Báo cáo khoa học: Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases docx

Activation of hepatocyte growth factor activator zymogen
(pro-HGFA) by human kallikrein 1-related peptidases
Shoichiro Mukai
1,2
, Tsuyoshi Fukushima
1
, Daiji Naka
3
, Hiroyuki Tanaka
1
, Yukio Osada
2
and
Hiroaki Kataoka
1
1 Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan
2 Department of Urology, Faculty of Medicine, University of Miyazaki, Japan
3 Mitsubishi Chemical Medience Corporation R&D and Business Development Segment, Tokyo, Japan
In the pericellular microenvironment of tumor tissues,
growth factors and proteases are critically important
for tumor progression. These factors enhance tumor
cell proliferation, survival, motility, invasion, and
angiogenesis. Proteolytic activities are also essential
for degrading components of the extracellular matrix
or initiating coagulation and fibrinolytic systems. In
addition, several growth factors require proteolysis to
gain full biological activity. Hepatocyte growth
factor ⁄ scatter factor (HGF ⁄ SF) is a multifunctional
Keywords
hepatocyte growth factor; HGF activator;
KLK4; KLK5; tissue kallikrein

Correspondence
H. Kataoka, Section of Oncopathology and
Regenerative Biology, Department of
Pathology, Faculty of Medicine, University
of Miyazaki, 5200 Kihara, Kiyotake,
Miyazaki 889-1692, Japan
Fax: +81 985 85 6003
Tel: +81 985 85 2809
E-mail:
(Received 9 December 2007, revised 29
December 2007, accepted 2 January 2008)
doi:10.1111/j.1742-4658.2008.06265.x
Hepatocyte growth factor activator (HGFA) is a serine protease and a
potent activator of prohepatocyte growth factor ⁄ scatter factor (pro-
HGF ⁄ SF), a multifunctional growth factor that is critically involved in tis-
sue morphogenesis, regeneration, and tumor progression. HGFA circulates
as a zymogen (pro-HGFA) and is activated in response to tissue injury.
Although thrombin is considered to be an activator of pro-HGFA, alterna-
tive pro-HGFA activation pathways in tumor microenvironments remain
to be identified. In this study, we examined the effects of kallikrein 1-
related peptidases (KLKs), a family of extracellular serine proteases, on the
activation of pro-HGFA. Among the KLKs examined (KLK2, KLK3,
KLK4 and KLK5), we identified KLK4 and KLK5 as novel activators of
pro-HGFA. Using N-terminal sequencing, the cleavage site was identified
as the normal processing site, Arg407–Ile408. The activation of pro-HGFA
by KLK5 required a negatively charged substance such as dextran sulfate,
whereas KLK4 could process pro-HGFA without dextran sulfate. KLK5
showed more efficient pro-HGFA processing than KLK4, and was
expressed in 50% (13 ⁄ 25) of the tumor cell lines examined. HGFA pro-
cessed by these KLKs efficiently activated pro-HGF ⁄ SF, and led to cellular

scattering and invasion in vitro. The activities of both KLK4 and KLK5
were strongly inhibited by HGFA inhibitor type 1, an integral membrane
Kunitz-type serine protease inhibitor that inhibits HGFA and other pro-
HGF ⁄ SF-activating proteases. These data suggest that KLK4 and KLK5
mediate HGFA-induced activation of pro-HGF ⁄ SF within tumor tissue,
which may thereafter trigger a series of events leading to tumor progression
via the MET receptor.
Abbreviations
ACT, human a
1
-antichymotrypsin; AT, a
1
-antitrypsin; CHO, Chinese hamster ovary; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HAI, hepatocyte growth factor activator inhibitor; HAI-1KD-1, secreted form of hepatocyte growth factor activator inhibitor type 1 consisting
of the first Kunitz domain; HGF ⁄ SF, hepatocyte growth factor ⁄ scatter factor; HGFA, hepatocyte growth factor activator; KLK, human
kallikrein 1-related peptidase; MDCK, Madin–Darby canine kidney; pAb, polyclonal antibody; PAI-1, plasminogen activator inhibitor-1;
PC50%, processing concentration 50%; a
2
-AP, a
2
-antiplasmin.
FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS 1003
growth factor known to play an important role in
tumor progression via its specific receptor tyrosine
kinase MET, the c-met proto-oncogene product [1,2].
HGF ⁄ SF is secreted primarily by stromal cells as an
inactive single-chain precursor (pro-HGF ⁄ SF) that
lacks biological activity and requires proteolytic cleav-
age to become the active, two-chain mature form [3].
To date, several proteases have been reported to be

HGF ⁄ SF-converting enzymes [3]. Hepatocyte growth
factor activator (HGFA) has been identified as a very
potent serum activator of pro-HGF ⁄ SF [4,5]. The
membrane-anchored, cellular surface serine proteases,
matriptase and hepsin, have also been reported as
cellular activators of pro-HGF ⁄ SF [6–8]. HGFA is
primarily synthesized by the liver, and circulates in
blood as an inactive zymogen (pro-HGFA) at a
concentration of approximately 40 nm [3,9]. Thrombin
proteolytically activates pro-HGFA through cleavage
of the Arg407–Ile408 bond in response to tissue
injury, generating the two-chain active form consisting
of a disulfide-linked 66 kDa long chain and 32 kDa
light chain [10,11]. The light chain exhibits the enzy-
matic activity, and the long chain is further cleaved
by proteases [10,11]. Activation can also occur in
tumor tissue, and we have reported enhanced activity
of HGFA and its involvement in the activation of
HGF ⁄ SF in tumors [12–15]. Although thrombin is
considered to be an activator of pro-HGFA [3,10],
alternative pathways in the activation of pro-HGFA
in the tumor microenvironment remain to be clarified.
As the endogenous inhibitor of HGFA, HGFA inhib-
itor type 1 (HAI-1), an integral membrane Kunitz-
type serine proteinase inhibitor, has been identified as
a cell surface regulator of HGFA activity [3,16,17].
Protein C inhibitor may also act as a serum inhibitor
of HGFA [18].
Human kallikrein 1-related peptidase (KLK) genes
consist of 15 homologous serine protease genes located

in tandem on chromosome 19q13.4 [19–21]. The KLK
proteins are translated as single-chain preproproteases,
with cleavage of the signal peptide prior to secretion.
An additional cleavage of the propeptide is required
for activation of KLKs. After activation, KLK3,
KLK7 and KLK9 have chymotrypsin-like activity,
whereas other KLKs have trypsin-like activity in pro-
teolysis [20].
Autoactivation of KLK2 and KLK5 has been
confirmed, and these enzymes can activate other
coexpressed KLKs [22]. Many members of the
KLK family are associated with various human
tumors, such as prostate, breast, ovary, colon, urothe-
lial and renal cancers [23–28]. For example, KLK3
(prostate-specific antigen) is a well-known tumor
marker for prostatic cancer [23], and the expression
of KLK5 is associated with unfavorable prognosis
and invasiveness in breast, ovary and urothelial can-
cers [26–28]. However, little is known regarding the
underlying biological significance of KLK expression
in tumors.
In this study, we examined the possible roles of
KLKs (KLK2, KLK3, KLK4, and KLK5) in the
HGF ⁄ SF signaling axis, focusing on their ability to
generate active HGF ⁄ SF in the pericellular microenvi-
ronment via activation of HGFA. We found that
KLK5 is an efficient activator of pro-HGFA, and
KLK4 also activates pro-HGFA to a lesser degree.
Results
Expression of KLKs in cancer cell lines

We characterized the expression of KLK2, KLK3,
KLK4 and KLK5 mRNAs in a panel of human tumor
cell lines. The expression of KLKs such as KLK4 and
KLK5 had already been extensively studied in ovarian
and breast cancers. For that reason, we examined cell
lines of human tumors that were derived from other
organs, in which activation of the HGF ⁄ SF signaling
axis has been reported [1–3,8,12,13]. Thus, we charac-
terized colon (RCM-1, HT29, CaCo-2, HCT116,
SW837, DLD-1, LoVo, Colo205), pancreas (S2-007,
SUIT-2, AsPC1, Panc1, MiaPaCa), lung (HLC1, LC-
2, LC-1, T3M11, LU139), kidney (MRT-1, Caki-1),
prostate (LNCap, PC3, DU145), and urinary bladder
(KU-1 and UMK-1). In addition, expression of MET,
HGF ⁄ SF, HGFA and HAI-1 was examined. As shown
in Fig. 1, KLK2 was expressed only by the prostatic
cancer cell line LNCap. The expression of KLK3 (also
known as PSA) was also limited, and was observed in
LNCap. Interestingly, DLD-1 derived from colon can-
cer also expressed low level of KLK3. KLK4 was
abundantly expressed by LNCap and MRT-1, and to
a lesser degree by DLD-1. On the other hand, low but
distinct expression of KLK5 was observed in 13 out of
25 cell lines examined. MET and HAI-1 were detect-
able in most (22 ⁄ 25 and 20 ⁄ 25, respectively) cell lines.
As the primer set for HAI-1 was designed to detect
both HAI-1 and its splicing variant with similar inhibi-
tory properties, HAI-1B [29], two PCR products (289
and 337 bp products for HAI-1 and HAI-1B, respec-
tively) were observed. None of the cell lines expressed

notable levels of HGF ⁄ SF mRNA. Although pro-
HGFA is produced mainly by the liver and can be
supplied from plasma in vivo [5,9], some tumor cell
lines, such as DLD-1, LU139, MRT-1, and LNCap,
also expressed endogenous HGFA.
Activation of HGFA by tissue kallikreins S. Mukai et al.
1004 FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS
Processing of pro-HGFA by KLKs
We directly tested the possibility that KLKs could
activate pro-HGFA. Before assaying, the enzymatic
activity of each recombinant KLK was confirmed with
synthetic chromogenic substrates (Fig. 2A). KLK3 and
KLK4 required activation by a processing protease,
whereas KLK2 and KLK5 were autoactivated [22]. As
reported previously [10], thrombin efficiently processed
pro-HGFA in the presence of a negatively charged
substance (dextran sulfate, 10 lgÆmL
)1
) (Fig. 2B).
Under the same assay conditions, KLK4 and KLK5
also processed pro-HGFA, generating a 32 kDa light-
chain band similar to thrombin-treated pro-HGFA
under reducing conditions (Fig. 2B). The N-terminal
amino acid sequences of the 32 kDa processed bands
generated by KLK4 and KLK5 were identified as
Ile-Ile-Gly-Gly-Ser. Therefore, thrombin, KLK4 and
KLK5 cleaved pro-HGFA at the same sites (Arg407–
Ile408) [10]. The processing activity of KLK2 was
weak, and generated a 34 kDa product similar to that
generated by plasma kallikrein (data not shown).

KLK3 did not show HGFA-processing activity
(Fig. 2B).
Thrombin, a known activator of pro-HGFA,
requires negatively charged substances such as dextran
Colon cancer
Pancreas
cancer
Lung
cancer
RCC
Prostate
cancer
KLK2
KLK3
KLK4
MET
KLK5
HGF/SF
HGFA
HAI-1
GAPDH
HCT116
MiaPaCa
Colo205
RCM-1
HT29
LU139
DU145
LNCap
DLD-1

SW837
Caco-2
LC-2
Panc1
T3M11
LC-1
HLC-1
AsPC1
SUIT-2
S2-007
LoVo
PC3
Caki-1
MRT-1
KU-1
UMK-1
Bladder
cancer
HAI-1B
HAI-1
Fig. 1. RT-PCR analyses for the expression
of KLKs and HGF-related molecules in vari-
ous human tumor cell lines.
Fig. 2. Cleavage of pro-HGFA by KLKs. (A)
Enzymatic activities of recombinant KLKs
measured with chromogenic substrates. (B)
Cleavage of pro-HGFA by KLKs. Pro-HGFA
(52 n
M) was incubated for 12 h at 37 °Cin
the presence of 10 lgÆmL

)1
dextran sulfate
with 5 n
M of one of the following: thrombin,
plasma kallikrein (p-kallikrein), KLK3, KLK4,
or KLK5. Recombinant KLK3 and KLK4 were
preincubated with 0.04 n
M thermolysin to
convert them to the active forms, and this
was followed by addition of 4 m
M phospho-
ramidon. Thermolysin also activated pro-
HGFA, and the addition of phosphoramidon
completely inhibited the activity of thermoly-
sin. Processing of pro-HGFA was deter-
mined by SDS ⁄ PAGE under reducing
conditions followed by immunoblot analysis
using a mAb to HGFA light chain (A-1). Both
KLK4 and KLK5 generated 32 kDa frag-
ments of HGFA. The N-terminal amino acid
sequences of the 32 kDa fragments were
identical (Ile-Ile-Gly-Gly-Ser) for KLK4-medi-
ated and KLK5-mediated processing.
S. Mukai et al. Activation of HGFA by tissue kallikreins
FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS 1005
sulfate [10]. Therefore, we checked the effect of dextran
sulfate on KLK4 and KLK5 activation of pro-HGFA.
In the presence of dextran sulfate, KLK5 processed
pro-HGFA more efficiently than did KLK4 (Fig. 3A).
The approximate concentrations of thrombin, KLK4

and KLK5 required to activate 50% of 52 nm pro-
HGFA in the presence of 10 lgÆmL
)1
dextran sulfate
after 12 h at 37 °C (PC50%) were 0.035 nm, 0.45 nm,
and 0.085 nm, respectively. Thus, KLK5 appeared to
be much more potent at activating pro-HGFA than
KLK4, and its specific activity was about half that of
thrombin. In the absence of dextran sulfate, the pro-
cessing of pro-HGFA by KLK5 and by thrombin was
markedly attenuated (Fig. 3B). In contrast, KLK4 acti-
vated pro-HGFA even in the absence of dextran sulfate
(PC50%: 0.60 nm and 0.45 nm in the absence and pres-
ence of dextran sulfate, respectively) (Fig. 3B). As
reported previously [10], an alternatively cleaved
80 kDa product of pro-HGFA (cleavage at the Arg88–
Ala89 bond) was generated by thrombin in the absence
of dextran sulfate. This 80 kDa product was not appar-
ent in the case of KLK5.
Further degradation of HGFA light chain was not
observed even in the presence of high concentrations
of KLK4 or KLK5, confirming that both KLKs are
activators of pro-HGFA (Fig. 3A,B). We also gener-
ated a time course for pro-HGFA processing by KLK.
In the presence of dextran sulfate, KLK5 was a more
efficient activator than KLK4 (Fig. 3C). Taken
together, these findings show that in the presence of
dextran sulfate, KLK5 was five to 10 times more
potent than KLK4 in the processing of pro-HGFA.
By using antibodies (A-1, N19, and C20) that recog-

nize different epitopes of HGFA, we further analyzed
the cleavage patterns of pro-HGFA by KLKs (Fig. 4).
The epitope of each antibody is indicated in Fig. 4B.
KLK5 cleaved the activation site in the first step to
generate the active light chain, and then cleaved the
Fig. 3. Dose-dependent and time-depen-
dent processing of pro-HGFA by KLK4 and
KLK5 and the effect of dextran sulfate. (A,
B) Pro-HGFA (52 n
M) was incubated with
various concentrations of KLK4 (0.1–10 n
M),
KLK5 (0.05–5 n
M) or thrombin (0.05–5 nM)
in the presence (A) or absence (B) of
10 lgÆmL
)1
dextran sulfate. The mixtures
were subjected to SDS ⁄ PAGE under reduc-
ing conditions, and analyzed by immunoblot
analysis. The extent of processing (%) is
shown. (C) Time course of pro-HGFA pro-
cessing in the presence of dextran sulfate.
Pro-HGFA (52 n
M) was incubated with 5 nM
KLK4, 5 nM KLK5 or 5 nM thrombin in the
presence of 10 lgÆmL
)1
dextran sulfate for
the indicated period at 37 °C. The mixtures

were subjected to SDS ⁄ PAGE under reduc-
ing conditions, and then subjected to immu-
noblot analysis.
Activation of HGFA by tissue kallikreins S. Mukai et al.
1006 FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS
heavy chain, generating 41–32 kDa fragments
(Fig. 4A). Similar, but less efficient, cleavage patterns
were also observed in KLK4 (not shown). A schematic
representation of each band observed in immunoblot is
indicated in Fig. 4A, and that of the cleavage sites is
shown in Fig. 4B.
Inhibition of KLK-mediated pro-HGFA activation
by serpins and HAI-1
The pro-HGFA-processing activity of KLK4 was
inhibited by plasma serine protease inhibitors such as
human a
1
-antichymotrypsin (ACT), a
1
-antitrypsin
(AT), and a
2
-antiplasmin (a
2
-AP), and to a lesser
degree by plasminogen activator inhibitor-1 (PAI-1)
(Fig. 5). On the other hand, KLK5 was inhibited
strongly by PAI-1 and a
2
-AP, but not by ACT and

AT (Fig. 5). Pro-HGFA processing by both KLKs
was potently inhibited by HAI-1KD1, a truncated
form of recombinant HAI-1 containing the first Kunitz
domain, which is the major functional inhibitor
domain against cognate proteases [29,30] (Fig. 5). As
mature HAI-1 is a membrane-anchored inhibitor
expressed on the surface of various epithelial cells and
Fig. 4. Analysis of cleavage sites of pro-HGFA by KLKs. (A) Time-dependent cleavage patterns of pro-HGFA (52 nM) were analyzed by using
three kinds of antibody to HGFA (A-1, C20, and N19) under reducing or nonreducing conditions. The epitope of each antibody is indicated in
(B). A schematic representation of each band in immunoblot analysis is also indicated (right and lower panels). The results obtained under
nonreducing conditions indicate the existence of multiple disulfide bonds in the heavy chain, as reported previously [5,10]. (B) Schematic rep-
resentation of the cleavage sites of pro-HGFA and epitopes of the antibodies. A-1 recognizes the light chain of active HGFA (hatched bar),
but the precise position of the epitope is not known. The intra-heavy-chain disulfide bonds are not indicated.
S. Mukai et al. Activation of HGFA by tissue kallikreins
FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS 1007
tumor cells [3,12,31,32], the activities of these KLKs
could be regulated by HAI-1 within the pericellular
microenvironment.
Degradation of pro-HGF ⁄ SF by KLK4 and KLK5
We examined whether KLK4 and KLK5 were able to
activate not only pro-HGFA but also pro-HGF ⁄ SF.
However, as shown in Fig. 6, pro-HGF ⁄ SF was
degraded by KLK4 and KLK5 in a dose-dependent
manner. Although, the physiological cleavage of pro-
HGF ⁄ SF may occur very inefficiently when the pro-
HGF ⁄ SF is incubated with a low concentration of
KLK5, pro-HGF ⁄ SF was almost completely degraded
into small fragments when the KLK5 ⁄ pro-HGF ⁄ SF
ratio was set higher than 0.1. Similar findings were
obtained with KLK4 (Fig. 6). On the other hand,

HGFA showed very efficient activation of pro-
HGF ⁄ SF (Fig. 6) without any degradation, even at
high enzyme ⁄ substrate ratios (data not shown).
Biological roles of KLK-dependent activation
of HGFA
We wanted to investigate whether KLK-mediated acti-
vation of pro-HGFA and subsequent processing of
pro-HGF ⁄ SF induced cellular responses via the MET
receptor tyrosine kinase. Thus, we examined the phos-
phorylation of MET. The addition of pro-HGF ⁄ SF,
which was preincubated with KLK5-treated pro-
HGFA, rapidly activated the cellular MET receptor
(Fig. 7A,B). We also examined the effects on cellular
scattering. Madin–Darby canine kidney (MDCK) cells
were treated with pro-HGF ⁄ SF in the presence or
absence of KLK5-treated pro-HGFA. Enhanced cellu-
lar scattering was observed at 12 h after the treatment
when the cells were treated concomitantly with pro-
HGF ⁄ SF and KLK5-activated HGFA (Fig. 7C).
Therefore, HGFA activated by KLK5 appears to be
functional. On the other hand, at 24 h after treatment,
cells treated with pro-HGF ⁄ SF and pro-HGFA also
showed cellular scattering. This may be due to process-
ing of pro-HGFA or pro-HGF⁄ SF by endogenous
protease of MDCK cells.
Finally, we used a KLK5-negative tumor cell line to
determine the effect of engineered expression of KLK5
on pericellular activation of the HGFA–HGF ⁄ SF axis.
For this purpose, we selected SUIT-2, because this cell
line did not express HGFA (Fig. 1) and the expression

levels of the HGF ⁄ SF activators such as matriptase
and hepsin were low (data not shown). As shown in
Fig. 8A, cellular KLK5 induced pro-HGF ⁄ SF activa-
tion via processing of pro-HGFA. It should be noted
that, although HGF ⁄ SF could be degraded by KLK5
in an in vitro tube assay (Fig. 6B), the cell-based assay
revealed that KLK5 ⁄ HGFA-mediated activation of
pro-HGF ⁄ SF worked without HGF ⁄ SF being
degraded. In a migration assay (Fig. 8B), pro-HGF ⁄ SF
enhanced migration of SUIT-2 cells even in the
absence of HGFA; this may be caused by a trace of
activated HGF ⁄ SF contaminating the pro-HGF ⁄ SF
preparation (as shown in the immunoblot in Fig. 8A)
or by endogenous pro-HGF ⁄ SF-activating protease of
SUIT-2. However, in the co-presence of pro-HGFA
and pro-HGF ⁄ SF, the expression of KLK5 signifi-
cantly upregulated cellular migratory capability as
compared with corresponding control cells (Fig. 8B).
The data suggested that pericellular pro-HGFA
derived from plasma or tumor cells may be activated
by tumor cell-derived KLK5, which may thereafter
trigger a series of events leading to cellular invasion
via HGF ⁄ MET signaling.
Discussion
In the present study, we showed that KLK5 activates
pro-HGFA, resulting in activation of pro-HGF ⁄ SF
and MET-mediated cellular responses. KLK4 also
Fig. 5. Inhibition of KLK-mediated pro-HGFA
activation by serpins and HAI-1. KLK4 or
KLK5 was preincubated with each protease

inhibitor (ACT, PAI-1, AT, a
2
-AP, or
HAI-1KD1) at a 1 : 10 molar ratio, and each
mixture was used for pro-HGFA activation
assay. The final concentrations of pro-HGFA,
KLK4 and KLK5 were 52 n
M,5nM, and
1n
M, respectively. The mixtures were
subjected to SDS ⁄ PAGE under reducing
conditions, and then subjected to immuno-
blot analysis.
Activation of HGFA by tissue kallikreins S. Mukai et al.
1008 FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS
showed pro-HGFA-processing activity, although the
specific activity was lower than that of KLK5. As neg-
atively charged substances, particularly dextran sulfate,
stimulated the activation of HGFA by thrombin [10],
we also examined the effect of dextran sulfate on
KLK-mediated HGFA activation. We found that
KLK5 required dextran sulfate to efficiently activate
pro-HGFA, whereas KLK4 activated pro-HGFA even
in the absence of dextran sulfate. The mechanism by
which dextran sulfate enhanced KLK5-mediated pro-
HGFA processing remains to be determined. As the
pericellular microenvironment is rich in negatively
charged substances such as glycosaminoglycans, and
both HGFA and HGF ⁄ SF also show affinity for nega-
tively charged substances [3], it is reasonable to postu-

late that, in tumors expressing KLK4 and ⁄ or KLK5,
efficient HGFA-activating machinery would be gener-
ated in the pericellular microenvironment. Pro-HGFA
is abundant in plasma [9], and is also expressed
by certain human cancers [12,14,33–35], whereas
pro-HGF ⁄ SF is produced by stromal cells and is
significantly increased in tumor tissues via interactions
between tumor cells and stromal cells [36]. Therefore,
these results reveal a novel mechanism in the control
of cellular invasiveness that involves an upstream
tumor cell-derived activator and downstream stromal
effectors in tumor tissue.
Kallikrein 1-related peptidases are expressed in vari-
ous tissues, and are implicated in several physiological
and pathological conditions. KLK5 is expressed in
normal skin as stratum corneum tryptic enzyme and in
the prostate [22,37]. In the epidermis, KLK5 activates
pro-KLK7 (known as stratum corneum chymotryptic
enzyme), and cleaves the components of corneodesmo-
somes, leading to desquamation in a coordinated man-
ner with KLK7 [38]. In the prostate, KLK5 is secreted
into the prostatic fluid. Self-activated KLK5 converts
KLK2 and KLK3 into their active forms. After ejacu-
lation, these KLKs degrade semenogelins, the compo-
nents of seminal clots, to release sperm [22]. KLK4
was first characterized as enamel matrix serine pro-
tease 1, and was reported to be involved in enamelo-
genesis by processing enamelin [39]. KLK4 is also
expressed abundantly in the prostate and secreted into
the prostatic fluid; however, its physiological function

in the prostate is not understood, except for the pro-
teolytic activation of KLK3.
Recently, KLKs have been studied in terms of their
diagnostic and prognostic values in some cancers [23].
KLK5 appears to be a potential biomarker of ovarian
and breast cancers [23,26,27], and may be involved in
the progression of prostate cancer [23–25]. Moreover,
overexpression of the KLK5 gene is associated with
invasiveness of urinary bladder carcinoma cells [28]. In
this study, KLK5 was expressed in more than 50% of
the tumor cell lines examined. However, the mecha-
nisms underlying the role of KLK5 in tumor progres-
sion are poorly understood. KLK5 and other KLKs
activated by KLK5 may degrade extracellular matrix
proteins such as fibronectin, laminin, and type IV col-
lagen, which facilitate the cellular invasiveness at the
invasion front [37]. Insulin-like growth factor-binding
proteins are also possible targets of KLKs [21,40].
Degradation of extracellular insulin-like growth
Fig. 6. Degradation of pro-HGF ⁄ SF by KLK4 and KLK5. (A, B) Pro-
HGF ⁄ SF (53 n
M) was incubated at various concentrations of KLK4
(A) or KLK5 (B) at 37 °C for 12 h, and the mixtures were subjected
to SDS ⁄ PAGE and analyzed by immunoblot. For positive control of
processing, the same amount of pro-HGF ⁄ SF was incubated with
0.05 n
M HGFA at 37 °C for 12 h and simultaneously analyzed. hc,
heavy chain of mature active form HGF ⁄ SF.
S. Mukai et al. Activation of HGFA by tissue kallikreins
FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS 1009

factor-binding proteins would increase the pericellular
concentration of insulin-like growth factor, and this
could eventually stimulate invasive growth of tumor
cells [21,40]. Our present observations offer another
means by which KLK5 could contribute to the malig-
nant progression of cancer cells, as the HGF ⁄ SF–MET
signaling axis is important for the invasive growth of
several types of tumors [1,2].
The activation of pro-HGF ⁄ SF is catalyzed by
serum and cellular proteases [3]. As a serum activator,
HGFA is the most potent HGF ⁄ SF-converting enzyme
[3–5]. Factor XIIa, factor XIa and plasma kallikrein
possess converting activity to lesser degrees [41]. On
the other hand, cellular surface serine proteases such
as matriptase and hepsin also show HGF ⁄ SF-convert-
ing activity, and serve as cellular activators of pro-
HGF ⁄ SF [6,7]. Clearly, there are several pathways for
the activatation and utilization of this multifunctional
growth factor in vivo, depending on the cellular type
and tissue microenvironment. In fact, knocking out
murine HGFA was not lethal for embryos [42],
whereas knocking out HGF ⁄ SF was lethal, due to
impaired development of the placenta and liver [43,44].
Therefore, another pro-HGF ⁄ SF-activating system
compensates for the loss of HGFA during tissue
development and morphogenesis. Moreover, both
A
Pro-HGF/SF
Pro-HGFA
KLK5

+
+
+
+
+
C
0 h
12 h
24 h
+ +
+
94
62
kDa
Pro-HGF/SF
hc
Pro-HGF/SF
Pro-HGFA
KLK5
+
+
+
+
+
+
+
+
145
145
kDa

p-MET
Total MET
ratio <0.1 <0.1 <0.1 <0.1<0.1 0.33
Incubation time (m)
0
0
0
101010
Pro-HGF/SF
Pro-HGFA
KLK5
+
+
+ +
+
+
+
+
+ +
+
+
B
Fig. 7. Biological activity of HGFA processed by KLK. (A) Processing of pro-HGF ⁄ SF by KLK5-treated pro-HGFA. Pro-HGF ⁄ SF (1660 nM) was
incubated with 1 n
M pro-HGFA pretreated (6 h at 37 °C) with 0.02 nM KLK5 for 1 h in the presence of 10 lgÆmL
)1
dextran sulfate in a final
volume of 20 lL. Control reaction mixtures lacking pro-HGFA, or KLK5, or both were also prepared. The mixtures were subjected to
SDS ⁄ PAGE under reducing conditions and analyzed by immunoblotting using a mAb to human HGF ⁄ SF. (B) Phosphorylation of MET induced
by HGF ⁄ SF processed by KLK5-treated pro-HGFA. PC3 cells were cultured in DMEM with 0.1% BSA for 48 h. Then, 1 l L of the mixture

described in (A) was added and incubated for 10 min. Cellular proteins were extracted at the indicated time points, and the samples were
analyzed by immunoblotting using antibody to human phosphorylated MET (p-MET). The same blot was analyzed with antibody to human
MET (total MET). The band densities were measured, and the ratio of p-MET to the corresponding total MET was calculated. (C) MDCK cells
were cultured in DMEM and 0.1% BSA for 48 h. Then, 1 lL of the mixture described in (A) was added, and the cells were further cultured
for 24 h. The culture morphology was photographed under phase-contrast microscopy at the indicated time points.
Activation of HGFA by tissue kallikreins S. Mukai et al.
1010 FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS
matriptase and hepsin knockout mice also failed to
show developmental anomalies, similar to HGF ⁄ SF
knockouts [45,46]. These observations obtained using
mutant mice indeed indicate the redundancy in the
pro-HGF ⁄ SF activation system in vivo.
Although complex and redundant, these processes
may be regulated by a single molecule, cell surface
HAI-1, as it potently inhibits all of these pro-
HGF ⁄ SF-converting proteases and is expressed on the
surface of epithelial and tumor cells [30,31]. Our results
revealed that KLK4 and KLK5 were also sensitive to
HAI-1. Therefore, HAI-1 might be a key molecule in
the regulation of pericellular HGF ⁄ SF bioactivity and
also of matrix remodeling and processing of other bio-
active molecules mediated by these proteases. In fact,
HAI-1 knockout mice cannot survive, due to impaired
development of placental tissue, indicating the critical
and nonredundant function of this cell surface protease
inhibitor [47–49]. Our data also showed that excessive
and prolonged exposure to KLK4 and KLK5 leads to
degradation of HGF ⁄ SF. However, our cell-based
assay indicates that this HGF ⁄ SF-degrading activity
does not affect the pericellular HGF ⁄ SF activity. We

speculate that, in the pericellular microenvironment,
the activity of KLKs might be regulated by HAI-1,
and the limited availability of KLKs is not enough to
degrade HGF ⁄ SF. On the other hand, as the process-
ing activity of HGFA on pro-HGF ⁄ SF is very potent,
only a trace amount of HGFA generated by KLKs
might be enough to generate active HGF ⁄ SF, which
thereafter triggers a series of events via the MET
receptor. Thus, regulation of KLKs, HGFA and other
HGF ⁄ SF-processing proteases by HAI-1 on the cell
surface may be important for optimal availability of
HGF ⁄ SF. We suggest that complex pericellular inter-
actions between proteases and inhibitors occur in vivo,
Fig. 8. Effects of endogenous KLK5 on the HGFA–HGF ⁄ SF activation cascade. (A) Effect of engineered KLK5 expression on HGFA-mediated
processing of pro-HGF ⁄ SF. SUIT-2 cells were transiently transfected with pCMV6-XL4-KLK5, and maintained with or without pro-HGFA
(1 ng ⁄ mL) for 3 h; this was followed by the addition of pro-HGF ⁄ SF (40 ngÆmL
)1
). After 8 h of cultivation, the processing of pro-HGF ⁄ SF
was analyzed by immunoblot analysis. Pro-HGF ⁄ SF incubated for the same period without any cells is shown as ‘cell free’. hc, heavy chain
of mature active form of HGF ⁄ SF. (B) Effects of engineered expression of KLK5 on cellular migration of SUIT-2. Values are mean num-
ber ± standard deviation of migrated cells per high-power field in triplicate experiments. Representative photographs of migrating cells are
also shown.
#
P < 0.001 as compared to corresponding mock control. *P < 0.001.
S. Mukai et al. Activation of HGFA by tissue kallikreins
FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS 1011
where they are required for tissue homeostasis. It is
likely that they also play important roles in pathologi-
cal phenomena. In Fig. 9, we summarize our hypo-
thesis regarding the pericellular activation system of

HGF ⁄ SF in tumors and the possible relationship of
KLKs to this system.
In summary, our results revealed possible roles for
KLK4 and KLK5 in the activation of pro-HGFA,
which activates the HGF ⁄ SF and HGF ⁄ SF–MET sig-
naling axis. This finding may shed light on novel func-
tions of these KLKs in pathophysiological conditions,
including tumor growth and progression.
Experimental procedures
Cell lines and reagents
Eight human colon cancer cell lines (RCM-1, HT29, CaCo-
2, HCT116, SW837, DLD-1, LoVo, Colo205), five human
pancreatic cancer cell lines (S2-007, SUIT-2, AsPC1, Panc1,
MiaPaCa), five human lung cancer cell lines (HLC1, LC-2,
LC-1, T3M11, LU139), two human renal cell carcinoma
cell lines (MRT-1, Caki-1), three human prostate cancer cell
lines (LNCap, PC3, DU145), the MDCK cell line and the
Chinese hamster ovary (CHO) cell line were used in this
study. RCM-1, LC-2, LC-1, MRT-1 and UMK-1 were
established in our laboratory. S2-007 and SUIT-2 were
kindly provided by T. Iwamura (Junwakai Memorial Hos-
pital, Miyazaki, Japan). KU-1 was kindly provided by
M. Oya (Keio University, Tokyo, Japan). DLD-1, LoVo,
T3M11, LU139 and Caki-1 were obtained from the Riken
Cell Bank (Tsukuba, Japan), and HT29, CaCo-2, SW837,
Colo205, HCT116, AsPC1, Panc1, MiaPaCa, LNCaP, PC-3
and DU145 were obtained from Dainihon Seiyaku (Osaka,
Japan). The cells were maintained in DMEM containing
10% fetal bovine serum at 37 °C in a humidified atmo-
sphere containing 5% CO

2
.
Preparations of recombinant pro-HGFA, pro-HGF ⁄ SF
and HAI-1KD1 have been described previously [4,10,30].
Dextran sulfate, Chaps, phosphoramidon and thrombin
Fig. 9. Hypothetical model for the pericellular activation of HGF ⁄ SF in tumors. Tumor cell–stroma interactions result in increased accumula-
tion of extracellular pro-HGF ⁄ SF in tumor tissue. There may be two pathways for the activation of pro-HGF ⁄ SF in the extracellular space.
One is mediated by membrane-bound serine proteinases (cellular activators), such as matriptase and hepsin [6–8]. The second pathway is
mediated by HGFA (serum activator), a very efficient activator of pro-HGF ⁄ SF [3–5]. Pro-HGFA is circulating in the blood, and is activated in
response to tissue injury by thrombin (serum pro-HGFA activator) [10,11]. Certain tumor cells also produce pro-HGFA by themselves [12,14].
Pericellular pro-HGFA may be activated by KLK4 or KLK5 (cellular pro-HGFA activator). HAI-1 might be an important regulator of pericellular
pro-HGF ⁄ SF activation.
Activation of HGFA by tissue kallikreins S. Mukai et al.
1012 FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS
were purchased from Sigma-Aldrich (St Louis, MO, USA).
ACT, AT, a
2
-AP and PAI-1 were obtained from
Calbiochem (San Diego, CA, USA). Chromogenic
substrates S-2586 (methoxysuccinyl-Arg-Pro-Tyr-p-nitroani-
line.HCl), S-2266 (D-Val-Leu-Arg-p-nitroaniline.2HCl) and
S-2302 (D-Pro-Phe-Arg-p-nitroaniline.2HCl) were pur-
chased from Chromogenix (Milan, Italy). Plasma kallikrein
and thermolysin were obtained from R&D Systems (Minne-
apolis, MN, USA).
A mouse mAb to human HGF ⁄ SF (P-1), which recog-
nized the a-chain of the two-chain active form of HGF ⁄ SF,
was used [14]. For detection of HGFA protein, a mAb to
human HGFA (A-1), which recognized the light chain of
the two-chain active form of HGFA [14], a goat polyclonal

antibody (pAb) C-20, which recognized the C-terminal
end of the HGFA light chain (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), and N-19, which recognized the
N-terminal end of the 34 kDa two-chain active form of
HGFA (Santa Cruz Biotechnology), were used. A rabbit
pAb to human MET (C-12) was obtained from Santa Cruz
Biotechnology, and a rabbit pAb to antiphosphorylated
MET (pYpYpY1230 ⁄ 1234 ⁄ 1235) was obtained from Bio-
source (Camarillo, CA, USA).
Preparation and activation of KLKs
Recombinant KLK3, KLK4 and KLK5 were purchased
from R&D Systems. For the preparation of pro-KLK2,
the cDNA encoding the whole coding region of human
pro-KLK2 was amplified by Pyrobest DNA polymerase
(Takara, Shiga, Japan), using cDNA generated from
normal human prostate (Invitrogen, Carlsbad, CA, USA).
The sequences of the primers were 5¢ -CAGGAATTCAG
CATGTGGGACCTG-3¢ (forward) and 5¢-ATACAGCTG
CACTCAGGGGTTGGC-3¢ (reverse). The product was
subcloned into pCRII (Invitrogen), sequenced, and used
as a template for subsequent PCR, using the Gateway
recombination system (Invitrogen) according to the manu-
facturer’s instructions, finally generating pcDNA-DEST40-
KLK2 with a histidine tag at the C-terminal end of KLK2.
The plasmid was linearized by ScaI and transfected into cul-
tured CHO cells by electroporation (MicroPorator MP-100;
NanoEn Tek, Korea). After transfection, colonies resistant
to G418 (Sigma, 0.5 mgÆmL
)1
) were selected and screened

for the expression of KLK2. For purification of recombi-
nant KLK2, KLK2-transfected CHO cells were cultured in
serum-free medium (CHO-S-SFM II; Invitrogen), and
recombinant KLK2 was affinity purified with a HisTrap
chelating column (GE Healthcare Bio-Science, Uppsala,
Sweden), according to the manufacturer’s instructions.
KLK3 and KLK4 were proforms, and required proteo-
lytic activation using thermolysin followed by addition of
phosphoramidon to inhibit excess thermolysin activity
before use. One microgram of recombinant pro-KLK3 was
incubated with 10 ng of thermolysin (molar ratio, 119 : 1)
in KLK activation buffer (50 mm Tris ⁄ HCl, 150 mm NaCl,
10 mm CaCl
2
, pH 7.5). The reaction was incubated at
37 °C for 3 min, and terminated by addition of phospho-
ramidone (4 mm, final concentration). Recombinant pro-
KLK4 was also mixed with thermolysin at the same molar
ratio as for KLK3 activation, and incubated at 37 °C for
2 h; this was followed by the addition of phosphoramidon.
KLK2 and KLK5 were autoactivated during incubation.
To confirm the enzymatic activity, activated KLKs were
incubated with the chromogenic substrates S-2586 (for
KLK3), S-2266 (KLK2, KLK4, and KLK5) or S-2302
(KLK2, KLK4, and KLK5). The chromogenic substrates
were diluted in 50 mm Tris ⁄ HCl, 0.05% Chaps (pH 7.5) to
a final concentration of 1 mm, and this was followed by the
addition of 100 ng of each KLK. The final volume of each
mixture was 150 lL. The enzymatic activity was monitored
by the release of p-nitroaniline (absorbance at 405 nm),

using a SpectraMax microplate reader (Applied Biosystems,
Foster City, CA, USA), at 37 °C for 50 min.
RNA extraction and RT-PCR analyses
Total cellular RNA was extracted from the cultured cells
with Trizol
TM
reagent (Invitrogen). For RT-PCR, 3 lgof
total RNA was reverse transcribed with a mixture of
oligo(dT) and random primers, using 200 units of Super-
Script reverse transcriptase (Invitrogen), and 1 ⁄ 30 of the
resultant cDNA was processed for each PCR with 0.1 lm
both reverse and forward primers and 2.5 units of HotStar
Taq DNA polymerase (Qiagen, Tokyo, Japan). For KLKs
and HAI-1, the following primers were designed: KLK2
forward, 5¢-CAGAGCCTGCCAAGATCACAGATG-3¢;
KLK2 reverse, 5¢-CCATTACAGACAAGTGGACCC
CCA-3¢; KLK3 forward, 5¢-ATGACGTGTGTGCGCAAG
TTCACCC-3¢; KLK3 reverse, 5¢-GATCCACTTCCGGTA
ATGCACCACC-3¢; KLK4 forward, 5¢-AATCATAAAC
GGCGAGGACTGCAG-3¢; KLK4 reverse, 5¢-TTAGCG
AGCAAGGGTCTGTTGTAC-3¢; KLK5 forward, 5¢-TGT
GCTCTGATCACAGCCTTGCTT-3¢; KLK5 reverse,
5¢-CCAGCATTTTAGCATTACTT-3¢; HAI-1 forward,
5¢-AAGAGTTTCGTTTATGGAGG-3¢; HAI-1 reverse,
5¢-TGTGCATATCGCAGTCGGATCCAT-3¢. For HGFA,
MET, HGF ⁄ SF and glyceraldehyde-3-phosphate dehydro-
genase (GAPDH), the following primer sequences were
used, as previously described [13]: HGFA forward,
5¢-CCACTTGGATGAGAACGTGA-3¢; HGFA reverse,
5¢-ATGATGCCGTAGAGGTAAGC-3¢; MET forward,

5¢-TTGCCAGAGACATGTATGATAAAGAATACT-3¢;
MET reverse, 5¢-TTGTCACTGGGGAAATGGAT-5¢;
HGF ⁄ SF forward, 5¢-GGGAAATGAGAAATGCAGCCA-
3¢; HGF ⁄ SF reverse, 5¢-AGTTGTATTGGTGGGTGC-3¢;
GAPDH forward, 5
¢-GTGAAGGTCGGAGTCAACG-3¢;
GAPDH reverse, 5¢-GGTGAAGACGCCAGTGGACTC-
3¢. The PCR products were analyzed by 1.5% agarose gel
electrophoresis.
S. Mukai et al. Activation of HGFA by tissue kallikreins
FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS 1013
Immunoblot analysis
The reaction samples were mixed with SDS ⁄ PAGE sample
buffer and heated for 15 min at 75 °C. SDS ⁄ PAGE was
performed under reducing conditions, using 4–12% gradi-
ent gels. After electrophoresis, the sample proteins were
transferred electrophoretically to Immobilon membranes
(Millipore; Billerica, MA, USA). After blocking of the non-
specific binding site with 5% nonfat dry milk in 50 mm
Tris ⁄ HCl (pH 7.5), 150 mm NaCl, and 0.05% Tween-20,
the membranes were incubated with primary antibody in
buffer containing 1% BSA at 4 °C overnight; this was fol-
lowed by four washes with the buffer, and incubation with
peroxidase-conjugated secondary antibody diluted in the
buffer with 1% BSA for 1 h at room temperature. The
labeled proteins were visualized with chemiluminescence
reagent (PerkinElmer Life Sciences, Boston, MA, USA).
Activation of pro-HGFA by KLK
Recombinant pro-HGFA (52 nm, final concentration) was
incubated with varying concentrations of one of the test pro-

teins (thrombin, plasma kallikrein, activated KLK2, KLK3,
KLK4, or KLK5) in 50 mm Tris ⁄ HCl and 0.05% Chaps
(pH 7.5) in the presence or absence of dextran sulfate
(10 lgÆmL
)1
) for 12 h at 37 °C. Final volumes of the reaction
mixtures were 20 lL. The reactions were subjected to
SDS ⁄ PAGE under reducing conditions, and then subjected
to immunoblot analysis for HGFA. The extent of the pro-
cessing was verified by image analysis using photoshop soft-
ware (Adobe Systems, San Jose, CA, USA). The specific
activity of each enzyme was expressed as the enzyme concen-
tration required for processing 50% of 52 nm pro-HGFA
and designated as PC50%. To analyze the effects of protease
inhibitors on KLK-mediated pro-HGFA activation, each
KLK was preincubated with a 10-fold greater concentration
of each protease inhibitor (ACT, AT, a
2
-AP, PAI-1, HAI-
1KD1) and used for the processing assay described above.
N-terminal amino acid sequences of cleaved
HGFA
Pro-HGFA (357 nm) was incubated with KLK4 (50 nm)or
KLK5 (37.5 nm)in50mm Tris ⁄ HCl and 0.05% Chaps
(pH 7.5) in the presence of 10 lgÆmL
)1
dextran sulfate at
37 °C for 12 h. The total volume of each reaction was
40 lL. After incubation, each sample was subjected to
SDS ⁄ PAGE. After electrophoresis, the sample proteins

were transferred electrophoretically to an Immobilon mem-
brane and stained with 0.1% Coomassie Brilliant Blue in a
water ⁄ methanol ⁄ acetic acid solution (4.5 : 4.5 : 1, v ⁄ v). The
cleaved HGFA protein band was cut and processed for
N-terminal amino acid sequences by automated Edman
degradation using a Procise 494 HT Protein Sequencing
System (Applied Biosystems).
Activation of pro-HGF ⁄ SF
The activation of HGFA by KLK treatment was verified
by a pro-HGF ⁄ SF activation assay as described previously
[10,17]. Briefly, 1.66 lm pro-HGF ⁄ SF was incubated for
12 h at 37 °C with 0.001 lm pro-HGFA that had been pre-
treated with KLK4 or KLK5 in 50 mm Tris ⁄ HCl and
0.05% Chaps (pH 7.5) for 2 h at 37 °C. The processing of
pro-HGF ⁄ SF was determined by immunoblot analysis
under reducing conditions, and the extent of processing was
semiquantified by photoshop software as described above.
To test the direct effect of KLK on the activation of pro-
HGF ⁄ SF, 53 nm pro-HGF ⁄ SF was incubated with various
concentrations of KLK4 or KLK5 in the assay buffer in a
final volume of 20 lL for 12 h at 37 °C. The reaction was
analyzed by immunoblot.
Cell scattering assay and phosphorylation of MET
Eight nanograms of pro-HGFA was incubated with 0.06 ng
of KLK5 in 50 mm Tris ⁄ HCl and 0.05% Chaps (pH 7.5)
for 6 h at 37 °C. Then, 3.2 lg of pro-HGF ⁄ SF was added
to the mixture and further incubated for 2 h in a final vol-
ume of 20 lL. Control reaction mixtures lacking pro-
HGFA or KLK5 were also prepared. For cell scattering
assays, MDCK cells were cultured in 25 cm

2
culture flasks
for 48 h in 4 mL of serum-free DMEM containing 0.1%
protease-free BSA at 37 °C. During the incubation, the cells
were washed with serum-free medium every 24 h. Then,
1 lL of the reaction mixture described above, which should
contain 160 ng of activated HGF ⁄ SF, was added to the
serum-free culture medium (i.e. 40 ngÆmL
)1
HGF ⁄ SF), and
the cells were further cultured for 24 h. The morphological
change of the cells was photographed under phase-contrast
microscopy at each desired time point. For the detection of
MET phosphorylation, PC3 cells (cultured under the same
conditions as MDCK cells) were treated with 1 lL of the
reaction mixture described above, equivalent to 160 ng of
activated HGF ⁄ SF in 4 mL of serum-free medium, for
10 min at 37 °C. Then, the cells were washed twice with
ice-cold NaCl ⁄ P
i
, and this was followed by the addition of
1.5 mL of 10% trichloroacetic acid on ice. The degenerated
cells were scraped and collected into microcentrifuge tubes,
and centrifuged at 21 480 g at 4 °C for 3 min. The
pellet was dissolved in the extraction solution, consisting of
7 m urea, 2% Triton X-100, and 5% 2-mercaptoethanol.
The extracted protein was analyzed by immunoblot
analyses.
Engineered expression of KLK5 and cell migration
assay

Engineered expression of human KLK5 was performed by
transient transfection of the KLK5 expression plasmid,
pCMV6-XL4-KLK5 (OriGene, Rockville, MD, USA),
Activation of HGFA by tissue kallikreins S. Mukai et al.
1014 FEBS Journal 275 (2008) 1003–1017 ª 2008 The Authors Journal compilation ª 2008 FEBS
using the SUIT-2 human pancreatic cancer cell line, which
lacks endogenous KLK5. To test the effect of engineered
KLK5 expression on HGFA-mediated processing of pro-
HGF ⁄ SF, the pCMV6-XL4-KLK5-transfected SUIT-2
cells (SUIT-2KLK5) were maintained in DMEM and
0.1% BSA with or without 1 ng ÆmL
)1
pro-HGFA for 3 h.
Then, pro-HGF ⁄ SF was added (final concentration in cul-
ture 40 ngÆmL
)1
), and cultivation was continued for 8 h.
The cellular proteins were extracted, and the processing of
pro-HGF ⁄ SF was analyzed by immunoblot analysis. The
migration capability of SUIT-2KLK5 cells was analyzed
using poly(vinyl pyrrolidone)-free polycarbonate filters
with a pore size of 8 lm (Chemotaxicells; Kurabo, Osaka,
Japan), which were coated with type IV collagen, as
described previously [15]. The cells were harvested from
culture by incubation with 0.125% trypsin and 0.5 mm
EDTA in NaCl ⁄ P
i
, and this was followed by neutraliza-
tion with an excess volume of 1% AT in DMEM. After
neutralization, the cells were collected by centrifugation at

55 g and rinsed three times in serum-free medium. The
cells (2 · 10
5
cells per 200 lL of serum-free medium, 0.1%
BSA, with or without 1 ngÆmL
)1
pro-HGFA) were then
added to the Chemotaxicells and incubated for 3 h. One
microliter of pro-HGF ⁄ SF solution (final concentration in
culture; 40 ngÆmL
)1
) or NaCl ⁄ P
i
was added and incubated
for an additional 21 h at 37 °Cin5%CO
2
. At that point,
the filters were fixed with 3.7% formaldehyde in NaCl ⁄ P
i
and stained with hematoxylin. The cells on the upper sur-
face of the filter were wiped off with a cotton swab.
Migration was quantified by counting the migrant cells on
the lower surface in 10 randomly selected high-power
fields (200-fold magnification). The cell count was per-
formed blind in triplicate Chemotaxicells. Three inde-
pendent experiments were performed to confirm the
tendency. Statistical analyses were carried out using
the statview 5.0 program (Brainpower, Calabass, CA,
USA). P-values less than 0.05 were considered statistically
significant.

Acknowledgements
This work was supported in part by Grant-in-Aid for
Scientific Research (B) No. 17390116 from the Minis-
try of Education, Science, Sports and Culture, Japan.
We are grateful to Dr S. Uchinokura, Department of
Neurosurgery, Faculty of Medicine, University of
Miyazaki, for his kind suggestions, and Ms Tobayashi
for her skillful technical assistance.
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S. Mukai et al. Activation of HGFA by tissue kallikreins
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