Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
In vitro characterization of TMPRSS2 inhibition in
IPEC-J2 cells
Erzsebet Pászti-Gere, Eszter Czimmermann, Gabriella Ujhelyi, Peter Balla,
Alexander Maiwald & Torsten Steinmetzer
To cite this article: Erzsebet Pászti-Gere, Eszter Czimmermann, Gabriella Ujhelyi, Peter Balla,
Alexander Maiwald & Torsten Steinmetzer (2016) In�vitro characterization of TMPRSS2 inhibition
in IPEC-J2 cells, Journal of Enzyme Inhibition and Medicinal Chemistry, 31:sup2, 123-129, DOI:
10.1080/14756366.2016.1193732
To link to this article: https://doi.org/10.1080/14756366.2016.1193732
Published online: 09 Jun 2016.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2016; 31(S2): 123–129
! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1193732
RESEARCH ARTICLE
In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells
Erzsebet Pászti-Gere1, Eszter Czimmermann1, Gabriella Ujhelyi2, Peter Balla3, Alexander Maiwald4, and
Torsten Steinmetzer4
1
Faculty of Veterinary Science, Department of Pharmacology and Toxicology, Szent István University, Budapest, Hungary, 2Faculty of Pharmacy,
Semmelweis University, Budapest, Hungary, 31st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest,
Hungary, and 4Faculty of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marburg, Germany
Abstract
Keywords
The transmembrane serine protease, TMPRSS2 is an important target in the treatment of
seasonal influenza infections and contributes to prostate carcinogenesis and metastasis. In this
study, the effect of the synthetic TMPRSS2 inhibitor I-432 on jejunal IPEC-J2 cell monolayers
cultured on membrane inserts was characterized. Using a fluorogenic substrate, it was found
that the apical addition of I-432 could suppress trypsin-like activity in the supernatants of IPECJ2 cells. The inhibition of TMPRSS2 did not affect physiologically produced hydrogen peroxide
levels in the apical and in basolateral compartments. Loss of expression of the TMPRSS2 serine
protease domain (28 kDa) was also observed when cells were pre-exposed to I-432. Partial
decrease in immunofluorescent signal intensities derived from the altered distribution pattern
of TMPRSS2 was detected after a 48 h long incubation of IPEC-J2 cells with the inhibitor
indicating the efficacy of TMPRSS2 inhibition via I-432 administration in vitro.
3-Amidinophenylalanine, extracellular H2O2,
fluorogenic AMC substrate, IPEC-J2 cells,
TMPRSS2, trypsin-like serine protease
Introduction
Cell surface-associated regulatory proteolytic processes appear to
be responsible for degradation of extracellular matrix components, and contribute to tumor invasion, metastasis, blood
coagulation, cell differentiation and apoptotic events. Type II
transmembrane trypsin-like serine proteases (TTSPs) have been
divided into four subgroups based on the phylogenetic analysis of
their serine protease domains and the domain pattern of the
extracellular stem region, including the human airway trypsin-like
protease (HAT)/differentially expressed in squamous cell carcinoma (DESC), the hepsin/transmembrane protease serine
(TMPRSS), the matriptase and the corin subfamilies1,2. All
TTSPs contain six conserved cysteine residues within their
catalytic domain, which form three intradomain disulfide bonds
as known for all serine proteases of the S1 fold. TTSPs have high
affinity towards substrates containing an Arg residue in the P1
position compared to Lys containing sequences and they may be
activated by other members of the family or via intermolecular
autoactivation, as shown in vitro for HAT, matriptase, matriptase2 and TMPRSS23–7. After activation, the C-terminal protease
domain remains covalently linked to N-terminal located domains
by a conserved disulfide bond and can develop their activity in
cellular compartments or on the cell surface8. Similarly to other
TTSPs, TMPRSS2 contains an extracellularly located C-terminal
serine protease domain as well as the stem region with a group A
Address for correspondence: Erzsebet Pászti-Gere, Department of
Pharmacology and Toxicology, Faculty of Veterinary Science,
Szent István University, Budapest, István u. 2. 1078, Hungary.
Tel: +36 1 4784100/8567. Fax: + 36 1 4784172. E-mail:
Gere.Erzsebet@aotk.szie.hu
History
Received 8 February 2016
Revised 8 May 2016
Accepted 17 May 2016
Published online 8 June 2016
scavenger receptor cysteine-rich domain (SRCR) and a lowdensity lipoprotein receptor class A domain (LDLRA)3.
TTSP-regulated proteolysis is a prerequisite for tissue homeostasis, however, uncontrolled expression and enhanced enzymatic
activity of cell surface-attached TTSPs can lead to different
disorders such as carcinogenesis9 and contributes to influenza and
other respiratory viral infections10. Moreover, TMPRSS2 accumulated in the glandular lumen of normal and cancerous prostate
tissues can be proteolytically cleaved and the rise in released
protease fragment levels can confirm the presence of cancer cells.
This can be used as diagnostic or prognostic marker for prostate
cancer7.
Expression of TMPRSS2 was detected in the luminal epithelial
cells of mouse and human prostate11, strong membranous protein
presence was found in renal tubules, epididymis and ducts of
pancreas, whereas moderate membranous staining was observed
in gastrointestinal tract7,12–14. However, the physiological role of
this TTSP has not been completely revealed so far. TMPRSS2
knockout mice showed normal growth and reached normal
adulthood without having abnormalities in organ histology and
alteration in protein levels of prostatic secretions. This suggests
that TMPRSS2 may maintain a specialized but nonvital function
that is apparent only in association with systemic homeostasis
perturbation15. TMPRSS2 is expressed on MDCK-TMPRSS2 cell
surface as full length zymogen (70 kDa) and in a processed form
(30 kDa). The truncated form containing the catalytic domain
can be shed in small amounts into supernatants, but interestingly,
only a minor enzymatic activity could be detected for the soluble
form of TMPRSS216.
TMPRSS2 present in the human airways is capable of cleaving
influenza virus hemagglutinin (HA) thus facilitating fusion
between viral and endosomal membranes. This suggests that
124
E. Pászti-Gere et al.
potent TMPRSS2 inhibitors could be potential drugs for the
treatment of influenza infections and virus spread. Suppression of
HA cleavage and the inhibition of influenza virus spread in
TMPRSS2-expressing MDCK cells by treatment with hydrophobic decanoylated peptide mimetic protease inhibitor, I-3 was
observed after exposure of cells to A/Hamburg/09 (H1N1)14,16.
I-432 was found to be one of the most potent 3-amidinophenylalanine-derived inhibitors of TMPRSS2 and matriptase17 and it
was also shown that I-432 at 50 mM did not affect cell viability in
IPEC-J2 cells after 48 h treatment18.
Enzymatic activity of TMPRSS2 was initially determined by
the trypsin substrate, Cbz-Gly-Gly-Arg- (aminomethyl) coumarin
(AMC). The amounts of matrix metalloproteases (MMP-2 and
MMP-9) responsible for metastasis of tumor cells were reported
to be elevated in prostate cancer upon activation of proteaseactivated receptor-2 (PAR-2), the substrate for TMPRSS2. By use
of the PAR-2 antagonist Phe-Ser-Leu-Leu-Arg-Tyr-NH2 it was
found that matriptase and TMPRSS2 can activate PAR2, which
suggests an additional link between a membrane-anchored serine
protease and prostate tumor metastasis. However, further studies
should be conducted if excessive amount of TMPRSS2 in
intracellular space may play a functional role in prostate
tumorigenesis in addition to cell surface protease activity9,13,19.
Another fluorogenic substrate, Boc-Leu-Gly-Arg-AMC was
used as TMPRSS2 substrate to assess the possibility of proteolytic
cleavage of the influenza virus surface glycoprotein HA by host
cell proteases16. Improved substrate properties were found for the
fluorogenic AMC derivative I-507 (Mes-D-Arg-Pro-Arg-AMC
KM ¼ 2.9 mM), which is more efficiently cleaved and was applied
for a first inhibitor screening. In that work various substrateanalogue structures containing a 4-amidinobenzylamide as P1
residue and several arylsulfonylated amides of 3-amidinophenylalanine could be identified as potent TMPRSS2 inhibitors17.
In this study, non-tumorigenic jejunal epithelial IPEC-J2 cells
grown on microporous membrane were used for mimicking in vivo
conditions to study the effects of a potent combined inhibitor I-432
(i) on enzymatic activity of this TTSP in cell membrane and in
soluble form using the fluorogenic substrate I-507 and (ii) on
production of physiological reactive oxygen species in apical and
basolateral compartments. In addition, the efficiency of TMPRSS2
inhibition was also investigated to see how the I-432 can affect this
trypsin-like transmembrane serine protease at protein level.
Methods
Cell lines and culture conditions
The IPEC-J2 cell line was derived from jejunal epithelia of a
neonatal piglet. The IPEC-J2 cell line was kindly provided by Dr.
Jody Gookin and Dr. Stephen Stauffer, Department of Clinical
Sciences, College of Veterinary Medicine, North Carolina State
University, Raleigh, NC, USA and it was used between passage 50
and 75. Cells form a differentiated layer and are attached to each
other via tight junctions. IPEC-J2 cells were seeded at a density of
1.5 105 per well on six-well plates with Transwell polyester
membrane inserts (pore size 0.4 mm; surface area 4.67 cm2;
Sigma-Aldrich, St. Louis, MO) coated with rat tail collagen
(Sigma-Aldrich) in a 1.5 ml apical and 2.6 ml basolateral volume.
Cells were maintained in complete medium containing 1:1
mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Nutrient Mixture (DMEM/F12) supplemented with 5% FBS,
5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenium, 5 ng/ml
epidermal growth factor and 1% penicillin-streptomycin (all from
Fisher Scientific, Pittsburgh, PA). Cell cultures were tested by
PCR and were found to be free of mycoplasma contamination.
Cells were allowed to adhere for 24 h before being washed and refed every other day until confluence was reached. Transepithelial
J Enzyme Inhib Med Chem, 2016; 31(S2): 123–129
Figure 1. Chemical structure of I-432.
electrical resistance (TEER) measurement of monolayers was
performed on alternate days after seeding, from day 5 to 21 of
culture, using an EVOM Epithelial Tissue Volt/Ohmmeter (World
Precision Instruments, Berlin, Germany). They were grown at
37 C in a humidified atmosphere of 5% CO2.
Exposure of IPEC-J2 cells to TMPRSS2 inhibitor
The stock solutions of dibasic TMPRSS2/matriptase inhibitor,
I-432 (Ki ¼ 0.9 nM for TMPRSS2 and Ki ¼ 2 nM for matriptase,
Figure 1) at 10 mM were prepared and it was kept at 20 C.
Before treatment, the confluent layers of IPEC-J2 cells were
washed twice with supplement-free plain medium. The solutions
of the TMPRSS2 inhibitor in phenol red free DMEM at 10, 25
and 50 mM were prepared freshly prior to enzyme activity assays
and immunofluorescence experiments from a 10 mM stock
solution. I-432 was added at the 50 mM in case of extracellular
H2O2 measurements and Western blot analysis. After 48 h
incubation, the cells were washed twice with plain medium
before being subjected to the subsequent procedures.
Protein extraction
For protein extraction, the IPEC-J2 cells were washed with
phosphate-buffered saline buffer (PBS, pH 7.4) and harvested with
cell scraper after adding 250 ml extraction buffer (20 mM Tris pH
7.4, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100 supplemented
with 10 ml/ml phosphatase inhibitors (Sigma-Aldrich) and 5 ml/ml
proteinase inhibitors (Sigma-Aldrich)). The pellet was collected in
1.5 ml Eppendorf tubes and lysed for 30 min on ice. Lysates were
then centrifuged at 12 000 rpm for 15 min at 4 C. The extracts
were mixed with 5 Laemmli sample buffer containing 5%
2-mercaptoethanol (BioRad Laboratories, Philadelphia, PA) and
heated to 95 C for 5 min. Protein concentration in the supernatant
was determined by the Bradford assay (BioRad Laboratories).
Western blot
Analysis of TMPRSS2 in cell extract was performed by Western
blotting. Equal amounts of protein (20 mg) were loaded and run on
a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) at 180 V for 1 h. After running, the proteins were
transferred onto Immobilion-P nitrocellulose membrane
(Millipore, Darmstadt, Germany) at constant 75 mA at 4 C
overnight. After blotting, the membrane was stained with Ponceau
S red (BioRad Laboratories) to visualize the transferred proteins.
Nonspecific binding of the antibody was blocked by incubation
with 5% nonfat milk (BioRad Laboratories) dissolved in 1 0.1
M Tris-buffered saline (TBS) containing 0.05% Tween 20 pH 7.4
(TBST) for 60 min at room temperature. The membrane was
washed five times for 5 min with 1 TBST, and incubated
overnight at 4 C with rabbit polyclonal anti-TMPRSS2 (1:500,
Sigma-Aldrich) antibody diluted in 1 TBST containing 3%
nonfat milk. Next day the membrane was washed 5 times for
In vitro characterization of TMPRSS2 inhibition
DOI: 10.1080/14756366.2016.1193732
5 min with TBST and incubated with horseradish peroxidase
(HRP) conjugated goat anti-rabbit secondary antibody (1:1000,
Cell Signaling Technology, Inc., Danvers, MA) diluted in 1
TBST containing 1% nonfat milk for 60 min at room temperature.
For loading control rabbit, anti-b-actin (1:2000, Cell Signaling
Technology, Inc.) antibody was used for 60 min. Detection was
performed by Super Signal West Pico ECL reagent for 10 min
(Pierce Biotechnology Inc., Rockford, IL). The molecular mass of
specific bands was determined comparing to the Precision Plus
Protein Standard (BioRad Laboratories) applied on the same gels.
Densitometric analysis of the blots was done with Kodak
Molecular Imaging Software 4.1 in a Kodak Image Station
4000 MM (Kodak, Rochester, NY).
Protease activity at the cell surface and in cell
supernatants
To determine the trypsin-like protease activity, IPEC-J2 cells
were cultured in 24-well plates. Prior to the protease activity
measurements, the cells were treated with I-432 at 10, 25 and
50 mM for 48 h. Cells were washed with phenol red-free DMEM
and the protease activity was assessed by incubation of cells with
the fluorogenic substrate Mes-D-Arg-Pro-Arg-AMC 2 TFA
(I-507)17. I-507 was used at the final concentration of 200 mM in
50 mM PBS (pH ¼ 7.4) for 30 min at 37 C. Hydrolysis of the
peptide was monitored by the measurements of fluorescence
intensity using a Victor X2 2030 fluorescence spectrometer at
ex ¼ 380 nm and em ¼ 460 nm.
To examine the enzymatic activity in the supernatants, IPECJ2 cells were grown in 24-well plates and were incubated with
I-432 at 10, 25 and 50 mM for 48 h. The supernatants were then
removed, cleared by low-speed centrifugation (3000 rpm, 10 min
at 4 C), and treated with the same fluorescence substrate at the
final concentration of 200 mM in the supernatant. After 30 min
incubation time at 37 C, fluorescence intensity was measured.
Extracellular H2O2 measurement by the Amplex
red method
Fluorescent ROS measurement of cell supernatant was based on
the detection of H2O2 using the Amplex Red Hydrogen Peroxide
Assay Kit (Invitrogen/Life Technologies, Eugene, OR). In the
presence of horseradish peroxidase (HRP), Amplex Red reacts
with H2O2 in a 1:1 stoichiometry producing a highly fluorescent
resorufin20. IPEC-J2 cells were treated with I-432 at 50 mM for
48 h in phenol red free DMEM and the H2O2 concentrations in the
medium were determined using a working solution of 100 mM
Amplex Red and 0.2 U/ml HRP. After 30 min incubation with the
dye at room temperature, the quantitative H2O2 contents of apical
and basolateral compartments were measured using a Victor X2
2030 fluorometer (ex ¼ 560 nm, em ¼ 590 nm).
Investigation of TMPRSS2 distribution via
immunfluorescent staining
Inserts were fixed in methanol for 5 min followed by bovine
serum albumin (BSA (5%), Sigma Aldrich) protein block for
20 min. Sections were incubated for 1 h in a humid chamber at
room temperature with anti-TMPRSS2 rabbit polyclonal primary
antibody (1:200, Sigma-Aldrich) and diluted in 5% BSA solution.
For secondary antibody Alexa546 (orange-red) anti-rabbit Ig-s
1:200 diluted in PBS were used for 1 h. Sialic acid residues in cell
membrane were stained with wheat germ agglutinin (WGA)
(1:200 diluted in PBS, WGA Alexa Fluor 488, InvitrogenMolecular Probes) for 10 min to visualize the localization
of cell membrane and cell nuclei were stained in blue
using 40 ,6-diamidino-2-phenylindole (DAPI) (1:500 diluted in
125
PBS, Invitrogen-Molecular Probes) for additional 10 min.
Between incubations, the slides were washed in PBS for
3 2 min. Membranes were attached on glass slides using
fluorescent mounting medium (DAKO, Glostrup, Denmark).
The samples were analyzed using a Nikon Eclipse E600
epifluorescent microscope (Nikon, Amsterdam, Netherlands)
with LUCIAÔ Citogenetics 2.5 software.
Statistical analysis
For statistical evaluation, R 2.11.1 software package (2010;
www.R-project.org) was applied. Differences between means
were evaluated by one-way analysis of variance (one-way
ANOVA) with post-hoc Tukey test, where data were of normal
distribution and homogeneity of variances was confirmed.
Results
Expression of TMPRSS2 in IPEC-J2 cells
Different protein species for TMPRSS2 in whole cell lysates from
IPEC-J2 cells untreated or exposed to I-432 analyzed by western
blotting can be seen in Figure 2. The serine protease domain of
TMPRSS2 was absent (missing 28 kDa band) in cell lysates when
IPEC-J2 cells were exposed to I-432 at 50 mM for 48 h
(p ¼ 0.02057). It was also observed that expression of truncated
fragments of 62 kDa was significantly reduced after I-432
administration (p ¼ 0.00357). However, full length form of
TMPRSS2 with molecular mass of 72 kDa can be found mainly
in I-432-treated samples and it can be detected only in trace
quantities in control IPEC-J2 cells (p ¼ 9.3535 105)
(Figure 2(A–C)).
Enzymatic activity of TMPRSS2
The cleavage rate of the substrate Mes-D-Arg-Pro-Arg-AMC was
determined in the absence and in the presence of I-432 in cellassociated forms and in supernatants of non-tumorigenic IPEC-J2
cells. I-432 could significantly decrease the activity of shed
TTSPs including that of TMPRSS2 of high potency towards I-507
thus lowering the extent of cleavage of applied fluorogenic
substrate. Significant differences can be seen in fluorescence
intensity values when IPEC-J2 cells were treated with I-432 at
10 mM (p ¼ 0.02039), at 25 mM (p ¼ 0.00113) and 50 mM
(p50.001) compared to those of controls (Figure 3A).
Tryptic activity of cell-associated TTSPs was also monitored
in intact IPEC-J2 cells. In accordance with the measurement of
soluble forms of proteins released from cells, we also detected
proteolytic activity of cell-associated TTSP (p40.05, Figure 3B).
Extracellular H2O2 produced by IPEC-J2 cells exposed
to I-432
After a 48 h incubation time with apically administered inhibitor,
I-432 at 50 mM fluorescence values of Amplex red were
determined in IPEC-J2 cells compared to nontreated control
cells (Figure 4). The results showed that there were not any
significant perturbations in redox balance in IPEC-J2 cells for the
50 mM concentration of the inhibitor compared to control values
independently of the sampling type (p ¼ 0.954 for apical
sampling, p ¼ 0.436 for basolateral sampling). It can be also
seen that prior to and after I-432 treatment extracellular hydrogen
peroxide levels did not differ in apical and in basolateral
compartments (p ¼ 0.795 and p ¼ 0.265, respectively).
Subcellular localization of TMPRSS2 in IPEC-J2 cells
IPEC-J2 cells were exposed to inhibitor I-432 at 10, 25 and 50 mM
for 48 h and cellular distribution of TMPRSS2 was analyzed by
126
E. Pászti-Gere et al.
J Enzyme Inhib Med Chem, 2016; 31(S2): 123–129
I-432
treated
IPEC-J2
Control
IPEC-J2
72 kDa
62 kDa
28 kDa
β-acn
(B)
***
10
8
6
4
2
0
(C)
1,4
**
TMPRSS2 28 KDA EXPRESSION
12
TMPRSS2 62 KDA EXPRESSION
TMPRSS2 72 KDA EXPRESSION
(A)
1,2
1
0,8
0,6
0,4
0,2
0
Co
I-432
Co
I-432
1,6
*
1,4
1,2
1
0,8
0,6
0,4
0,2
0
Co
I-432
Figure 2. Expression of TMPRSS2 in IPEC-J2 cells. IPEC-J2 cells were untreated or exposed to I-432 for 48 h at 50 mM in phenol red free DMEM at
pH ¼ 7.4 at 37 C. Cell lysates were prepared and analyzed by immunoblotting using polyclonal TMPRSS2 Ab. The full length of TMPRSS2 present in
IPEC-J2 cells appeared as a 72 kD band, the truncated form as a 62 kDa band and serine protease domain as 28 kDa band on the blot, whereas b-actin
(42 kDa) was used as reference housekeeping protein on the same blot. (A) Relative expression levels for full length form with apparent molecular mass
of 72 kDa in I-432 treated samples versus control (p ¼ 9.3535 105). (B) Relative expression levels for truncated form with apparent molecular
mass of 62 kDa in I-432 treated samples versus control (p ¼ 0.00357). (C) Relative expression levels for serine protease domain with apparent
molecular mass of 28 kDa in I-432 treated samples versus control (p ¼ 0.02057). Relative expression levels of detected protein products were
compared in I-432 and in mock (Co) IPEC-J2 samples based on densitometric quantification of blot bands. Data are presented as average relative
expression intensities ± SDs. Results shown are representative of three independent experiments.
epifluorescent miscroscopy. In control cells, TMPRSS2 (show in
red) can be found on the cell surface with a more intense staining
pattern at cell-cell contacts and within the IPEC-J2 cells where the
staining was rather diffuse. Immunfluorescence analysis revealed
that in control samples wheat germ agglutinin (WGA, shown in
green) was co-localized with TMPRSS2 confirming the cell
membranous positivity in addition to perinuclear presence of
TMPRSS2 under physiological conditions. With increasing concentration of I-432 only smaller amounts of TMPRSS2 could be
detected (Figure 5), presumably at the site of the synthesis with a
visible decrease in the membrane presence of TMPRSS2.
Discussion
Prophylactic vaccination is widely accepted to stop seasonal flu
epidemics, however, the vaccine has to be reformulated annually
due to the alteration of the virus genome causing changes in virus
surface proteins. Currently, there are only few approved therapeutic options with limited efficacies in the treatment of acute
influenza virus infections, including administration of M2 ion
channel blockers or neuraminidase inhibitors21. Very recently, the
pyrazinecarboxamide RNA polymerase inhibitor, favipiravir was
approved in Japan against influenza22.
Proteolysis of influenza virus HA by host cell proteases is a
prerequisite for viral infectivity, but the precise protease
responsible for HA cleavage has not been completely elucidated
yet. It was previously shown that TMPRSS2 allows HA cleavage
thus promoting viral spread. Knockout of TMPRSS2 could
prevent the propagation of H1N1, H7N9 viruses into the lungs
of mice, however, TMPRSS2 seemed to play only marginal role in
H3N2-caused infection23,24.
Development of synthetic inhibitors of TMPRSS2 can offer
also possibilities in the therapy of human metapneumovirus
(HMPV) infections by reduction in cleavage of the fusion protein
F25,26 or in the treatment of severe acute respiratory syndrome
coronavirus, where TMPRSS2 is involved in the spike protein S
activation27,28. Calu-3 cells infected with H1N1 or H3N2
influenza viruses were treated with I-432 and related TMPRSS2
inhibitors at 50 mM for 48 h incubation time and it was found that
the viral replication was significantly reduced. So far, the most
potent inhibitor for suppression of H1N1 and H3N2 influenza
viral spread in Calu-3 cells is I-432, which was also applied in our
present study17.
IPEC-J2 behave similarly to human colon adenocarcinoma
cells (Caco-2 and T84 cells) with the advantage of not being
cancerous, and their glycolysation pattern, proliferation rate and
colonization ability are closer to physiological functioning of
enterocytes29. Influenza A viruses in pigs can lead to both
economic losses and can present permanent risks of cross-species
transmission of new strains to humans30. It was also reported that
In vitro characterization of TMPRSS2 inhibition
DOI: 10.1080/14756366.2016.1193732
(A) 35000
control
25 µM I-432
10 µM I-432
50 µM I-432
TREATMENT TYPE
127
IF STAINING OF TMPRSS2
Fluorescence intensity
30000
*
25000
**
***
Control
20000
15000
10000
5000
10 µM I-432
0
cell supernatant
(B) 50000
control
25 µM I-432
10 µM I-432
50 µM I-432
Fluorescence intensity
40000
25 µM I-432
30000
20000
10000
50 µM I-432
0
cell-associated
Figure 3. Measurements of enzymatic TMPRSS2 activity using the
fluorescence substrate I-507 at 200 mM in presence of inhibitor I-432
apically applied at 10, 25 and 50 mM for 48 h in IPEC-J2 cell system.
(A) Enzymatic activity assay of shed TMPRSS2 in the supernatant of
IPEC-J2. The data are indicated as average fluorescence intensities ± SDs
(n ¼ 3). Significant differences can be seen between IPEC-J2 cells
exposed to I-432 at 10 mM (p ¼ 0.02039), at 25 mM (p ¼ 0.00113), at
50 mM (p50.001) for 48 h compared to those in untreated samples.
(B) Activity measurements of cell-associated TMPRSS2 in control and in
inhibitor-treated IPEC-J2, the data are indicated as average fluorescence
intensities ± SDs. No significant differences were found in control and
inhibitor-treated IPEC-J2 cells (n ¼ 3, p40.05).
50000
Apical I-432 treatment
Amplex red fluorescence
Apical sampling
Basolateral sampling
40000
30000
20000
10000
0
Control
I-432
Figure 4. Detection of extracellular H2O2 production when IPEC-J2 cells
were treated with 50 mM I-432 for 48 h apically using apical and
basolateral sampling. Data are presented as average fluorescence
intensities ± SDs. Apical treatment with I-432 did not result in significant
alterations in peroxide level independently of the sampling type
(p ¼ 0.954 for apical sampling, p ¼ 0.436 for basolateral sampling, n ¼ 3).
Figure 5. Immunostaining of TMPRSS2 expression pattern in IPEC-J2
cells cultured on polyester membrane inserts treated with inhibitor I-432
at 10, 25 and 50 mM for 48 h. Cell nuclei are stained blue (DAPI), cell
membranes are labeled with wheat germ agglutinin (Alexa 488, green). In
control samples, TMPRSS2 (Alexa 564, red) is colocalized with wheat
germ agglutinin. 48 h long treatment of I-432 induced significant
fluorescent signal loss of TMPRSS2. TMPRSS2 occurence was mainly
observed around synthesis sites in I-432-treated IPEC-J2 cells. 600
magnification. The scale bar (white) indicates 20 mm (n ¼ 3).
cleavage of HA in human colon adenocarcinoma cells appeared to
take place intracellularly31 and reverse transcription-PCR analyzes revealed that Caco-2 cells express TMPRSS216. Further
studies are still ongoing to ascertain the abundance and cellular
localization of TMPRSS2 in different cell types and tissues. This
is the first study in which expression of TMPRSS2 was
determined and confirmed at protein level in a non-tumorigenic
intestinal cell line, IPEC-J2 using Western blot technique.
In this work, we used the potent TMPRSS2 inhibitor I-432 to
elucidate how this drug candidate can influence physiological
parameters such as reactive oxygen species production as a key
indicator of redox status. It was previously shown by us that I-432
at 50 mM applied for 48 h did not induce cell death compared to
control cells18. It was found, however, that various inhibitors
including I-432 could weaken intestinal barrier integrity by
lowering transepithelial electrical resistance in vitro32.
This is the first study in which the inhibitory effects of a potent
3-amidinophenylalanine-derived serine protease inhibitor was
characterized on TMPRSS2 expression at protein level in
IPEC-J2 cell line. Only the enzymatic activity of TTSP in cell
supernatants could be suppressed in a concentration-dependent
manner when cells were exposed to I-432 at 10, 25 and 50 mM.
Under untreated conditions the baseline enzymatic activities were
comparable in case of supernatants and membrane associated
128
E. Pászti-Gere et al.
forms, but I-432 could not suppress cleavage of I-507 by
cell-associated proteases suggesting that trypsin-like serine proteases other than TMPRSS2 could be present in the studied
samples. Similarly to the previous results in MDCK cells16,
enzymatic activity of TMPRSS2 at the cell surface and the soluble
forms released from the plasma membrane were found to be very
low. Evaluation of host cell contribution to influenza viral
pathogenesis as a consequence of host-virus infection in swine,
IPEC-J2 cell line seems to be a better in vitro model as an
alternative to MDKC cells which differ significantly from either
human or porcine cells33. The further advantage of the application
for IPEC-J2 cell line originated from small intestine of unsuckled
pigs is that it enables the replacement of animal infection models
and supports the in-depth investigation of cross-species transmission and rare, but existing extrapulmonary effects of viral spread
in digestive system34. Based on the fact that expression of
TMPRSS2 in stomach and in colon was confirmed, it can be
assumed that this serine protease might be responsible for putative
extrapulmonary spread of influenza viruses14,35.
To evaluate the side effect of this potential drug candidate, the
oxidative stress-inducing properties of I-432 was also monitored.
The extracellular hydrogen peroxide level detected by Amplex red
fluorescent probe was not altered when I-432 was used at 50 mM for
48 h incubation time. Protein species such as serine protease
domain with molecular mass 28 kDa and the full length form of
TMPRSS2 (72 kDa) were also identified7,16 in addition to 62 kDa
truncated fragment. It was also confirmed that treatment
with 50 mM I-432 induced complete loss of the band for
serine protease domain on the blot. Another consequence of
TMPRSS2 blockage via I-432 was the reduction in immunfluorescent signals indicating the lowered expression of this TTSP when
the inhibitor was used at micromolar concentrations for 2 days.
Taken together, it was ascertained that suppression of TMPRSS2
via I-432 resulted in dysregulated enzymatic activity and modified
target protein expression profile compared to physiological serine
protease functioning in non-tumorigenic jejunal cell monolayers.
Conclusion
Based on our data, it can be concluded that I-432 as potential drug
candidate against influenza virus infection possesses favorable
side effect profile considering the complete lack of oxidative
stress induction after its administration thus allowing the maintenance of physiological redox status. The ability of this
compound for TMPRSS2 inhibition was confirmed at protein
level in vitro indicated by the lack of expression of serine protease
domain and the decreasing membranous positivity of TMPRSS2
immunofluorescent signals.
Acknowledgements
We are indebted to Dr. Jody Gookin and Dr. Stephen Stauffer, Department
of Clinical Sciences, College of Veterinary Medicine, North Carolina
State University, Raleigh, NC, USA for providing IPEC-J2 cells and for
the valuable advice on handling them and to Dr. Nóra Meggyesházi,
Semmelweis University, 1st Department of Pathology and Experimental
Cancer Research for contributing to taking fluorescent images of the
IPEC-J2 samples. This project was supported by the János Bolyai
Research Scholarship of the Hungarian Academy of Sciences and by the
Hungarian Scientific Research Fund [grant number 115685].
Declaration of interest
The authors report no declaration of interest. The authors alone are
responsible for the content and writing of this article. This research was
supported by the 9877–3/2015/FEKUT grant of the Hungarian Ministry
of Human Resources.
J Enzyme Inhib Med Chem, 2016; 31(S2): 123–129
References
1. Antalis TM, Bugge TH, Wu Q. Membrane-anchored serine proteases in health and disease. Prog Mol Biol Transl Sci 2011;99:1–50.
2. Szabo R, Bugge TH. Type II transmembrane serine proteases in
development and disease. Int J Biochem Cell Biol 2008;40:
1297–316.
3. Hooper JD, Clements JA, Quigley JP, Antalis TM. Type II
transmembrane serine proteases. Insights into an emerging class of
cell surface proteolytic enzymes. J Biol Chem 2001;276:857–60.
4. Bugge TH, Antalis TM, Wu Q. Type II transmembrane serine
proteases. J Biol Chem 2009;284:23177–81.
5. Takeuchi T, Harris JL, Huang W, et al. Cellular localization of
membrane-type serine protease 1 and identification of proteaseactivated receptor-2 and single-chain urokinase-type plasminogen
activator as substrates. J Biol Chem 2000;275:26333–42.
6. Velasco G, Cal S, Quesada V, et al. Matriptase-2, a membranebound mosaic serine proteinase predominantly expressed in human
liver and showing degrading activity against extracellular matrix
proteins. J Biol Chem 2002;277:37637–46.
7. Afar DE, Vivanco I, Hubert RS, et al. Catalytic cleavage of the
androgen-regulated TMPRSS2 protease results in its secretion by
prostate and prostate cancer epithelia. Cancer Res 2001;61:1686–92.
8. Szabo R, Wu Q, Dickson RB, et al. Type II transmembrane serine
proteases. Thromb Haemost 2003;90:185–93.
9. Ko CJ, Huang CC, Lin HY, et al. Androgen-induced TMPRSS2
activates matriptase and promotes extracellular matrix degradation,
prostate cancer cell invasion, tumor growth and metastasis. Cancer
Res 2015;75:2949–60.
10. Esumi M, Ishibashi M, Yamaguchi H, et al. Transmembrane serine
protease TMPRSS2 activates hepatitis C virus infection. Hepatology
2015;61:437–46.
11. Vaarala MH, Porvari K, Kyllönen A, et al. The TMPRSS2 gene
encoding transmembrane serine protease is overexpressed in a
majority of prostate cancer patients: detection of mutated TMPRSS2
form in a case of aggressive disease. Int J Cancer 2001;94:705–10.
12. Lin B, Ferguson C, White JT, et al. Prostate-localized and androgenregulated expression of the membrane-bound serine protease
TMPRSS2. Cancer Res 1999;59:4180–4.
13. Lucas JM, True L, Hawley S, et al. The androgen-regulated type II
serine protease TMPRSS2 is differentially expressed and mislocalized in prostate adenocarcinoma. J Pathol 2008;215:118–25.
14. Bertram S, Glowacka I, Blazejewska P, et al. TMPRSS2 and
TMPRSS4 facilitate trypsin-independent spread of influenza virus in
Caco-2 cells. J Virol 2010;84:10016–25.
15. Kim TS, Heinlein C, Hackman RC, Nelson PS. Phenotypic analysis
of mice lacking the TMPRSS2-encoded protease. Mol Cell Biol
2006;26:965–75.
16. Böttcher-Friebertshäuser E, Freuer C, Sielaff F, et al. Cleavage of
influenza virus hemagglutinin by airway proteases TMPRSS2 and
HAT differs in subcellular localization and susceptibility to protease
inhibitors. J Virol 2010;84:5605–14.
17. Meyer D, Sielaff F, Hammami M, et al. Identification of the first
synthetic inhibitors of the type II transmembrane serine protease
TMPRSS2 suitable for inhibition of influenza virus activation.
Biochem J 2013;452:331–43.
18. Pászti-Gere E, McManus S, Meggyesházi N, et al. Inhibition of
matriptase activity results in decreased intestinal epithelial monolayer integrity in vitro. PLoS One 2015;10:e0141077.
19. Wilson S, Greer B, Hooper J, et al. The membrane-anchored serine
protease, TMPRSS2, activates PAR-2 in prostate cancer cells.
Biochem J 2005;388:967–72.
20. Mohanty JG, Jaffe JS, Schulman ES, Raible DG. A highly sensitive
fluorescent micro-assay of H2O2 release from activated human
leukocytes using a dihydroxyphenoxazine derivative. J Immunol
Methods 1997;202:133–41.
21. Steinmetzer T, Hardes K, Böttcher-Friebertshäuser E, Garten W.
Strategies for the development of influenza drugs: basis for new
efficient combination therapies. Top Med Chem 2015;15:143–82.
22. Furuta Y, Gowen BB, Takahashi K, et al. Favipiravir (T-705),
a novel viral RNA polymerase inhibitor. Antiviral Res 2013;100:
446–54.
23. Tarnow C, Engels G, Arendt A, et al. TMPRSS2 is a host factor that
is essential for pneumotropism and pathogenicity of H7N9 influenza
A virus in mice. E J Virol 2014;88:4744–51.
DOI: 10.1080/14756366.2016.1193732
24. Garten W, Braden C, Arendt A, et al. Influenza virus activating host
proteases: identification, localization and inhibitors as potential
therapeutics. Eur J Cell Biol 2015;94:375–83.
25. Schowalter RM, Smith SE, Dutch RE. Characterization of human
metapneumovirus F protein-promoted membrane fusion: critical
roles for proteolytic processing and low pH. J Virol 2006;80:
10931–41.
26. Shirogane Y, Takeda M, Iwasaki M, et al. Efficient multiplication of human metapneumovirus in vero cells expressing the
transmembrane serine protease TMPRSS2. J Virol 2008;82:
8942–6.
27. S, Nagata N, Shirato K, Kawase, et al. Efficient activation of
the severe acute respiratory syndrome coronavirus spike protein
by the transmembrane protease TMPRSS2. J Virol 2010;84:
12658–64.
28. Glowacka I, Bertram S, Müller MA, et al. Evidence that
TMPRSS2 activates the severe acute respiratory syndrome
coronavirus spike protein for membrane fusion and reduces
viral control by the humoral immune response. J Virol 2011;85:
4122–34.
In vitro characterization of TMPRSS2 inhibition
129
29. Cencic A, Langerholc T. Functional cell models of the gut and their
applications in food microbiology – a review. Int J Food Microbiol
2010;141:S4–14.
30. Peitsch C, Klenk HD, Garten W, Böttcher-Friebertshäuser E.
Activation of influenza A viruses by host proteases from swine
airway epithelium. J Virol 2010;84:5605–14.
31. Zhirnov OP, Vorobjeva IV, Ovcharenko AV, Klenk HD. Intracellular
cleavage of human influenza a virus hemagglutinin and its
inhibition. Biochemistry Mosc 2003;68:1020–6.
32. Pászti-Gere E, Barna RF, Ujhelyi G, Steinmetzer T. Interaction
exists between matriptase inhibitors and intestinal epithelial cells.
J Enzyme Inhib Med Chem 2015. [Epub ahead of print]. doi:
10.3109/14756366.2015.1060483.
33. Sun Z, Huber VC, McCormick K, et al. Characterization of a porcine
intestinal epithelial cell line for influenza virus production. J Gen
Virol 2012;93:2008–16.
34. Shinde V, Bridges CB, Uyeki TM, et al. Triple-reassortant swine
influenza A (H1) in humans in the United States 2005-2009. N Engl
J Med 2009;360:2616–25.
35. Kuiken T, Taubenberger JK. Pathology of human influenza
revisited. Vaccine 2008;26:59–66.