Tohoku J. Exp. Med., 2004, 203, 313-318 RIG-I in T24 Cells
313
Upregulation of Retinoic Acid-Inducible Gene-I in T24
Urinary Bladder Carcinoma Cells Stimulated with
Interferon-γ
TADAATSU I MAIZUMI , N ORITO YAGIHASHI , 1 M ASAHARU H ATAKEYAMA , K OJI
YAMASHITA , A KIRA I SHIKAWA , K AGEAKI TAIMA , 2 H IDEMI YOSHIDA , S OROKU
YAGIHASHI1 and KEI SATOH
Department of Vascular Biology, Institute of Brain Science, 1Department of
Pathology, and 2Department of Internal Medicine, Hirosaki University
School of Medicine, Hirosaki 036-8562
IMAIZUMI, T., YAGIHASHI, N., HATAKEYAMA, M., YAMASHITA, K., ISHIKAWA, A.,
TAIMA, K., YOSHIDA, H., YAGIHASHI, S. and SATOH, K. Upregulation of Retinoic AcidInducible Gene-I in T24 Urinary Bladder Carcinoma Cells Stimulated with Interferon-γ . Tohoku J. Exp. Med., 2004, 203 (4), 313-318 ── Urinary bladder epithelial
cells play an important role in the host defense against urinary tract infections. Interferon-γ (IFN-γ ) is a potent cytokine that regulates immune responses by inducing
multiple genes in many types of cells including urinary bladder epithelial cells. Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH-box family, which is
involved in various reactions related to RNA metabolism, and is induced in leukemic
cells by retinoic acid or in endothelial cells by lipopolysaccharide. We have studied
the expression of RIG-I in T24 cells, a cell line derived from human urinary bladder
epithelial carcinoma cells. IFN-γ stimulated T24 cells to express RIG-I mRNA and
protein in concentration- and time-dependent manners. Immunohistochemical analysis revealed the expression of RIG-I in the urinary bladder epithelium from a patient
with chronic urinary tract infection and in a bladder epithelial carcinoma. We conclude that RIG-I may play some role in inflammatory reactions in the urinary tract
epithelium. ──── RIG-I; IFN-γ ; T24 cells; urinary bladder epithelial cells
© 2004 Tohoku University Medical Press
Received March 2, 2004; revision accepted for publication June 7, 2004.
Address for reprints: Dr. Tadaatsu Imaizumi, Department of Vascular Biology, Institute of Brain Science,
Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
e-mail: [email protected]
313
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T. Imaizumi et al.
Epithelial cells serve as a first physical barrier against infectious agents, and urinay bladder
epithelial cells are an important cellular component in a host-defense mechanism in urinary tract
infections. Various cytokines stimulate epithelial
cells to express key molecules for immunological and inflammatory reactions, and interferonγ (IFN-γ ) is a major cytokine that plays a pivotal
role in the regulation of these reactions. IFN-γ
exerts its biological activities through the control
of multiple genes (Decker et al. 1997; Stark et al.
1998), and urinary bladder epithelial cells are one
of the targets of IFN-γ (Nouri et al. 1994; Park et
al. 1997; Lung et al. 1998).
Retinoic acid-inducible gene-I (RIG-I) was
first identified as a gene induced by retinoic acid
in promyelocytic leukemia cells (Sun 1997).
Melanoma-differentiation associated gene-5 is
highly homologous to RIG-I and induced during
differentiation of a melanoma cell line (Kang et
al. 2002); and RIG-I may be involved in the control of cellular differentiation. RIG-I is a member
of the DExH box family protein and regarded
as a putative RNA helicase from its amino acid
sequence; however, details of its biological functions are not known. RIG-I is also expressed in
vascular endothelial cells stimulated with lipopolysaccharide (Imaizumi et al. 2002), and porcine homologue of RIG-I, RNA helicase induced
by virus (RHIV-1), is induced in macrophages
infected with porcine reproductive and respiratory syndrome virus (Zhang et al. 2000). In the
present study, we have addressed the expression
of RIG-I in T24 urinary bladder carcinoma cells,
which has been used as a model of urothelium
(Kato et al. 1978; Byrne et al. 1989; Shiono and
Ike 1999). We also examined the expression of
RIG-I in the bladder epithelium and carcinoma in
vivo.
MATERIALS AND METHODS
Reagents
Recombinant human (r[h]) IFN-γ was
obtained from Roche Diagnostics GmbH
(Mannheim, Germany), and r(h) interleukin-4
(IL-4) from R&D Systems (Minneapolis, MN,
USA). An RNeasy total RNA isolation kit
and Taq DNA polymerase were from Qiagen
(Hilden, Germany). RPMI1640, fetal bovine
serum (FBS), M-Mulv reverse transcriptase
and primer oligo(dT)12-18 were from InVitrogen
(Carlsbad, CA, USA). A rabbit anti-RIG-I antibody was prepared as described (Imaizumi et al.
2002). An anti-rabbit IgG antibody labeled with
HRP was from Kirkegaard Perry (Gaithersburg,
MD, USA). Superblock blocking buffer and a
SuperSignal West Pico chemiluminescence substrate were from Peirce (Rockford, IL, USA).
Oligonucleotide primers were synthesized by
Greiner Japan (Atsugi).
Cell culture
T24 is a cell line derived from human
urinary bladder transitional epithelial cell carcinoma (Bubenik et al. 1970), and was a generous
gift from the Department of Urology, Hirosaki
University School of Medicine. The cells were
cultured using RPMI1640 supplemented with
10% (v/v) FBS.
Reverse transcription-polymerase chain reaction
Total RNA was extracted from the cells using an RNeasy kit, and first strand cDNA was synthesized from 1 μ g of total RNA using M-Mulv
reverse transcriptase and primer oligo(dT)12-18.
Primers for RIG-I, intercellular adhesion molecule-1 (ICAM-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows:
RIG-I-F (5´-GCA TAT TGA CTG GAC
GTG GCA-3´),
RIG-I-R (5´-CAG TCA TGG CTG CAG
TTC TGT C-3´),
ICAM-1-F (5´-CAC AGT CAC CTA TGG
CAA CG-3´),
ICAM-1-R (5´-TTC TTG ATC TTC CGC
TGG C-3´),
GAPDH-F (5´-CCA CCC ATG GCA AAT
TCC ATG GCA-3´),
and GAPDH-R (5´-TCT AGA CGG CAG
RIG-I in T24 Cells
GTC AGG TCC ACC-3´).
The reaction condition for RIG-I was 1
×(94°C, 1 minute), 30×(94°C, 1 minute; 58°
C, 1 minute; 72°C, 1 minute), and 1×(72°C,
10 minutes). The condition for ICAM-1 and
GAPDH was 1×(94°C, 1 minute), 30×(94°C, 1
minute; 55°C, 1 minute; 72°C, 1 minute), and 1×
(72°C, 10 minutes). The products were analyzed
by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The size of the PCR products for RIG-I, ICAM-1 and GAPDH was 644 bp,
742 bp and 696 bp, respectively. Because all of
these primer pairs were designed from different
exons, the products with the expected size were
amplified from single-strand cDNA but not from
contaminating genomic DNA. The PCR products
were confirmed to be specific to each cDNA by
sequencing.
Western blotting
Western blot analysis of RIG-I protein was
performed as described previously (Imaizumi et
al. 2002). Briefly, cells were washed twice with
cold phosphate-buffered saline (pH 7.4) and lysed
with Laemmli reducing sample buffer. The lysate
was subjected to electrophoresis in a 6-9% gradient polyacrylamide gel. Proteins were transferred
to a PVDF membrane, and RIG-I protein was
detected using rabbit anti-RIG-I antibody, HRPlabeled anti-rabbit IgG, and a SuperSignal West
Pico chemiluminescent substrate.
Immunohistochemistry
Expression of RIG-I in the urinary bladder
tissues from two patients was examined by immunohistochemical staining. Case 1; the bladder
tissue from 76 years old male with suspected
malignancy was obtained by biopsy. Non-specific
chronic inflammatory change was observed. Case
2; bladder tumor was resected from 71 years old
male, and it was diagnosed as urothelial carcinoma. Tissue sections mounted on APS-coated
glass slides were deparaffinized, and the slides
were immersed, for 30 minutes, in methanol
containing 0.3% hydrogen peroxide to block en-
315
dogenous peroxidase activity. The sections were
incubated with 1% goat serum for 30 minutes at
room temperature and then with anti-RIG-I antibody (1:1000) or non-immune rabbit IgG overnight at 4°C. The samples were incubated with
biotinylated anti-rabbit IgG and HRP-streptavidin,
and peroxidase reaction was performed by incubating with a DAB/H2O2 solution. The nuclei of
the tissue were counterstained with hematoxyline
solution. The histological specimens are retrieved
from archives of materials in the Department
of Pathology under the guideline issued from
Japanese Society of Pathology.
RESULTS
IFN-γ upregulated the expression of RIG-I
transcript and protein in T24 cells in a concentration-dependent manner (Figs. 1A and 1B). The
expression of RIG-I mRNA reached maximal
level 8-16 hours of stimulation with IFN-γ (Fig.
2A), and the protein expression was somewhat
lagged behind the mRNA expression (Fig. 2B).
IFN-γ -induced expression of mRNA for ICAM-1
was also confirmed (Fig. 2A). Cotreatment of the
cells with IL-4 did not affect the IFN-γ -induced
RIG-I expression (Fig. 3).
Immunohistochemical identification of
RIG-I in urinary bladder tissue is shown in Fig.
4. In case 1, RIG-I immunoreactivity was clearly
observed in the epithelial layer, and accumulation
of mononuclear cells in the subepithelial area was
consistent with the existence of chronic inflammatory changes in the bladder wall. The staining
was also seen in the stroma as well. In case 2,
diffuse expression of RIG-I was detected in bladder carcinoma cells. Staining of RIG-I in normal
mucosa was faint (not shown).
DISCUSSION
IFN-γ is a highly-ranked molecule in the
hierarchy of network for the regulation of immunological and inflammatory responses. In urinary
bladder epithelial cells, IFN-γ has been known to
stimulate the expression of immuno-regulatory
factors such as major histocompatibility antigen
316
T. Imaizumi et al.
Fig. 1. IFN-γ stimulates RIG-I expression in T24
cells in a concentration-dependent manner.
(A) T24 cells were incubated with 0.1-10
ng/ml IFN-γ for 16 hours, and the cells were
subjected to total RNA extraction. Singlestrand cDNA was synthesized from 1 μg of
total RNA, and RT-PCR analyses of mRNAs
for RIG-I and GAPDH were performed. (B)
T24 cells were treated with IFN-γ for 24 hours.
Cells were lysed, and lysates were subjected
to SDS-PAGE. Proteins were transferred to
a PVDF membrane. RIG-I protein with a
molecular weight of 101 K was detected using
a rabbit anti-RIG-I antibody and HRP-labeled
anti-rabbit IgG.
Fig. 2. RIG-I is expressed in T24 cells stimulated
with IFN-γ in a time-dependent fashion. (A)
Cells were stimulated with 10 ng/ml IFN-γ
for up to 24 hours, and RT-PCR analyses for
RIG-I, ICAM-1 and GAPDH were performed.
(B) Cell lysate was prepared and subjected to
western blot analysis for RIG-I.
Fig. 3. Effect of IL-4 on the expression of mRNAs
for RIG-I in T24 cells. T24 cells were cotreated with 10 ng/ml IL-4 and/or 10 ng/ml IFNγ for 16 hours. Total RNA was extracted and
RT-PCR analyses for RIG-I and GAPDH were
performed.
RIG-I in T24 Cells
317
Fig. 4. Immunohistochemical detection of RIG-I in urinary bladder epithelial cells. Case 1; Bladder tissues obtained by biopsy were stained with a RIG-I antibody (A) or a control antibody (B). Nuclear
counterstaining was performed. Note that epithelial cells were stained positively in (A), but not in
(B). Case 2; Bladder epithelial carcinoma tissue resected was stained as well. Bar=30 μ m.
complex class II (Nouri et al. 1994) and ICAM-1
(Park et al. 1997). In the present study, we found
the expression of RIG-I in T24 cells stimulated
with IFN-γ , and RIG-I was also detected in the
epithelium of urinary bladder tissue along with
the evidence of chronic inflammatory reactions.
RIG-I or its homologue are induced in response to
bacterial endotoxin or viral infection (Zhang et al.
2000; Imaiuzmi et al. 2002) and these results suggest that RIG-I is implicated in cellular responses
against infectious agents. In addition to its probable role in cellular differentiation (Sun 1997),
RIG-I may function as one of the mediators of
immune and inflammatory reactions. In this regard, it is noteworthy that RIG-I overexpression
in T24 cells resulted in the induction of cyclooxygenase-2, another key moleculte involved in
inflammatory responses (Imaizumi et al. 2002).
IFN-γ is generally regarded as a major cytokine produced by Th1 lymphocytes, which are
important in host defense, while IL-4 is produced
by Th2 lymphocytes involved mainly in allergic
reactions. IL-4 by itself, or in combination with
IFN-γ , had no effect on the expression of RIG-I,
and RIG-I expression may mainly contribute to
Th1 type reaction.
RIG-I belongs to the DExH-box family,
which is involved in various reactions related to
RNA metabolism such as pre-mRNA splicing,
RNA transport, ribosome biogenesis and RNA
degradation (Gatfield et al. 2001; Schwer 2001).
Results of the present study suggest that RIG-I
plays a role in RNA metabolisms in inflammatory
reactions, although details of RIG-I bioactivities
are still unknown.
In summary, IFN-γ upregulates the expression of RIG-I in T24 cells, and RIG-I is detected
in the urinary bladder epithelium and in the urothelial carcinoma. These results suggest the existence of a novel molecular mechanism by which
IFN-γ regulates inflammatory reactions in the
urinary tract.
Acknowledgements
The authors thank Drs. Stephen Prescott,
Thomas McIntyre and Guy Zimmerman of the University of Utah for critical discussions. The authors
also thank Takanobu Itoi, Naoto Uto, Tomohiro
318
T. Imaizumi et al.
Tanno and Kumiko Munakata for assistences.
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