSS13 was highly expressed in this system
To activate pro-TMPRSS13, we treated the protein with enterokinase The immunoblot analysis of the reaction product using the anti-c-Myc IgG showed a band of 37 kDa under reducing conditions (Fig 1B), and that of 67 kDa under nonreducing conditions (Fig 1C) The 37 kDa band probably corresponded to the protease domain of TMPRSS13, suggesting the proteolytic activation of the pro-protein Detection of the 67 kDa band suggests that the pro-protein was cleaved at a single site, and the cleaved protein is a two-chain form linked by a disulfide bond The prote-ase domain of TMPRSS13 was quantified by scanning densitometry of the immunoblot, using the protease domain of the TMPRSS13 expressed in E coli as a standard The protease activity of the enterokinase-treated pro-TMPRSS13 was measured using a synthetic substrate (Pyr–RTKR–MCA), which has been shown as
an efficient substrate of the protease [13] This substrate was not cleaved by enterokinase itself, or by the untreated pro-TMPRSS13 The enterokinase-treated
TM LDLA SRCR SPD
SS
Pro-TMPRSS13 (wild-type)
AMTGR 325 I 326 VGG
N myc-His- C
SS
DDDDKIVGG
Recombinant Pro-TMPRSS13
N
myc-His- C
SS Enterokinase
(kDa)
100
50 37
25
IB: anti-c-Myc
63
250
1 2
IB: anti-c-Myc
1 2
150 75
(kDa)
100
50 37
25 63
250 150 75
Fig 1 Production and activation of the recombinant pro-TMPRSS13 (A) Schematic representation of the structure of pro-TMPRSS13 (wild-type), the recombinant pro-TMPRSS13 and the enterokinase-cleaved pro-TMPRSS13 The wild-type pro-TMPRSS13 comprises 567 amino acids The amino acid numbering starts from the putative N-terminus of the protein The domain structures are indicated in pro-TMPRSS13 (wild-type) TM, transmembrane domain; LDLA, LDL receptor class A domain; SRCR, scavenger receptor cysteine-rich domain; SPD, serine protease domain The predicted disulfide linkage is shown as SS The putative activation cleavage site (indicated by an arrow) and its sur-rounding sequence are shown in pro-TMPRSS13 (wild-type) The recombinant pro-TMPRSS13 is a secreted form in which the cytoplasmic domain and transmembrane domain (Met1-Gln186) are replaced with the mouse immunoglobulin j-chain signal peptide In addition, AMTGR325 in the wild-type protein is replaced with the enterokinase recognition sequence (DDDDK, underlined) for cleavage in vitro before Ile326 (activation cleavage) The recombinant pro-TMPRSS13 is tagged at the C-terminus with myc-His The enterokinase-cleaved recombi-nant TMPRSS13, the disulfide-linked two-chain form, is illustrated at the bottom (B, C) Immunoblot analysis of the recombirecombi-nant pro-TMPPRSS13 produced in COS-7 cells, and its enterokinase-treated product Samples of pro-TMPRSS13 (lane 1) and enterokinase-treated pro-TMPRSS13 (lane 2) were separated by SDS ⁄ PAGE under reducing conditions (B) or under nonreducing conditions (C), and analysed
by immunoblotting with the anti-c-Myc IgG The protease domain of TMPRSS13 was quantified by scanning densitometry of the immunoblot (B, lane 2) with NIH IMAGEJ software using the protease domain of TMPRSS13, which was expressed in E coli, as a standard.
Trang 4pro-TMPRSS13 efficiently cleaved the substrate, and
thus was used for an assay of inhibition by HAI-1
Inhibition of TMPRSS13 protease activity by
soluble HAI-1
Inhibition of the protease activity of TMPRSS13 was
assessed using recombinant soluble forms of HAI-1,
HAI-1–NK1 and HAI-1–NK1LK2 HAI-1 is first
pro-duced as a 66 kDa transmembrane form, and
subse-quent ectodomain shedding releases two major soluble
forms of 40 and 58 kDa from the cell surface into the
extracellular space [14] HAI-1–NK1, which
corre-sponds to the 40 kDa form, consists of the N-terminal
region (N) and one Kunitz domain (K1), whereas
HAI-1–NK1LK2, corresponding to the 58 kDa form,
consists of the N-terminal region (N), two Kunitz
domains (K1 and K2), and the low-density lipoprotein
(LDL) receptor class A domain (L) between the
Kunitz domains (Fig 2A) Inhibition by aprotinin was
compared with that by HAI-1, because aprotinin has
been shown to efficiently inhibit the protease activity
of TMPRSS13 [12] TMPRSS13 (100 pm) was
incu-bated with various concentrations of HAI-1–NK1,
HAI-1–NK1LK2 and aprotinin, and protease activity
was measured using the synthetic substrate Figure 2C
shows the dose dependence of the inhibitory activities
HAI-1–NK1 had the most potent inhibitory effect
(IC50= 2.18 ± 0.18 nm) HAI-1–NK1LK2 and
apro-tinin showed much weaker inhibitory activity than
HAI-1–NK1
Hepatocyte growth factor activator inhibitor type 2
(HAI-2), also known as placental bikunin, is also a
transmembrane Kunitz-type serine protease inhibitor
[15,16] HAI-2 has been shown to inhibit matriptase
and hepsin [9,10,17] Thus, we examined the effect of a
soluble form of HAI-2 (Fig 2B) on the protease
activ-ity of TMPRSS13 HAI-2 inhibited TMPRSS13
(IC50= 1.54 ± 0.01 nm) (Fig 2C), and the IC50 was
similar to that of HAI-1–NK1 However, the
inhibi-tion curves were quite different: the inhibiinhibi-tion curve of
HAI-2 was sigmoidal, whereas that of HAI-1–NK1
was not (Fig 2C)
Formation of complexes of TMPRSS13 and
HAI-1–NK1
To confirm the inhibitory effect of HAI-1–NK1 on
TMPRSS13, we examined the formation of complexes
by the protease–inhibitor pair 1–NK1 and
HAI-1–NK1LK2 were incubated with the activated
TMPRSS13 at different molar ratios The samples
were boiled or not boiled, and subjected to an
immu-noblot analysis Immuimmu-noblotting with an anti-HAI-1 IgG showed that increasing concentrations of TMPRSS13 shifted the HAI-1–NK1 band (40 kDa) to
a higher molecular mass species (70 kDa) when the samples were not boiled (Fig 3A) This shift was con-firmed by an immunoblot analysis with an anti-TMPRSS13 IgG (Fig 3B) When samples were boiled, the band did not shift (Fig 3A,B) These results indi-cate the formation of TMPRSS13ÆHAI-1–NK1 com-plexes On the other hand, the HAI-1–NK1LK2 band (58 kDa) did not shift to a high molecular mass species even in the presence of a high concentration of
B
Hepatocyte growth factor activator inhibitor Type-2 (HAI-2)
myc-His-N
C
Inhibitor concentration (n M )
.
.
.
SP N K1 LDLA K2 N C HAI-2 NK1 Aprotinin NK1LK2 (2) N
(1) N C
0 10 20 30 40 50 60 70 80 90 100
Fig 2 Dose dependence of the inhibitory activity of soluble forms of HAI-1 and HAI-2 against the protease activity of TMPRSS13 (A) Schematic representation of the structure of the full-length HAI-1 (1) and soluble forms of HAI-1, HAI-1–NK1LK2 (2) and HAI-1–NK1 (3), tagged at the C-terminus with myc-His.
SP, signal peptide; N, N-terminal region; K1, Kunitz domain 1; LDLA, LDL receptor class A domain; K2, Kunitz domain 2; TM, transmembrane domain (B) Schematic representation of the structure of the full-length HAI-2 (1) and a soluble form of HAI-2 tagged at the C-terminals with myc-His (2) (C) Dose dependence
of the inhibitory activity of soluble forms of HAI-1 and HAI-2 against the protease activity of TMPRSS13 TMPRSS13 was incubated with various concentrations of HAI-1–NK1 (•), HAI-1– NK1LK2 (j), aprotinin (m) or HAI-2 (r) Then, Pyr-RTKR-MCA was added, and after further incubation, the fluorescence of the reaction mixtures was measured Data show the mean ± stan-dard deviation for three separate experiments and are expressed
as a percentage of TMPRSS13 activity.
Trang 5TMPRSS13 (Fig 3A,B), which is consistent with data
showing weak inhibitory activity of HAI-1–NK1LK2
against the protease activity of TMPRSS13
Proteolytic activation of pro-HGF by TMPRSS13
Pro-HGF is proteolytically activated by matriptase
and hepsin, and the protease activity is inhibited by
HAI-1 [9,10,18] Therefore, we examined whether
TMPRSS13 also functions as an HGF-converting
pro-tease The single-chain pro-HGF (2 lm) was incubated
with various concentrations of TMPRSS13 The
reac-tion products were separated by SDS⁄ PAGE under
reducing conditions and stained with Coomassie
Bril-liant Blue The incubation generated two main bands
of 60 and 32 kDa (Fig 4A) The sizes corresponded
to the heavy chain and light chain of activated HGF, suggesting that pro-HGF is activated by TMPRSS13 The intensity of the pro-HGF band on the gel was quantified by scanning densitometry, and the percent-age of HGF processed was calculated Pro-HGF was almost completely converted to the two-chain form by
54 nm TMPRSS13 (Fig 4B)
We then analysed the effect of HAI-1–NK1 on the pro-HGF converting activity of TMPRSS13 The sin-gle-chain pro-HGF (2 lm) was incubated with TMPRSS13 (54 nm) pretreated with or without HAI-1– NK1 (5 lm) The pretreatment of TMPRSS13 with HAI-1–NK1 did not generate the 60 and 32 kDa bands (Fig 4C), indicating that HAI-1–NK1 inhibits the pro-HGF converting activity of TMPRSS13
A
TMPRSS13 (n M ) TMPRSS13 (n M )
50
37
75
(kDa)
50
37
75
(kDa)
50 75 100
(kDa)
50 75 100
(kDa)
NK1 (n M )
Not boiled
NK1LK2 (n M )
Not boiled
2 20 200
20
2 20 200
TMPRSS13 (n M ) TMPRSS13 (n M )
2 20 200
20
2 20 200
B
TMPRSS13 (n M ) TMPRSS13 (n M )
50
37
75
50 37
75 100 NK1 (n M )
Not boiled
NK1LK2 (n M )
Not boiled
2 20 200
20
2 20 200
TMPRSS13 (n M ) TMPRSS13 (n M )
50
37
75
(kDa)
50 37
75 100 NK1 (n M )
Boiled
NK1LK2 (n M )
Boiled
2 20 200
20
2 20 200
(kDa) IB: HAI-1
IB: TMPRSS13
*
* *
*
Fig 3 TMPRSS13 forms complexes with HAI-1–NK1 TMPRSS13
at the indicated concentrations was incubated with 20 n M HAI-1–
NK1 and HAI-1–NK1LK2 at 37 C for 2 h After the addition of SDS
sample buffer with 100 m M dithiothreitol, each sample was boiled
or not boiled (as indicated) Samples were separated by SDS ⁄ PAGE
under reducing conditions, and analysed by immunoblotting with
anti-HAI-1 IgG (A) or anti-TMPRSS13 IgG (B) The asterisks indicate
complexes of TMPRSS13 and HAI-1–NK1.
100 75 50 37 25 (kDa)
100 75 50 37 25 (kDa)
Pro-HGF HGF heavy-chain HGF light-chain
Pro-HGF HGF heavy-chain
HGF light-chain HAI-1-NK1
B
Protease concentration (n M )
0 20 40 60 80 100
C
HAI-1-NK1 (µ M )
5 0
Fig 4 Proteolytic conversion of pro-HGF by TMPRSS13 and its inhi-bition by HAI-1–NK1 (A) Pro-HGF (2 l M ) was incubated with various concentrations of TMPRSS13 The reaction mixtures were separated
by SDS ⁄ PAGE under reducing conditions The gel was stained with Coomassie Brilliant Blue (B) The intensity of the band of pro-HGF was quantified with NIH IMAGEJ software and the percentage of HGF processed was calculated (C) Pro-HGF (2 l M ) was incubated with TMPRSS13 (54 n M ) pretreated with or without HAI-1–NK1 (5 l M ) The reaction mixtures were analysed as described in (A).
Trang 6Biological activities of HGF converted by
TMPRSS13
To examine the biological activities of the HGF
con-verted by TMPRSS13, we used the human
hepatocellu-lar carcinoma cell line HepG2 HGF induces a
scattering of cell colonies and inhibition of
serum-dependent proliferation in HepG2 cells [19] These
bio-logical responses to HGF are transduced through the
activation of a high affinity receptor, the c-met
proto-oncogene product (c-Met), and also require strong
activation of the extracellular signal-regulated kinase
(ERK) [20] Therefore, we first analysed the activation
of c-Met by assessing its tyrosine phosphorylation
HepG2 cells were treated with the TMPRSS13-cleaved
pro-HGF, and tyrosine phosphorylation of c-Met was
analysed by immunoblotting using an
anti-phospho-c-Met IgG The tyrosine phosphorylation was induced in
HepG2 cells treated with the TMPRSS13-cleaved
pro-HGF at a level comparable with that in cells treated
with the purified active HGF, whereas it was not
induced in HepG2 cells treated with the uncleaved
pro-HGF (Fig 5A) Treatment of the cells with
TMPRSS13 itself did not induce the phosphorylation
(Fig 5A)
We then analysed the activation of ERK by
assess-ing its phosphorylation Immunoblottassess-ing usassess-ing an
anti-phospho-ERK1⁄ 2 IgG showed that the
phosphor-ylation of ERK1⁄ 2 was more enhanced in HepG2 cells
treated with the TMPRSS13-cleaved pro-HGF than in
HepG2 cells treated with the uncleaved pro-HGF or
with TMPRSS13 (Fig 5B) Finally, we analysed the
biological response of HepG2 cells by observing their
scattering phenotype Treatment with the
TMPRSS13-cleaved pro-HGF induced a scattering of cell colonies,
whereas no scattering was observed in the cells treated
with the uncleaved pro-HGF or with TMPRSS13
(Fig 5C) These results indicate that TMPRSS13
con-verts the inactive pro-HGF into the active two-chain
form of HGF
Co-expression of TMPRSS13 and HAI-1 mRNA in
cultured cell lines
Because TMPRSS13 and HAI-1 are both
transmem-brane proteins, HAI-1 is probably co-expressed with
TMPRSS13 in the same cells to function as a
physio-logical inhibitor of the protease We examined the
co-expression of TMPRSS13 and HAI-1 mRNA in
cultured cell lines by RT-PCR We analysed five
human carcinoma cell lines: a lung carcinoma cell line
A549, a colon carcinoma cell line LoVo, stomach
car-cinoma cell lines MKN45 and MKN74, and HepG2
A549 and LoVo cells have been shown to express TMPRSS13 mRNA [13] MKN45 cells were used for identification of HAI-1 proteins [4] MKN74 and HepG2 cells have been shown to respond to HGF [20] TMPRSS13 mRNA was detected in MKN45 and MKN74 cells, but not in A549, LoVo and HepG2 cells On the other hand, HAI-1 mRNA was detected
in LoVo, MKN45, MKN74 and HepG2 cells (Fig 6) These results indicate that HAI-1 mRNA is co-expressed with TMPRSS13 mRNA in MKN45 and MKN74 cells
A
B
C Pro-HGF
Active HGF
TMPRSS13
IB: Phospho-c-Met
IB: Phospho-ERK1/2 IB: ERK1/2
IB: c-Met (Tyr1234/1235)
Pro-HGF + TMPRSS13
Pro-HGF TMPRSS13 TMPRSS13 Active HGF
Pro-HGF +
Pro-HGF TMPRSS13 TMPRSS13 Active HGF
Pro-HGF +
Fig 5 Biological activity of HGF converted by TMPRSS13 Cells were treated with reaction mixtures of pro-HGF alone (Pro-HGF), TMPRSS13 alone (TMPRSS13) or pro-HGF and 2000 ngÆmL)1 TMPRSS13 (Pro-HGF + TMPRSS13) at 50 ngÆmL)1pro-HGF Cells were also treated with purified active HGF at 50 ngÆmL)1 (Active HGF) (A) Cells were cultured for 5 min Lysate of the cells was immunoblotted with the anti-phospho-c-Met IgG (upper panel) and anti-c-Met IgG (lower panel) (B) Cells were cultured for 5 min Lysate of the cells was immunoblotted with the anti-phospho-ERK1 ⁄ 2 IgG (upper panel) and anti-ERK1 ⁄ 2 IgG (lower panel) (C) Cells were cultured for 4 days The morphology of the cells was analysed by light microscopy.
Trang 7In this study, we tested the inhibitory effect of HAI-1
on the protease activity of several members of the
TTSP family using enzymes expressed in E coli We
found that the protease activity of TMPRSS13 was
inhibited by HAI-1, but that of TMPRSS11A,
like 4 and like 5 was not TMPRSS11A,
HAT-like 4, and HAT-HAT-like 5 belong to the HAT⁄ DESC
subfamily [2] Mouse DESC1, also of the HAT⁄ DESC
subfamily, forms stable inhibitory complexes with
plas-minogen activator inhibitor-1 and protein C inhibitor
[21] Thus, these serpins might be endogenous
inhibi-tors of TMPRSS11A, HAT-like 4 and HAT-like 5
The protease activity of TMPRSS13 expressed in
E coli was weak, probably because of incorrect
pro-tein folding Thus, we expressed the enzyme in
mam-malian cells To obtain an active TMPRSS13 in
mammalian cells, we constructed an expression vector
encoding a recombinant protein with two
modifica-tions, and transfected COS-7 cells with the vector One
modification was that we deleted the N-terminal
cyto-plasmic and transmembrane domains and tagged the
C-terminus with six His sequences, to simply purify
the protein from the conditioned medium of the
trans-fected cells by one-step column chromatography The
other modification was that we replaced the putative
activation cleavage sequence with the enterokinase
rec-ognition sequence, because the molecular mechanism
of the proteolytic activation of pro-TMPRSS13 is
unknown The purified pro-enzyme did not show any
protease activity, and the enterokinase treatment
gen-erated an active enzyme (Fig 1) Using this active
TMPRSS13, we demonstrated that HAI-1–NK1 had
inhibitory activity against the protease (Fig 2) The
activity was much stronger than that of aprotinin,
which was previously described as an inhibitor of
TMPRSS13 [12] The inhibitory activity of HAI-1– NK1 against TMPRSS13 was confirmed by in vitro binding assays HAI-1–NK1 formed complexes with the active TMPRSS13 (Fig 3) HAI-1–NK1 consists
of the N-terminal region and the first Kunitz domain, and corresponds to the 40 kDa form of HAI-1 gener-ated from a transmembrane form by extracellular shedding [4] TMPRSS13 mRNA is expressed in a variety of human adult tissues, and predominantly in lung, placenta, pancreas and prostate [12] HAI-1 mRNA is also highly expressed in placenta, pancreas and prostate [4] Thus, the 40 kDa form of HAI-1 could function as an endogenous regulator of TMPRSS13 in these tissues
HAI-1–NK1LK2 had a much weaker inhibitory effect against TMPRSS13 than HAI-1–NK1 (Fig 2) Moreover, no complex of HAI-1–NK1LK2 and TMPRSS13 was detected in the in vitro binding assays (Fig 3) These results indicate that HAI-1–NK1LK2 only weakly associates with TMPRSS13 HAI-1– NK1LK2 consists of the N-terminal region, the first Kunitz domain, the LDL receptor class A domain, and the second Kunitz domain, and corresponds to the
58 kDa form of HAI-1 identified in the conditioned medium of cultured carcinoma cells [14] Weaker inhibitory activity of HAI-1–NK1LK2 against HGFA and matriptase was also observed, and an idea that the second Kunitz domain may obstruct the protease-bind-ing site of the first Kunitz domain was proposed [22,23] The present results indicate that this idea may also apply to TMPRSS13 The weaker inhibitory activ-ity of HAI-1–NK1LK2 was prominent against TMPRSS13, compared with that against HGFA and matriptase Thus, the presence of the second Kunitz domain may more strongly affect the binding of the first Kunitz domain to TMPRSS13
A soluble form of HAI-2, another Kunitz-type inhibitor, also inhibited the protease activity of TMPRSS13, with an IC50 similar to that of HAI-1– NK1 (Fig 2C) HAI-2 mRNA is highly expressed in various human adult tissues [15], some of which also express TMPRSS13 mRNA, suggesting that HAI-2 could be an endogenous inhibitor of TMPRSS13 in these tissues
The inhibition curve of HAI-1–NK1 was not sigmoi-dal, which is unusual, compared with the sigmoidal curve of HAI-2 Moreover, a high concentration of HAI-1–NK1 was needed for full inhibition of the pro-tease activity of TMPRSS13 (Fig 2C) These results suggest the characteristic association of HAI-1–NK1 with TMPRSS13, the mechanism of which remains to
be investigated The in vitro binding assays showed that only small portions of HAI-1–NK1 and
TMPRSS13
HAI-1
GAPDH
HAI-1B HAI-1
No template control A549 LoVo MKN45 MKN74 HepG2
Fig 6 RT-PCR analysis of TMPRSS13 and HAI-1 mRNA in human
carcinoma cell lines Total RNA was isolated from cultured A549,
LoVo, MKN45, MKN74 and HepG2 cells, and subjected to RT-PCR
analysis The primers for HAI-1 generate two PCR products of HAI-1
and its splice variant (HAI-1B) [35] GAPDH mRNA was used as an
internal control.
Trang 8TMPRSS13 formed complexes (Fig 3) The weak
complex formation may be related to the characteristic
association of the protease–inhibitor pair
In the present study we have shown that
TMPRSS13 converted the single-chain pro-HGF to a
two-chain form in vitro (Fig 4) We proved that the
two-chain form of HGF is biologically active, by three
assessments Its treatment of HepG2 cells induced the
tyrosine phosphorylation of c-Met, enhanced the
phos-phorylation of ERK, and induced the scattering
phenotype (Fig 5) Thus, the proteolytic cleavage of
pro-HGF by TMPRSS13 generates a biologically
active HGF The concentration for half-maximal
activ-ity of TMPRSS13 was 15 nm (Fig 4B) This value was
0.17 nm for HGFA under similar reaction conditions
[24] Thus, the specific activity of TMPRSS13 is
approximately 90-fold lower than that of HGFA
TMPRSS13 preferentially recognizes cleavage sites
consisting of paired basic amino acid residues (RR or
KR at positions P2 and P1) In addition, the presence
of a basic amino acid residue (R or K) at position P4
enhances the efficiency of cleavage [13] The HA protein
of an HPAI virus strain with the KKKR motif at the
cleavage site was efficiently converted to mature HA
subunits in TMPRSS13-transfected cells [13],
suppor-ting the preference for the cleavage sequences in
sub-strates of TMPRSS13 Pro-HGF has the KQLR motif
at the cleavage site [25] Thus, the nonbasic amino acid
residue at position P2 may cause the low specific activity
of TMPRSS13 for the conversion of pro-HGF
HGF is a pleiotropic factor that functions as a
mito-gen, motogen and morphogen for a variety of cells,
particularly epithelial cells [25,26] HGF is thought to
play a crucial role in the regeneration of various
tissues following injury [27] HGF is a mesenchymal
cell-derived heparin-binding glycoprotein that is
secreted as an inactive single-chain precursor The
secreted HGF normally remains inactive, probably
associated with the extracellular matrix in the tissues
producing it In response to tissue injury, such as
hepatic and renal injury, the inactive single-chain HGF
is converted to a two-chain form exclusively in
the injured tissue This conversion is mediated by
ser-ine protease activity, which is induced in the injured
tissue [28] The two-chain form is required for the
bio-logical activity of HGF [29,30] Thus, the biobio-logical
effects of HGF in injured tissue are regulated through
proteolytic processing by a serine protease HGFA is a
serum-derived serine protease that efficiently converts
the single-chain HGF to the biologically active
two-chain form in vitro [31] The role of HGFA in the
pro-teolytic activation of HGF in vivo was determined by
analysing knockout mice In HGFA-deficient mice,
regeneration of the injured intestinal mucosa and the activation of HGF were impaired, but the injured liver was completely regenerated, suggesting that HGFA is responsible for the activation of HGF in the injured intestinal mucosa, but not in other injured tissues [32] Thus, other serine proteases are probably involved in the activation of HGF in these tissues
Several serine proteases have been shown to convert pro-HGF to the active form in vitro They include serine proteases involved in blood coagulation, such as plasma kallikrein, and coagulation factors XIa and XIIa [24,33] These serine proteases might be responsible for the activation of HGF in injured tissues Matriptase and hepsin, members of the TTSP family, also convert pro-HGF to the active form [9,10,15] Thus, it is possi-ble that these TTSPs function as HGF-converting pro-teases in injured tissue A two-step model for the activation of HGF in injured tissues has been proposed When tissue injury occurs, circulating plasma serine proteases, such as HGFA, are activated in response to the activation of the coagulation cascade and inflamma-tion The activated proteases convert pro-HGF to the active form (the first step) Subsequently, the activated HGF functions as a mitogen for the epithelial cells The proliferating epithelial cells produce TTSPs, such as matriptase The TTSPs convert pro-HGF to the active form (the second step) The activated HGF is involved
in further proliferation of the epithelial cells [32] TMPRSS13 might also function as an HGF-converting protease in the second step, because it appears to be expressed in epithelial cells [13] The specific activity of the HGF conversion of TMPRSS13 is much lower than that of HGFA as described above However, TMPRSS13 localizes to the cell surface, and thus could function in the pericellular activation of HGF
The pro-HGF converting activity of TMPRSS13 was inhibited by HAI-1–NK1 (Fig 4C), suggesting that HAI-1 functions as a regulator for the activation
of HGF in injured tissues RT-PCR analysis showed that TMPRSS13 mRNA is co-expressed with HAI-1 mRNA in MKN45 and MKN74 carcinoma cells (Fig 6) Thus, the pericellular activation of HGF by TMPRSS13 could be regulated by HAI-1 produced in the same cells Further characterization is required to clarify the roles of TMPRSS13 and HAI-1 in regulat-ing the activation of HGF in vivo
Experimental procedures
DNA constructs
The cDNA clones for the protease domains with short pro-sequences of TTSPs were obtained from appropriate human
Trang 9cDNA libraries (Takara, Kyoto, Japan) by PCR, and
inserted into an E coli expression vector, pMAL-c2X (New
England BioLabs, Ipswich, MA, USA) The putative
acti-vation cleavage sequences were replaced with the
enteroki-nase recognition sequence (DDDDK) using a QuikChange
site-directed mutagenesis kit (Stratagene, La Jolla, CA,
USA)
The cDNA clone for the full-length TMPRSS13 was
obtained from a human placenta cDNA library (Takara)
by PCR The PCR product was further amplified by PCR
using a primer containing an EcoRI restriction site and a
primer containing an XbaI site, which also had a point
mutation replacing the stop codon with a Leu codon The
PCR product was subcloned into a mammalian expression
vector, p3xFLAG-CMV14 (Sigma, St Louis, MO, USA)
To construct an expression vector encoding
pro-TMPRSS13 lacking the cytoplasmic and transmembrane
domains, a cDNA sequence encoding amino acid residues
187–567 was amplified by PCR using
p3xFLAG-CMV14-TMPRSS13 as a template The PCR product was
subcloned into the EcoRI and PstI sites of a mammalian
expression vector, pSecTag2C (Invitrogen, Carlsbad, CA,
USA) The activation cleavage site (A321MTGR325) was
replaced with the enterokinase recognition sequence as
described above
To construct an E coli expression vector encoding
pro-TMPRSS13, the cDNA sequence was excised by digestion
with HindIII and XbaI from
p3xFLAG-CMV14-TMPRSS13, and subcloned into an expression vector,
pcDNA3.1⁄ myc-His-A (Invitrogen) The activation cleavage
site was replaced with the enterokinase recognition
sequence as described above A cDNA sequence encoding
amino acid residues 315–567 with the C-terminally tagged
myc-His sequence was amplified by PCR, and subcloned
into the EcoRI and PstI sites of an E coli expression
vector, pMAL-c2X
To construct an E coli expression vector encoding
HAI-1–K1, the cDNA sequence encoding amino acid residues
241–305 was amplified by PCR using cDNA of HAI-1 [4]
as a template The PCR product was subcloned into the
BamHI and XbaI sites of the vector, pcDNA3.1⁄
myc-His-A The cDNA sequence encoding HAI-1–K1 with the
C-terminally tagged myc-His sequence was amplified by
PCR, and subcloned into the NdeI and NotI sites of an
E coliexpression vector, pET30a (EMD Chemicals,
Gibbs-town, NJ, USA)
To construct expression vectors encoding HAI-1–NK1
and HAI-1–NK1LK2, cDNA sequences encoding amino
acid residues 1–314 and 1–436 were amplified by PCR using
cDNA of HAI-1 [4] as a template The PCR products were
subcloned into the HindIII and XbaI sites of pcDNA3.1⁄
myc-His-A
To construct an expression vector encoding HAI-2, the
cDNA sequence encoding amino acid residues 1–194 was
amplified by PCR using cDNA of HAI-2 [15] as a template
The PCR product was subcloned into the HindIII and XbaI sites of pcDNA3.1⁄ myc-His-A
Preparation and activation of pro-TTSPs expressed in E coli
Escherichia coli cells were transformed with the expression vectors encoding pro-TTSPs The cells were lysed by soni-cation, and the lysate was applied to an amylose resin (New England BioLabs) After the resin was washed with phosphate-buffered saline (PBS), bound proteins were eluted with 1 mm maltose in PBS The eluted fraction was treated overnight with enterokinase (EMD Chemicals) at
2 unitsÆ100 lL)1
Cell culture
COS-7 cells, A549 cells and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium, CHO cells and Lovo cells were cultured in Ham’s F12 medium, and MKN45 cells and MKN74 cells were cultured in RPMI1640 med-ium, supplemented with 10% fetal bovine serum, 100 unitsÆmL)1 penicillin and 100 lgÆmL)1 streptomycin at
37C in a humidified atmosphere containing 5% CO2
Preparation and activation of pro-TMPRSS13 expressed in COS-7 cells
Cells were seeded on eight 100 mm collagen-coated plates (Iwaki, Chiba, Japan) at a density of 1·106
cellsÆplate)1 The cells were transfected with the expression vector encod-ing the secreted form of pro-TMPRSS13 at 6 lgÆplate)1 using the FuGENE-6 reagent (Roche Diagnostics, India-napolis, IN, USA) After 24 h, the medium was replaced with serum-free medium, and cells were further cultured for
3 days The conditioned medium was applied to a nickel nitrilotriacetic acid resin (EMD Chemicals), and the proteins bound to the resin were eluted with nickel nitrilotriacetic acid buffer (EMD Chemicals) The eluted fraction was treated overnight with enterokinase at 2 unitsÆ100 lL)1
Quantification of TMPRSS13
The enterokinase-treated pro-TMPRSS13 was quantified by immunoblotting using the protein expressed in E coli as a standard The protease domain with its short pro-sequence and the enterokinase recognition sequence of TMPRSS13 fused at the N-terminus to maltose-binding protein and tagged at the C-terminus with myc-His was expressed in
E coli Preparation of the cell lysate, purification of the proteins, and treatment with enterokinase were carried out
as described above for TTSPs expressed in E coli The enterokinase-treated pro-TMPRSS13 was separated by SDS⁄ PAGE under reducing conditions, and stained by
Trang 10Coomassie Brilliant Blue The intensity of the band of the
protease domain was quantified using bovine serum
albu-min (BSA) as a standard
The enterokinase-treated pro-TMPRSS13 obtained from
COS-7 cells was separated by SDS⁄ PAGE under reducing
conditions In parallel, various amounts of the
enteroki-nase-treated pro-TMPRSS13 obtained from E coli were
separated by SDS⁄ PAGE After electrophoresis, the
sam-ples were subjected to an immunoblot analysis with the
anti-c-Myc IgG The intensity of the band of the protease
domain was quantified using the protease domain of the
protein obtained from E coli
Enzyme inhibition assay
HAI-1–K1 was prepared as follows Escherichia coli cells
were transformed with the expression vector encoding
HAI-1–K1 The cells were lysed by sonication The lysate was
centrifuged, and the pellet was dissolved in urea (6 m) To
refold proteins, glutathione (oxidized form, 5 mm),
glutathi-one (reduced form, 1 mm) and arginine (100 mm) were
added to the solution, and the final concentration of urea
was adjusted to 0.5 m The refolded HAI-1–K1 was purified
by column chromatography using a nickel nitrilotriacetic
acid resin, followed by dialysis against PBS The
enteroki-nase-treated pro-TTSPs were mixed with HAI-1–K1
(0.67 lm) and incubated in the assay buffer (50 mm
Tris⁄ HCl pH 7.5, 150 mm NaCl, and 0.05% Brij 35) for
10 min at 37C Then each substrate was added to the
mixture at a final concentration of 100 lm After
incuba-tion for 3 h at 37C, the amount of
7-amino-4-methyl-coumarin liberated from the substrate was determined
fluorimetrically with excitation and emission wavelengths of
355 and 460 nm, respectively, using a fluorometer (1420
ARVOsx; Perkin Elmer Life Science, Boston, MA, USA)
HAI-1–NK1, HAI-1–NK1LK2 and HAI-2 were prepared
as follows The expression vectors encoding HAI-1–NK1,
HAI-1–NK1LK2 and HAI-2 were introduced into CHO
cells using Superfect transfection reagent (Qiagen, Hilden,
Germany) Transfected cells were cultured at 37C
over-night The medium was replaced with fresh medium
con-taining Geneticin (G418) Neomycin-resistant colonies were
selected and further cultured in a roller bottle When the
cells became confluent, the medium was replaced with
serum-free medium, and the cells were further cultured for
5 days The proteins were purified from the conditioned
medium by column chromatography using nickel
nitrilotri-acetic acid and anti-c-Myc IgG resins Aprotinin was
obtained from Nakarai Tesque (Kyoto, Japan) The
entero-kinase-treated pro-TMPRSS13 (100 pm) and a series of
concentrations of inhibitors were mixed and incubated in
the assay buffer (50 mm Tris⁄ HCl pH 8.0, 150 mm NaCl,
and 0.05% Brij 35) for 10 min at 37C Then,
Pyr-RTKR-MCA (Peptide Institute, Osaka, Japan) was added to the
mixture at a final concentration of 100 lm The final
volume of each mixture was 200 lL After incubation for
1 h at 37C, the amount of 7-amino-4-methylcoumarin lib-erated from the substrate was determined as described above The enzymatic activity without inhibitors was used
as an uninhibited control The IC50was defined as the con-centration of inhibitor that inhibited the enzymatic activity
by 50% compared with the uninhibited control The per-centage value relative to the uninhibited control was plotted against the log of inhibitor concentrations The IC50value was calculated using the graphpad prism software (Graph-Pad Software, San Diego, CA, USA)
Binding assay
HAI-1–NK1 or HAI-1–NK1LK2 was mixed with various concentrations of TMPRSS13 in the assay buffer The mix-ture was incubated at 37C for 2 h and SDS sample buffer (20 mm Tris⁄ HCl pH 6.8, 0.5% SDS, 5% glycerol and 0.002% bromophenol blue) with 100 mm dithiothreitol was added Some of the samples were boiled for 5 min Twenty microlitres of each sample was analysed by immunoblotting
HGF-converting activity of TMPRSS13
The recombinant pro-HGF was prepared as described pre-viously [34] Pro-HGF (2 lm) was mixed with various con-centrations of TMPRSS13 in 20 lL of 20 mm sodium phosphate (pH 7.3) containing 100 mm NaCl and 0.01% Chaps and incubated at 37C for 2 h The reaction mixture was separated by SDS⁄ PAGE under reducing conditions Proteins in the gel were stained with Coomassie Brilliant Blue The intensity of the pro-HGF band was quantified by scanning densitometry using NIH imagej software
To examine the inhibitory effect of HAI-1–NK1 on the HGF-converting activity of TMPRSS13, TMPRSS13 (54 nm) was incubated with HAI-1–NK1 (5 lm) in 20 mm sodium phosphate (pH 7.3) containing 100 mm NaCl and 0.01% Chaps at 37C for 10 min Then, pro-HGF (2 lm) was added to the mixture The final volume of the mixture was 20 lL After incubation at 37C for 2 h, the reaction mixture was analysed by SDS⁄ PAGE, as described above
Preparation of cell lysate
HepG2 cells were seeded at 1·106
cellsÆ100 mmÆplate)1 They were treated with reaction mixtures of the assay for HGF-converting activity of TMPRSS13 or with purified active HGF (provided by the Research Center of Mitsubi-shi Chemical Corp., Yokohama, Japan) for 5 min The cells were washed twice with ice-cold PBS, and lysed with lysis buffer (137 mm NaCl, 8.1 mm Na2HPO4Æ12H2O, 2.68 mm KCl, 1.47 mm KH2PO4, 1 mm Na3VO4, 5 mm EDTA, 1% Nonidet-P40, 0.5% sodium deoxycholate, 1 lgÆmL)1