IL-22 - Kyushu University Library

Interleukin-22-induced STAT3 signal
transduction in oral squamous cell
carcinoma
2010
LUTFUN NAHER, B.D.S
Department of Oral and Maxillofacial Oncology,
Division of Maxillofacial Diagnostic and Surgical Sciences,
Faculty of Dental Science, Kyushu University,
Fukuoka, Japan
1
Interleukin-22-induced STAT3 signal
transduction in oral squamous cell
carcinoma
2010
Submitted as a partial fulfillment of the requirements for the degree of
Doctor of Philosophy (Ph.D) in Dental Science in Kyushu University by
LUTFUN NAHER, B.D.S.
Department of Oral Pathology and Medicine,
Division of Maxillofacial Diagnostic and Surgical Sciences
Faculty of Dental Science, Kyushu University
Department of Oral and Maxillofacial Oncology,
Division of Maxillofacial Diagnostic and Surgical Sciences,
Faculty of Dental Science Kyushu University
Supervisors :
Professor Hidetaka Sakai
Professor Seiji Nakamura
Instructor:
Assistant Professor Tamotsu Kiyoshima
2
List of Submitted Paper and Presentation
<Submitted Paper>
Lutfun Naher, Tamotsu Kiyoshima, Ieyoshi Kobayashi, Hiroaki Fujiwara,
Yukiko Ookuma, Kengo Nagata, Seiji Nakamura, Satoru Ozeki, and
Hidetaka Sakai.
Interleukin-22-induced STAT3 signal transduction in oral squamous
cell carcinoma
Submitted to the American Journal of Pathology (2009).
<Presentation>
L. NAHER, T. KIYOSHIMA, H. FUJIHARA, I. KOBAYASHI, K.
NAGATA, K. FUKIWAKE, Y. OOKUMA, S. NAKAMURA, and H.
SAKAI. Kyushu University, Fukuoka, Japan
STAT3 signal transduction in squamous cell carcinoma stimulated by
IL-22
IADR 87th General Session and Exhibition, April 2009 (U.S.A)
3
List of Contents
List of Submitted Paper and Presentation
List of Contents
…………………………………………3
……………………………………………………………………4
LIST OF ABBREVIATION …………………………………………………………6
Abstract …………………………………………………………………………………7
Introduction ……………………………………………………………………………9
Materials and Methods ………………………………………………………………12
Reagents …………………………………………………………………………12
Cell Lines and Culture Conditions ………………………………………………12
Semiquantitative RT-PCR Analysis for IL-22 receptor mRNA Expression ……13
Real-time Quantitative PCR Analysis for mRNA Expression of the STAT3
downstream genes in Oral SCC cells after IL-22 stimulation …………………13
Immunoblotting and Inhibitor Treatment ………………………………………14
Immunocytochemistry for STAT3 ………………………………………………15
Cell proliferation assay
…………………………………………………………16
Construction of NF-κ B-response Luciferase reporter Vector and Luciferase
Assay ………………………………………………………………………………17
Real-time Quantitative PCR Analysis for mRNA Expression of the keratinocyte
differentiation-related genes in Oral SCC cells after IL-22 stimulation ………17
Statistical Analysis ………………………………………………………………18
Table 1
…………………………………………………………………………19
Table 2
…………………………………………………………………………20
Table 3
…………………………………………………………………………21
Results
………………………………………………………………………………22
4
Human carcinoma cell lines express IL-22 receptor chains
…………………22
MISK81-5, squamous cell carcinoma cells are responsive to IL-22
IL-22 Transiently Activates JAK1 and Tyk2 in MISK81-5
…………23
……………………26
IL-22 induces translocation of pSTAT3 into the nucleus in MISK81-5 cells …28
IL-22 promotes to express anti-apoptotic and mitogenic genes in MISK81-5 …32
IL-22 Activates MAPK Pathways in MISK81-5 …………………………………34
IL-22 induces tumor cell proliferation in vitro …………………………………36
IL-22 subtly induces the cellular NF-κ B activation status
……………………37
IL-22-induced Phosphorylation of STAT3 and ERK1/2 is inhibited by
preincubation with AG490 ………………………………………………………39
IL-22 reduces the expression of differentiation-related genes …………………40
IL-22-induced MMP-2 is inhibited by preincubation with AG490
……………41
Discussion ……………………………………………………………………………44
ACKNOWLEDGEMENTS …………………………………………………………50
REFERENCES ……………………………………………………………………… 52
5
LIST OF ABBREVIATION
α-MEM: alpha-minimal essential medium
β-actin: beta–actin
BSA: Bovine serum albumin
DMEM: Dulbecco's Modified Eagle's medium
ECL: Enhanced chemiluminescence
EGF: Epidermal growth factor
FBS: Fetal bovine serum
HRP: Horseradish peroxidase
IL-6: Interleukin 6
IL-10: Interleukin 10
IL-10R2: Interleukin 10 receptor 2
IL-22: Interleukin 22
IL-22R1: Interleukin 22 receptor 1
JAK1: Janus tyrosine kinase 1
MAPK: Mitogen-activated protein kinase
OSCC: oral squamous cell carcinoma
PBS: Phosphate buffered saline.
PBST: Phosphate buffered saline Tween-20
pS :Serine phosphorylation
pY: Tyrosine phosphorylation
RPMI: Roswell Park Memorial Institute medium
RT-PCR: Reverse transcription polymerase chain reaction
SDS: Sodium dodecyl sulfate
STAT3: Signal Transducers and Activator of Transcription 3
6
Abstract
Cytokines or cytokine-related mediators influence the cellular behavior of malignant
tumors. Interleukin-22 (IL-22) is a member of the IL-10 family. Its main targets are
epithelial cells, but not immune cells. The objectives of this study were to examine
IL-22 signal transduction in oral squamous cell carcinoma (OSCC) cells. RT-PCR
showed that human OSCC cells, MISK81-5 and HSC-3 cells, expressed IL-22 receptor
chains. Immunoblotting showed that IL-22 induced a transient tyrosine phosphorylation
of STAT3 (pY705-STAT3) in MISK81-5 cells after IL-22 stimulation. The change in
the serine phosphorylation of STAT3 was subtle during the examination periods.
Meanwhile pY705-STAT3 activation in HSC-3 cells was undetectable after IL-22
stimulation. The immunocytochemistry demonstrated that IL-22 induced translocation
of phosphorylated STAT3 into the nucleus of MISK81-5 cells. IL-22 temporarily
up-regulated the expression of anti-apoptotic and mitogenic genes such as Bcl-xL,
survivin and c-Myc as well as SOCS3. IL-22 transiently activated ERK1/2 and induced
a delayed phosphorylation of p38 MAP kinase. ERK1/2 phosphorylation was decreased
below the control level after 30 min. IL-22 negligibly involved activation of NF-κB as
detected by using MISK81-5 cells stably transfected with pGL4.26[luc2/minP/Hygro]
encompassed four tandem copies of the NF-κB consensus sequence. MISK81-5 cells
treated with IL-22 showed mild cellular proliferation and down-regulation of the
keratinocyte differentiation-related genes in comparison to unstimulated cells. IL-22
also increased MMP-2 protein in MISK81-5 cells, but not MMP-9. An unexpected
7
finding was that the phosphorylation of STAT3 was attenuated in MISK81-5 cells
pretreated with AG490, a specific JAK2 inhibitor, even after IL-22stimulation. The
mechanism remained unclear. These results indicated that IL-22 differentially activated
the STAT3 signaling system depending on the type of OSCC. IL-22 may play roles in
the tumor growth, cell differentiation and progression through the activation of the
STAT3 pathway. The blockade of JAK/STAT signals may provide a novel therapeutic
strategy for OSCC.
8
Introduction
More than 90% of malignant epithelial tumors arising in the oral cavity are
squamous cell carcinomas (1, 2). Over 250,000 cases of oral squamous cell carcinoma
(OSCC) globally occurred in the year 2000. This represents 5% of all cancers for males
and 2% for females. OSCC is one of the most common malignancies in humans.
However, the overall survival rates have not substantially improved for decades (3). In
addition, a significantly increased incidence of OSCC in young subjects has been
reported in recent decades (4, 5). Increased understanding of the biology of OSCC
carcinogenesis could lead to new designs of new perspectives and therapies for OSCCs.
In general, varying degrees of inflammatory cells infiltration are observed around
malignant tumors containing squamous cell carcinoma. In this situation, it is thought
that inflammatory cells aggregate to attack, damage and remove the carcinoma cells.
However, cytokines or cytokine-related mediators have direct proliferative and
anti-proliferative effects on the tumor cells, and influence the cellular behavior of
malignant tumors and the natural course of cancer (6, 7, 8).
IL-22 is a new member of the IL-10 family, and is expressed mainly in activated T
cells, mast cells and NK cells (9, 10). In addition, a subset of helper T cells abundantly
produces IL-22 and may play important roles in skin homeostasis and pathology (11).
The IL-22 receptor is a heterodimeric receptor of class II cytokine and consists of two
chains, IL-22R1 and IL-10R2. IL-22R1 is expressed in a variety of non-immune tissues:
skin, lung, small intestine, liver, colon, kidney, and pancreas (12), unlike IL-10R2 that
9
is ubiquitously expressed in various organs and cell types including immune cells. Since
IL-22 does not act between immune cells but rather from the immune cells to the
non-immune cell compartment, IL-22 therefore appears to be unique among cytokines.
Although only a small number of studies have so far addressed the roles of IL-22 in
malignant cell proliferation and apoptosis, there are inconsistencies in the findings.
IL-22 induces the activation of the three major MAPK pathways in hepatoma cells: the
ERK, JNK, and p38 kinase pathways (13) and the expression of many anti-apoptotic
and mitogenic proteins following activation of STAT3 (14). The tumorigenicity of
hepatoma cells is induced by constitutive overexpression of IL-22 (14). In human colon
adenocarcinoma cells, IL-22 can accelerate the expression of inducible nitric-oxide
synthase that is regarded as potential promoter of tumor growth in a variety of human
neoplasia (15). IL-22 protects human lung non-small cell carcinoma cells against
chemotherapy via the activation of anti-apoptotic proteins (16). In contrast, IL-22
treatment induces cell cycle arrest of murine breast adenocarcinoma EMT6 through
inhibition of the ERK1/2 and AKT phosphorylation (17). While neither tumor growth
nor metastasis was influenced by IL-22 expressed in murine colon carcinoma Colon 26
cells, the survival of the mice with IL-22 expressing Colon 26 cells was significantly
prolonged in comparison to the control mice, suggesting that IL-22 might play a
protective role in the hosts with tumors (18). Although IL-22 appears to exert variable
functions in different carcinoma cells, it is conceivable that IL-22 might play roles
during tumor genesis because IL-22 stimulates signaling pathways that are involved in
the regulation of cell growth, cell proliferation, and cell cycle control. However, so far
10
there is little knowledge about the potential roles of IL-22 in the cell biology of oral
squamous cell carcinoma cells. These findings raised our interest of how IL-22 induces
STAT signal transduction in oral squamous cell carcinoma.
The aim of this study was to analyze the signal transduction and genes induced in
OSCC cells by IL-22, and to comprehensively evaluate the potential biologic activity of
IL-22 in OSCC. In addition, this study examined the cell differentiation in
IL-22-mediated OSCC cells.
11
Materials and Methods
Reagents
Recombinant human IL-6, IL-22 and TNF-α(Wako, Osaka, Japan), and mouse EGF
(Sigma, St. Louis, MO, USA) were used. Antibodies reactive to total STAT3,
phosphorylated STAT3 (pY705, pS727), JAK1, Tyk2, ERK1, phosphorylated ERK1/2
(pT202/pY204), p38α and phosphorylated p38 (pT180/pY182) were purchased from
BD Biosciences (Franklin Lakes, NJ USA). Antibodies for phosphorylated JAK1
(pJAK1), phosphorylated Tyk2 (pTyk2), MMP-2 and MMP-9 were obtained from Santa
Cruz Biotechnology, Inc (Calif., USA). Antibody for total STAT3 (Santa Cruz
Biotechnology) was used in the immunocytochemistry. AG490 were obtained from
Calbiochem (La Jolla, Calif., USA). Except where indicated, all other materials were
obtained from either Wako or Sigma chemical (St. Louis, MO, USA) companies.
Cell Lines and Culture Conditions
Human oral SCC cell lines, MISK81-5, sMISK (19) and HSC-3 (supplied by the
Japanese Cancer Research Resources Bank, JCRB), human gastric adenocarcinoma cell
lines, MKN45 and MKN74 (supplied by the JCRB), and a human keratinocyte cell line,
HaCaT, were used in this study. The oral SCC cell lines were grown in α-MEM
(Invitrogen, Carlsbad, Calf., USA), supplemented with 10% fetal bovine serum (Filtron,
Brooklyn, Australia), 100 IU/ml penicillin and 100 µg/ml streptomycin. Gastric
adenocarcinoma cell lines were grown in RPMI 1640 medium (Invitrogen) with 10%
12
fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin (Invitrogen).
HaCaT was maintained in D-MEM (Invitrogen) with 10% fetal bovine serum and 10
µg/ml gentamicin (Invitrogen).
Semiquantitative RT-PCR Analysis for IL-22 receptor mRNA Expression
The cells were seeded in a 60mm dish (BD Falcon, NJ, USA) at 5 x 105 cells/dish. After
24 h, the culture medium was changed a fresh medium with 10% serum or without. The
next day, total RNAs from cell lines in the culture medium with 10% serum and without
were isolated using the SV Total RNA Isolation System (Promega, Madison, Wisc.,
USA) according to the manufacturer’s protocol. cDNA was generated from isolated
total RNA by reverse transcription (RT) with SuperScript VILO cDNA Synthesis Kit
(Invitrogen). Semiquantitative PCR was amplified with Advantage 2 (Clontech,
Mountain View, Calif., USA). Target genes were IL-22R1 and IL-10R2. β-actin was
used as endogenous marker. The used specific primer sets are shown in Table 1. The
PCR-reaction conditions were carefully chosen to stop the reaction condition during the
exponential phase of the amplification of each gene. The PCR products were separated
by electrophoresis on agarose gels and were then visualized with ethidium bromide.
Real-time Quantitative PCR Analysis for mRNA Expression of the STAT3
downstream genes in Oral SCC cells after IL-22 stimulation
MISK81-5 cells were seeded in a 60mm dish (BD Falcon) at 5 x 105 cells/dish. After 24
h, the culture medium was changed a fresh medium without serum. The next day, cells
13
were stimulated with new medium containing or not containing IL-22 (20 ng/ml). Total
RNAs were isolated after various periods of time. Real-time quantitative PCR was
performed using Thermal Cycler Dice® Real Time System (TaKaRa, Shiga, Japan) with
SYBR® Premix Ex TaqTM II (TAKARA) according to the manufacturer’s instructions.
The specific primer sets used for Bcl-xL (BCL2L1), survivin (BRIC5), c-Myc (MYC),
cyclin D1 (CCND1) and suppressor of cytokine signaling 3 (SOCS3), are shown in
Table 2. The specificity of products generated for each set of primers was determined
using a melting curve and/or gel electrophoresis. The referable gene was determined
through the examination of expression stability of house keeping genes; transferring
receptor
(TFRC),
β-actin
(ACTB),
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and β-2-microglobulin (B2M). The specific primer sets are shown in Table 2.
The relative expression levels of each targeted gene were normalized using the ∆∆CT
comparative method, based on the referable gene threshold cycle (CT) values (20). The
reference
gene
was
selected
using
the
geNorm
system
(http://medgen.ugent.be/~jvdesomp/genorm/).
Immunoblotting and Inhibitor Treatment
MISK81-5, HSC-3 and MKN74 cells were seeded in a 60mm dish (BD Falcon) at 5 x
105 cells/dish. After 24 h, the culture medium was changed to a fresh medium without
serum. The next day, cells were stimulated with new medium containing or not
containing IL-6 (20 ng/ml) or IL-22 (20 ng/ml). When indicated, MISK81-5 cells were
preincubated for 12 h with 100 µM AG490. After incubation for 5, 15, 30, 60 or 120
14
min, proteins were extracted from cells lysed in ice-cold RIPA buffer (Sigma),
containing 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1.0% Igepal CA-630, 0.5% sodium
deoxycholate and 0.1% sodium dodecyl sulfate (SDS) with protease inhibitors; 2%
protease inhibitor cocktail (Sigma), 20 µM lactacystin, 25 mM β-glycerophosphate, 1
mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM Na4VO3. The proteins were
separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then
transferred to an Immun-Blot® PVDF Membrane (Bio-Rad, Hercules, CA, USA). The
membranes were blocked with 10% non-fat skim milk in Tris-buffered saline with
Tween 20 (TBST) (10 mM Tris–HCl, pH 7.6; 150 mM NaCl; 0.5% Tween 20), then
incubated with the indicated antibodies. Bound antibodies were visualized with
HRP-conjugated secondary antibodies with the use of enhanced chemiluminescence
(ECL) plus detection system (Amersham, Piscataway, N.J., USA). The amounts of
loaded proteins were estimated using a Micro BCA Protein Assay Kit (Pierce, Rockford,
IL., USA). All the membranes were also probed with antibody for β-actin (Sigma) to
normalize the relative activities.
Immunocytochemistry for STAT3
MISK81-5 and HSC-3 cells were plated at a density of 3 x 103 cells/well in an 8-well
culture slide coated with poly-D-lysine (BD Biosciences, San Jose, Calif., USA). After
24 h, the culture medium was changed to a new medium without serum. The cells were
fixed at the described time after stimulation of EGF, IL-6 or IL-22. The fixative was
either 4% paraformaldehyde in phosphate-buffered saline (PBS) or acetone-ethanol for
15
10 min. They were permeabilized by incubation in 0.1% Tween-20 in PBS for 10 min.
Non-specific binding of the antibodies was blocked with normal goat or rabbit serum
(Nichirei, Tokyo, Japan) for 15 min. The cells were incubated with primary antibody for
1 h at room temperature. After washing three times in PBS, the cells were incubated in
Alexa Fluor® 568 goat anti-rabbit IgG or Alexa Fluor® 594 rabbit anti-mouse IgG
(Invitrogen)
for
30
min.
The
nuclei
were
counterstained
with
4’,
6-diamidino-2-phenylindole dihydrochloride (DAPI; Dojindo, Kumamoto, Japan) at 37
O
C. The cells were washed in PBS in the intervals between the steps. All of these
procedures were performed at room temperature if not mentioned otherwise. The
stained cells were then observed and photographed under fluorescent microscopy
(Olympas, Tokyo, Japan).
Cell proliferation assay
IL-22-treated cell proliferation was quantified using a homogeneous assay based on
quantification of the ATP present level with CellTiter-Glo® Luminescent Cell Viability
Assay (Promega). The culture medium was changed to a new medium without serum at
24 h after cell seeding (3 x 103 cells/well) in 96-well plate (BD Falcon). After
performing the non-serum culture for 6 h, the cells were stimulated with 20 ng/ml of
IL-22 every 24 h twice in the culture period for 48 h. Luminescence was measured in a
Microplate Luminometer (Turner Biosystem, Sunnyvale CA, USA). Each ratio
represents the mean ± SD (bars) of three values from one representative experiment.
16
Construction of NF-κ B-response Luciferase reporter Vector and Luciferase Assay
Four tandem copies of the NF-κB consensus sequence were encompassed upstream of
the minimal promoter (minP) in pGL4.26[luc2/minP/Hygro] to reduce the background
reporter. This vector was transfected in MISK81-5 cells. After clonal selection of stably
transfected cells with hygromycin, the MISK-pGL4-NF-κB cells were generated. The
cells were seeded (1 x 104 cells/well) in 96-well plate (BD Falcon), and the culture
medium was changed to a new medium without serum after 24 h. The cells were
stimulated with IL-6, IL-22 or TNF-α, or unstimulated after the non-serum culture for 6
h. Luminescence was measured the following day using the One-Glo luciferase system
(Promega) in the Microplate Luminometer (Turner Biosystem). The activity is indicated
as the relative fold to the unstimulated samples. Each ratio represents the mean ± SD
(bars) of three values from one representative experiment.
Real-time Quantitative PCR Analysis for mRNA Expression of the keratinocyte
differentiation-related genes in Oral SCC cells after IL-22 stimulation
MISK81-5 cells were seeded (5 x 105 cells/well) in a 60mm dish (BD Falcon) and the
medium was changed to a new medium without serum after 24 h. After 6 h, the cells
were stimulated with 20 ng/ml of IL-22. Total RNAs were isolated the next day. A
real-time quantitative PCR and analysis were performed as described. The targets in
keratinocyte differentiation-related genes were; loricrin (LOR), involucrin (IVL),
keratin 1 (KRT1), keratin 5 (KRT5), keratin 10 (KRT10) and keratin 14 (KRT14).
These specific primer sets are shown in Table 3.
17
Statistical Analysis
All the experiments were independently repeated at least three times. A statistical
analysis was performed by Student’s t-test. A p value of less than 0.05 or 0.01 was
considered to be statistically significant.
18
Table 1
Primer sets used for semiquantitative RT-PCR in the present study
Target
IL-22R1
IL-10R2
β-actin
Sequence
(sense)
5’-CTC CAC AGC GGC ATA GCC T-3’
(antisense)
5’-ACA TGC AGC TTC CAG CTG G-3’
(sense)
5’-GGC TGA ATT TGC AGA TGA GCA-3’
(antisense)
5’-GAA GAC CGA GGC CAT GAG G-3’
(sense)
5’-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG
CG-3’
(antisense)
5’-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT
GC-3’
19
Table 2
Primer sets used for quantitative RT-PCR in the present study
Target
Bcl-xL
Survivin
c-Myc
cyclin D1
SOCS3
TFRC
β-actin
GAPDH
B2M
Sequence
(sense)
5’-TAG GGT GGC CCT TGC AGT TC-3’
(antisense)
5’-GTG AGG CAG CTG AGG CCA TAA-3’
(sense)
5’-TTC TCA AGG ACC ACC GCA TC-3’
(antisense)
5’-GCC AAG TCT GGC TCG TTC TC-3’
(sense)
5’-CGG ATT CTC TGC TCT CCT CGA C-3’
(antisense)
5’-CCT CCA GCA GAA GGT GAT CCA-3’
(sense)
5’-GTG CAT CTA CAC CGA CAA CTC CA-3’
(antisense)
5’-TGA GCT TGT TCA CCA GGA GCA-3’
(sense)
5’-CCC AAG GAC GGA GAC TTC GAT-3’
(antisense)
5’-GAA ACT TGC TGT GGG TGA CCA T-3’
(sense)
5’-GCG AGC ACT GAC CAG ATA AGA ATG-3’
(antisense)
5’-TCC CGA TAA TGT GTT AGG ATT GTG A-3’
(sense)
5’-TGG CAC CCA GCA CAA TGA A-3’
(antisense)
5’-CTA AGT CAT AGT CCG CCT AGA AGC A-3’
(sense)
5’-GCA CCG TCA AGG CTG AGA AC-3’
(antisense)
5’-TGG TGA AGA CGC CAG TGG A-3’
(sense)
5’-CGG GCA TTC CTG AAG CTG A-3’
(antisense)
5’-GGA TGG ATG AAA CCC AGA CAC ATA G-3’
20
Table 3
Primer sets used for quantitative RT-PCR in the present study
Target
loricrin
involucrin
keratin 1
keratin 5
keratin 10
keratin 14
Sequence
(sense)
5’-TCA TGA TGC TAC CCG AGG TTT G-3’
(antisense)
5’-TGC AAA TTT ATT GAC TGA GGC ACT G-3’
(sense)
5’-TAA CCA CCC GCA GTG TCC AG-3’
(antisense)
5’-ACA GAT GAG ACG GGC CAC CTA-3’
(sense)
5’-AGA TCA CTG CTG GCA GAC ATG G-3’
(antisense)
5’-TGA TGG ACT GCT GCA AGT TGG-3’
(sense)
5’-GAT AGC ATC ATC GCT GAG GTC AAG-3’
(antisense)
5’-AGC CTC TGG ATC ATC CGG TTC-3’
(sense)
5’-AGG CTG GCA GCT GAT GAC TTC-3’
(antisense)
5’-CAG GGT CAG CTC ATC CAG CA-3’
(sense)
5’-ACT TCA AGA CCA TTG AGG ACC TGA G-3’
(antisense)
5’-CAG GGT CAG TTC GTC CAG CA-3’
21
Results
Human carcinoma cell lines express IL-22 receptor chains—
Since IL-22 is a ligand, it is necessary to know whether the used cell lines express a
receptor for IL-22. The receptor for IL-22 consists of a heterodimer of IL-22R1 and
IL-10R2, and therefore the mRNA expression of IL-22R1 and IL-10R2 was
investigated with semiquantitative RT-PCR analysis to demonstrate that squamous cell
carcinoma cell lines have the capability to respond to IL-22. mRNA expression was also
examined in these cells under non-serum condition in order to analyze IL-22 biological
activity in the squamous cell carcinoma cell lines. IL-22R1 and IL-10R2 were
detectable in all tested carcinoma cell lines in the medium with serum (Figure 1, left
panel) and without (Figure 1, right panel), even though the expression intensity varied
among them. HaCaT keratinocytes served as positive control for expression of both
IL-22 receptor chains (21).
Figure 1. MISK81-5 cells express IL-22 receptor chain mRNA similar to other
human carcinoma lines tested.
IL-10R2 and IL-22R1 mRNA expression were examined by semi-quantitative RT-PCR.
The expression was detectable in all these cell lines, but the expression intensity varied
22
among them. β-actin was used as internal control.
MISK81-5, squamous cell carcinoma cells are responsive to IL-22—
The kinetics of STAT3 phosphorylation in MISK81-5 and HSC-3 cells stimulated with
IL-22 for various periods of time was examined because studies have reported the
activation of STAT3 signaling in other cell lines by IL-22 (13, 17, 22). Two important
phosphorylation sites in STAT3, pY705 and pS727, were examined by immunoblotting
and compared with IL-6 stimulation. Although IL-6 is structurally different from IL-22,
which is a member of IL-10 family, IL-6 leads to the phosphorylation of STAT3
through JAK1 activation (23). Moreover, IL-6 also induces the activation of the MAPK
pathways (24).
IL-22 induced tyrosine-phosphorylation of STAT3 (pY705-STAT3) in MISK81-5
cells, within 15 min, peaking at 30 min (Figure 2A). This phosphorylation was transient,
and was decreased toward the baseline and to barely detectable levels for STAT3 after
120 min. The change in the serine-phosphorylation of STAT3 (pS727-STAT3) in
MISK81-5 treated with IL-22 was subtle within the tested periods. Meanwhile,
pY705-STAT3 increased within 5 min and still remained detectable in MISK81-5 cells
at least 1 h after IL-6 stimulation. IL-6 showed a subtle change in pS727-STAT3 within
the tested periods as well as IL-22. On the other hand, IL-6 similarly activated
pY705-STAT3 in HSC-3, but the activation of pY705-STAT3 by IL-22 was not
detectable during the tested periods (Figure 2B). As shown in Figure 2C,
23
pY705-STAT3 was not detectable in MKN74 cells even after the treatment of IL-22 and
IL-6. The protein level of total STAT3 remained unchanged during the tested periods in
both cells treated with IL-22 or IL-6.
24
Figure 2. IL-22 induces transient STAT3 phosphorylation in MISK81-5, squamous
cell carcinoma cells.
(A and B) MISK81-5 (A), HSC3 (B) and MKN74 (C) cells were incubated with IL-22
(20 ng/ml) or IL-6 (20 ng/ml) for varying times up to 120 min. The blots of the cellular
content protein were stained using antibody for phospho-STAT3 (pY705, pS727) or
total STAT3. A, IL-22 and IL-6 induced tyrosine phosphorylation of STAT3
25
(pY705-STAT3) in MISK 81-5 cells within 15 and 5 min, respectively. IL-22 induced
transient tyrosine phosphorylation of STAT3 in MISK81-5 cells with similar time
kinetics to IL-6. IL-6 and IL-22 induced subtle change in pS727-STAT3 within the
tested periods. B, in HSC-3 cells, pY705-STAT3 was undetectable after IL-22
stimulation, although transient pY705-STAT3 was induced after IL-6 stimulation. IL-22
and IL-6 induced transient pS727-STAT3. C, in MKN74 cells, pY705-STAT3 was
undetectable even after IL-22 and IL-6 stimulations. Meanwhile, pS727-STAT3 was not
changed after the stimulation. All findings represent the results of three independently
performed experiments.
IL-22 Transiently Activates JAK1 and Tyk2 in MISK81-5—
Janus kinase (JAK) family is known to be related to the phosphorylation of STAT
proteins in response to extracellular signals and oncogenes. There are four members of
JAK family such as JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2). Out of them,
JAK1 is associated with IL-22R1, and Tyk2 is associated with IL-10R2 (25).
Therefore, the activation of JAK1 and Tyk2 by IL-22 was examined because the
JAK-STAT pathway is responsible for cytokine signals. MISK81-5 and HSC-3 cells
were either stimulated with IL-22 for various periods, or were unstimulated. An
immunoblot analysis with these lysates was performed using specific antibodies for
phosphorylated JAK1 (pJAK1) and Tyk2 (pTyk2). As shown in Figure 3A, IL-22
stimulation of MISK81-5 cells induced the transient and slight phosphorylation of Tyk2
26
and JAK1. After 15 min, however, both pJAK1 and pTyk2 were decreased. At 120 min,
the total and phosphorylated proteins were barely detectable. Meanwhile the cells
treated with IL-6 showed a slight increase of pJAK1 and pTyk2 at 60 and 15 min,
respectively (Figure 3A). Meanwhile, phosphorylation of JAK1 and Tyk2 in HSC-3
cells was similar to that in MISK81-5 (Figure 3B).
Figure 3. IL-22 induces transient phosphorylation of JAK1 and Tyk2.
5 x 105 MISK81-5 cells were stimulated with IL-22 for 5, 15, 30, 60, and 120 min or
unstimulated as a control. The membrane was immunoblotted with each antibody and
reprobed repeatedly. IL-22 treatment resulted in transient and slight phosphorylation of
JAK1 and Tyk2 at 5 min, and decreased to barely detectable levels after 15 and 60 min,
27
respectively. Meanwhile, JAK1 and Tyk2 activation in MISK81-5 cells was slightly
increased at 60, 15 min after IL-6 stimulation, respectively.
IL-22 induces translocation of pSTAT3 into the nucleus in MISK81-5 cells—
IL-22 stimulation induced STAT3 phosphorylation in MISK81-5 cells, but not in
HSC-3 cells. Therefore the intracellular localization of STAT3 and its phosphorylated
forms in SCC cell lines after IL-6 or IL-22 stimulation were examined using
immunocytochemistry. STAT3 was noted in both nucleus and cytoplasm of MISK81-5
before IL-6 stimulation. Most of the STAT3 was observed in the nucleus of MISK81-5
cells within 5 min after IL-6 stimulation. STAT3 was also detectable in the cytoplasm
after 30 min, suggesting the relocation of STAT3 from the nucleus into the cytoplasm
(Figure 4A). The nuclear translocation of pSTAT3 in MISK81-5 was observed at 30
min after IL-22 treatment as well as IL-6 (Figure 4B), whereas no increased signal of
pSTAT3 in the nucleus was detected in HSC-3 treated with IL-22 (Figure 4C). IL-22
treatment resulted in transient STAT tyrosine phosphorylation and nuclear translocation
from the cytoplasm in MISK81-5 cells (Figures 2A and 4B), thus suggesting that the
STAT3-mediated pathways are able to promote the transcriptional activity in MISK81-5
cells in response to IL-22.
28
29
30
Figure 4. IL-22 translocates pSTAT3 into the nucleus in MISK81-5 cells.
A, MISK81-5 cells were either untreated as a control (left) or treated with IL-6 for 5, 15,
and 30 min. The cells were immunostained for total STAT3 protein after fixation with
4% paraformaldehyde. IL-6 quickly translocated most of STAT3 into the nucleus of
MISK81-5 cells within 5 min after IL-6 stimulation. After 30 min, STAT3 was also
noted in the cytoplasm. B and C, MISK81-5 (B) or HSC-3 (C) cells were either
untreated as a control (left) or treated with IL-22 or IL-6 for 30 min. These cells were
immunostained for pSTAT3 (pS727) protein after acetone-ethanol fixation for 10 min.
B, translocation of pSTAT3 into the nucleus was noted in MISK81-5 cells treated with
IL-22 as well as with IL-6 or EGF. C, in HSC-3 cells, the translocation of pSTAT3 into
the nucleus was not clear after IL-22 stimulation, but the findings were induced by IL-6.
31
Alexa Fluor® 568 goat anti-rabbit IgG (A) or Alexa Fluor® 594 rabbit anti-mouse IgG
(B and C) was used as the secondary antibody (red); Nuclei were counter-stained with
DAPI (blue); EGF was used as control stimulation for the activation of STAT3.
IL-22 promotes to express anti-apoptotic and mitogenic genes in MISK81-5—
The expression of STAT3-downstream genes was examined with real-time PCR to
further explore the mechanism underlying IL-22-STAT3 signaling. GAPDH, β-actin,
β2M and TFRC, which are used as housekeeping genes in many studies, were assessed
in order to evaluate the expression of STAT3-associated genes after IL-22 stimulation
more accurately since cytokine stimulation can induce instability in the housekeeping
gene expression (26, 27, 28). β2M was selected as the internal control among them
using geNorm system (http://medgen.ugent.be/~jvdesomp/genorm/)(data not shown).
As shown in Figure 5, the expression of anti-apoptotic proteins, Bcl-xL (BCL2L1)
and survivin (BRIC5), mitogenic proteins, c-Myc (MYC) and cyclin D1 (CCND1) and
the suppressor for STAT3, SOCS3, genes were examined in MISK81-5 treated with
IL-22. The expression of Bcl-xL and c-Myc genes exhibited a 2.4-fold increase as peak
at 30, 60 min after IL-22 stimulation, respectively (Figures 5A and C). However, the
expression of c-Myc gene was dramatically decreased to 40% at 120 min. SOCS3
expression was markedly induced at 30 min after IL-22 stimulation, exhibited a 37-fold
increase at 60 min and decreased at 120 min (Figure 5E). IL-22 significantly increased
32
the gene expression of survivin at 60 and 120 minutes. Meanwhile, the gene expression
of cyclin D1 was significantly decreased at 30 and 120 minutes (Figures 5B and D).
(D) cyclin D1
(A) Bcl-xL
100
10
10
*
**
1
**
1
0.1
0.1
0
5
15
30
60
120 (min)
(B) survivin
0
5
15
30
60
120 (min)
(E) SOCS3
10
100
*
1
*
*
**
10
**
1
0.1
0.1
0
5
15
30
60
120 (min)
0
5
15
30
60
120 (min)
0
5
15
30
60
120 (min)
(F) B2M
(C) c-Myc
10
10
*
*
1
1
**
0.1
0.1
0
5
15
30
60
120 (min)
Figure 5. IL-22 transiently induces the expression of anti-apoptotic and mitogenic
genes in MISK81-5.
MISK81-5 cells were treated with IL-22 (20 ng/ml) for various times as indicated.
RNAs were then prepared and analyzed by real-time RT-PCR. Bcl-xL (A), survivin (B),
c-Myc (C), cyclin D1 (D) and SOCS3 (E) were targeted in the STAT3-downstream
genes. Bcl-xL, c-Myc and SOCS3 exhibited a peak expression at 30, 60 and 60 min
33
after IL-22 stimulation, respectively. However, c-Myc gene expression was
significantly decreased at 120 minutes. IL-22 significantly increased the gene
expression of survivin at 60 and 120 minutes and decreased cyclin D1 at 30 and 120
minutes. Significant differences between the stimulated and unstimulated samples are
indicated with single or double-asterisks (p<0.05; *, p<0.01; **). Β2M (F) was used as
a reference gene.
IL-22 Activates MAPK Pathways in MISK81-5—
As shown in Figure 6A, IL-22 induced phosphorylation of ERK1/2 in MISK81-5 cells
within 5 min, but slightly decreased at 15 min. This phosphorylation was decreased
under the control level after 30 min. IL-22 also induced a delayed phosphorylation of
p38 MAP kinases after 60 min. Although the peak of pERK1/2 was noted at 15 min,
similar results obtained in MISK81-5 treated with IL-6. On the other hand, the
activation of ERK1/2 and p38 MAP kinases was undetectable in HSC-3 cells after
IL-22 treatment (Figure 6B). IL-6 showed similar activation of ERK1/2 and p38 MAP
kinases to that in MISK81-5 treated with IL-22 or IL-6. The protein levels of
unphosphorylated ERK and p38 MAP kinases remained unchanged in the tested periods
in both cells treated with IL-22 or IL-6.
34
Figure 6. IL-22 induces the phosphorylation of several members of the MAPK
pathways.
A and B, MISK81-5 (A) and HSC-3 (B) cells (5 x 105 cells/well) were seeded in a 60
mm dish. At 24 h after cell seeding, the medium was changed to fresh one without
serum. The next day, the cells were treated with recombinant IL-22 (20 ng/ml) or IL-6
(20 ng/ml) for 5, 15, 30, 60 or 120 min or unstimulated. Total lysates were analyzed by
immunoblotting with an anti-phospho-ERK1/2 antibody. The membranes were
repeatedly reprobed and immunoblotted with anti-p-p38, anti-ERK and anti-p38
antibodies in order. A, IL-22 induced a transient activation of ERK1/2 in MISK81-5
cells. IL-22 also induced a delayed phosphorylation of p38 MAP kinase as well as IL-6.
B, the activation was undetectable in HSC-3 cells after IL-22 treatment, whereas the
cells stimulated by IL-6 showed similar activation of ERK1/2 and p38 MAP kinases to
that in MISK81-5 treated with IL-6 or IL-22. However,
35
IL-22 induces tumor cell proliferation in vitro—
IL-22 induced phosphorylation of ERK1/2, which is one of important mediators for cell
proliferation, thus the effect of IL-22 on the cell proliferation of squamous cell
carcinoma cell lines was examined in vitro. The effect of IL-22 on proliferation of
MISK81-5 and HSC-3 cells in vitro was determined using the CellTiter-Glo®
Luminescent Cell Viability Assay (Promega). MISK81-5 cells treated with IL-22
showed a 1.3-fold increase in the cell viability in comparison to control samples. A
significant difference was demonstrated between the IL-22-treated cells and controls
(p<0.05; Figure 7). Meanwhile, there was no significant difference in the cell viability
in HSC-3 cells between the IL-22-treated cells and controls (Figure 7). This result
indicated that IL-22 shows a cell proliferative effect on MISK81-5 cells, thus
suggesting that STAT3 tyrosine phosphorylation plays an important role in the
induction of tumor cell proliferation.
viability
㻍㻃
(fold)
1.0
㻓㻑㻓㻃
IL-22
-
-
+
HSC-3
+
MISK81-5
36
Figure 7. IL-22 shows a proliferative effect on MISK81-5 cells in vitro.
3 x 103 MISK81-5 and HSC-3 cells per well were seeded into a 96-well plate. These
cells were incubated with or without IL-22 (20 ng/ml) for 48 h. Cell viability of
MISK81-5 cells treated with IL-22 was increased to 1.3-fold in comparison to the
control cells. Significant difference was noted between the stimulated and control
samples (p<0.05). Meanwhile, IL-22 did not significantly affect cell proliferation of
HSC-3 cells.
IL-22 subtly induces the cellular NF-κ B activation status—
The activation of p38 leads to the activation of multiple transcription factors that induce
expression of many different genes. Luciferase assays with MISK-pGL4-NF-κB cells
were examined to confirm that IL-22-induced NF-κB activation correlated with
transcriptional activation. The construct transfected in MISK81-5 cells is regulated by
four tandem copies of the NF-κB consensus sequence. MISK-pGL4-NF-κB cells were
stimulated with TNF-α as a positive control for NF-κB activation. As shown in Figure 8,
the cells stimulated with 50 and 100 ng/ml TNF-α demonstrated 3.6-fold and 3.8-fold
increases in luciferase activity, respectively, in comparison to the unstimulated
MISK-pGL4-NF-κB cells. Significant differences were observed between the
stimulated and control samples (p<0.01). However, the effects of IL-22 and IL-6 were
only subtle or negligible. No significant difference was noted between the IL-22- or
IL-6-stimulated and control samples (Figure 8).
37
fold
induction
㻍㻍㻃
4.0
㻍㻍㻃
3.0
2.0
1.0
㻓㻑㻓㻃
0
control
50
100
20
TNF-α
50
IL-22
20
50
(ng/ml)
IL-6
Figure 8. IL-22 is negligibly associated with up-regulation of the cellular NF-κB
activation.
The stably transfected cells, MISK-pGL4-NF-κB cells, were seeded in 21 wells of a
96-well plate. The next day, the cells were incubated in medium without serum. The
cells were stimulated with TNF-α (50 or 100 ng/ml), IL-6 (20 or 50 ng/ml) or IL-22 (20
or 50 ng/ml) or with control medium at 6 h after the medium was changed. A luciferase
assay was performed after 24 h. Each ratio is indicated as relative fold to the
unstimulated control samples, and indicates the mean ± SD (bars) of three values from
one representative experiment. Significant differences in the transcription activity are
indicated with double-asterisks (p<0.01; **).
38
IL-22-induced Phosphorylation of STAT3 and ERK1/2 is inhibited by preincubation
with AG490—
IL-22-exposed MISK81-5 cells were pre-incubated with AG490, a JAK2-specific
inhibitor. The phosphorylation levels of STAT3 and ERK1/2 were determined by
immunoblotting. MISK81-5 cells were pre-treated with AG490 following the treatment
with IL-22 for 15 min since STAT3 and ERK1/2 were phosphorylated in response to
treatment with IL-22 for 15 min (Figures 2A and 6A). In spite of no detectable change
in the STAT3 level in MISK81-5 cells with AG490-pre-treatement or without,
pY705-STAT3 was unexpectedly attenuated pre-treatment of cells with AG490 (Figure
9, lanes 3 and 4). Similar results were observed in the ERK1/2 phosphorylation. No
detectable changes in the pY705-STAT3 and pERK1/2 were observed in
IL-22-unstimulated MISK81-5 cells pre-treated with or without AG490 (Figure 9, lanes
1 and 2).
39
Figure 9. IL-22-induced phosphorylation of STAT3 and ERK1/2 is attenuated by
AG490.
5 x 105 MISK81-5 cells were seeded in a 60 mm dish one day before stimulation with
IL-22 (20 ng/ml) for 15 min (lanes 3 and 4) or without stimulation (lanes 1 and 2).
Some cells were pre-incubated for 12 h with 100 µM AG490 (lanes 2 and 4). Total
lysates
were
analyzed
by
immunoblotting
with
an
antibody
against
the
tyrosine-phosphorylated STAT3 (pY705-STAT3). The membrane was repeatedly
reprobed and immunoblotted with an anti-pERK1/2 antibody, with anti-total STAT3
antibody, with an anti-ERK antibody and then with an anti-β-actin antibody.
IL-22 reduces the expression of differentiation-related genes—
The expression of keratinocyte differentiation-associated genes was examined in
MISK81-5 cells after IL-22 stimulation to further approach the biological effect of
IL-22 on the differentiation of squamous cell carcinoma cells. The targeted genes were;
loricrin (LOR), involucrin (IVL), keratin 1 (KRT1), keratin 5 (KRT5), keratin 10
(KRT10) and keratin 14 (KRT14). The quantitative real-time PCR analysis
demonstrated that the expression of IVL, and KRT1 genes was significantly
down-regulated to ~20% and ~5% by IL-22 treatment, respectively (p<0.01). Although
the expression of LOR, KRT5 and KRT14 genes was also decreased to ~80%, ~80%,
and ~85% in the IL-22-treated samples, respectively, there was no significant difference
40
between the stimulated and control samples. The expression of KRT10 gene remained
unchanged in MISK81-5 treated with or without IL-22 (Figure 10).
1
0
1
**
**
0.
1
0.0
1 IL-22
-
+
B2M
Figure
10.
-
+
LOR
IL-22
-
IVL
treatment
-
+
+
KRT1
reduces
the
-
+
KRT10
-
+
KRT5
expression
of
-
+
KRT14
keratinocyte
differentiation-related genes.
5 x 105 MISK81-5 cells were seeded in a 60 mm dish one day before stimulation. The
expression of loricrin (LOR), involucrin (IVL), keratin 1 (KRT1), keratin 10 (KRT10),
keratin 5 (KRT5) and keratin 14 (KRT14) was compared between MISK81-5 cells with
IL-22 stimulation for 24 h and unstimulated cells. Significant differences in the gene
expression are indicated with single or double-asterisks (p<0.05; *, p<0.01; **).
IL-22-induced MMP-2 is inhibited by preincubation with AG490—
Whether IL-22 induces MMP-2 and MMP-9 protein expression in MISK81-5 cells was
41
examined because the matrix metalloproteinases (MMPs) are involved in metastatic and
invasive potentials of malignant cells. Then, the effect of AG490 on protein expression
of MMP-2 and MMP-9 was also examined in MISK81-5 cells after IL-22 stimulation,
since AG-490 inhibited STAT3 activation as well as ERK1/2 activation, as shown in
Figure 9.
IL-22-stimulated samples showed higher intensity in comparison to unstimulated
samples, as shown in Figure 11. Then the samples preincubated with AG490 showed
lower intensity after IL-22 stimulation. MMP-2 protein induced by IL-22 was
attenuated by pre-treatment with AG490 (Figure 11, lane 6). Similar result was seen in
MISK81-5 cells after IL-6 stimulation (Figure 11, lanes 3 and 4). No detectable change
in the MMP-2 protein level in MISK81-5 cells with or without AG490-pre-treatement
(Figure 11, lanes 1 and 2). Meanwhile, the levels of MMP-9 protein remained
undetectable in all samples examined in this study.
Figure 11. MMP-2 protein expression induced by IL-22 is attenuated by
preincubation with AG490.
42
5 x 105 MISK81-5 cells were seeded in a 60 mm dish one day before stimulation with
IL-22 (20 ng/ml; lanes 5 and 6) or IL-6 (20 ng/ml; lanes 3 and 4) for 30 min or without
stimulation (lanes 1 and 2). Half of samples were pre-incubated for 12 h with 100 µM
AG490 (lanes 2, 4 and 6). Extracted proteins were subjected to immunoblotting for
MMP-2 protein. The membrane was repeatedly reprobed and immunoblotted for
MMP-9 protein, and then for β-actin as an internal control.
43
Discussion
This study analyzed the signal transduction activated by IL-22 in human OSCC cells.
IL-22 induced the transient phosphorylation of STAT3 (pY705 and pS727) and the
phosphorylation of JAK1 and Tyk2. The phosphorylated STAT3 was translocated into
the nucleus by IL-22 as well as by IL-6. IL-22 induced the activation of the ERK and
p38 MAPK pathways, but negligibly activates NF-kB. IL-22 affected on the
proliferation of OSCC cells with mild stimulation. IL-22 down-regulated the gene
expression of proteins that regulate the terminal differentiation of keratinocytes. These
results suggested IL-22 plays important roles in the cell biology of OSCC cells.
IL-22 induces activation of STAT3 in carcinoma cells with functional IL-22
receptors. No study has addressed the relationship between IL-22 and OSCC cells. The
current study therefore examined how OSCC cells respond to IL-22 stimulation, and
how IL-22 induces the phosphorylation of STAT3 in OSCC cells. Y705 and S727 of
STAT3 were targeted as important phosphorylation sites, since only a few studies have
examined phosphorylation sites by IL-22. The expression of two IL-22 receptor chains
was confirmed in MISK81-5 and HSC-3 cells of OSCC by RT-PCR. However, IL-22
stimulation induced STAT3 phosphorylation in MISK81-5 cells, but not in HSC-3 cells.
This result indicated that IL-22 receptors were functional in MISK81-5 cells, and that
IL-22 did not activate STAT3 signaling in HSC-3 cell lines.
MISK81-5 cells showed transient phosphorylation of STAT3 at Y705 by IL-22
stimulation. Similar results were previously reported for IL-22 stimulation (13, 29).
44
This
result
coincided
with
the translocation
of
pS727-STAT3
protein
in
immunocytochemistry. Although the immunoreactivity of pS727-STAT3 was subtly
changed after IL-22 stimulation, a transient translocation of pS727-STAT3 into the
nucleus was observed in MISK81-5 cells. Meanwhile, the translocation of
pS727-STAT3 was not noted in HSC-3 treated with IL-22 (Figures 4 B and C). When
pY705-STAT3 was decreased, STAT3 was detected in the cytoplasm as shown in the
unstimulated cells (Figures 2A and 4A). Together with up-regulation of the STAT3
downstream genes after IL-22 stimulation as shown in Figure 5, the results suggested
that pY705 mediated translocation of pSTAT3. This finding was supported by the study
of Zhong et al. (30), in which the phosphorylation of STAT3 at Y705 involves the
translocation of STAT3 into the nucleus, thereby activating multiple target gene
transcription.
On the other hand, the change in pS727-STAT3 was subtle in this study. IL-22
signaling did not seem to involve the phosphorylation of STAT3 at S727 in OSCC cells.
This tendency was also noted in the immunocytochemistry for pS727-STAT3 in
MISK81-5 cells (Figure 4B). The phosphorylation of STAT3 at S727 in OSCC cells
was different from that in the study of Lejeune et al. (13) who also showed transient
increased pS727-STAT3 in hepatoma cells by IL-22. pS727 plays a regulatory role in
STAT3
activation
for
its
maximal
transcriptional
activity
(31).
Although
erythropoietin-induced pS727-STAT3 is regulated by ERK signaling cascades (32), the
function and mechanism of pS727 remained unclear in this study.
45
The activity of MAP kinases such as ERK and p38 after IL-22 stimulation in this
study (Figure 6) are partly reminiscent of those on hepatoma cells (13, 14). IL-22 can
activate kinases such as ERK1/2 and p38, activation of which are key events leading to
cell proliferation (33). While IL-22 transiently activated ERK1/2 and induced a delayed
phosphorylation of p38 MAP kinase, ERK1/2 phosphorylation was decreased under the
control level after 30 min. A similar result was seen in the IL-22 treatment of murine
breast adenocarcinoma EMT6, in which ERK1/2 phosphorylation was inhibited by
IL-22, thus leading to cell cycle arrest (17). Indeed, IL-22 mildly stimulated the cell
proliferation of MISK81-5 cells in this study. This stimulation may be due to a
complicated synergistic effect between the transiently increased activity of ERK1/2 and
the expression of c-Myc gene, and/or the inhibition of ERK1/2 phosphorylation.
The transient activation of STAT3 also involved the transient up-regulation of
SOCS3 expression in MISK81-5 cells (Figure 5E). The transient up-regulation of
SOCS3 expression may affect the transient activation of STAT3 and STAT3-associated
factors in MISK81-5 cells, because SOCS3 plays a role as suppressor against STAT
signaling, while SOCS3 is one of the downstream genes of STAT3. In other words,
transient STAT3 phosphorylation would be down-regulated by up-expressed SOCS3,
which in turn may be decreased after down-regulation of STAT3 in the feedback system.
It is possible that this cascade is repeated in MISK81-5 cells after IL-22 stimulation. In
addition, IL-22 may constitutively contribute to the activation of STAT3 and expression
of anti-apoptotic and mitogenic genes and be implicated in the tumorigenesis of
squamous cell carcinoma with the SOCS3 methylation since the methylation of the
46
SOCS3 promoter was found in both head and neck SCCs and various SCC cell lines (23,
34).
Surprisingly pretreatment with AG490, a specific JAK2 inhibitor attenuated the
phosphorylation of STAT3 and ERK1/2 in MISK81-5 cells stimulated by IL-22,
although IL-22R1 and IL-10R2 are thought to associate with selective activators of
JAK1 and Tyk2, respectively (25). Similar finding was found in other studies. AG490
inhibits not only JAK2 but also JAK1 phosphorylation in human colorectal cancer (35).
Neilson et al. (36) recently reported that AG490 inhibited prolactin (PRL) activation of
JAK2 and also inhibited PRL activation of JAK1, although PRL receptors had been
considered selective activators of JAK2 but not JAK1, JAK3, or Tyk2, thus suggesting
that the tyrosine phosphorylation of JAK1 was induced in the JAK2-dependent
mechanism after binding of PRL to its receptor. Together, these results suggested that
JAK2 phosphorylation might be associated with IL-22-STAT3 signaling in MISK81-5
cells. However, the nonspecific inhibition of Jak1 may have occurred in the cells
pretreated with AG490. In addition, Dumoutier et al. (37) revealed that the IL-22
receptor recruits the coiled-coil domain of STAT3 in a tyrosine-independent manner.
Therefore, further studies are also needed to elucidate the detailed mechanism and roles
of the IL-22 receptor.
Keratinocytes are thought to show changes in the expression and synthesis of
cytoskeletal proteins after exposure to proliferative or inflammatory cytokines (38). IVL,
LOR, KRT1 and KRT10 are the characteristic markers of normal suprabasal
keratinocytes (39). IL-22 reduced the expression of IVL, LOR, KRT1 and KRT10 genes
47
in MISK81-5 cells. The result indicated IL-22 could regulate the terminal differentiation
of OSCC cells as well as keratinocytes. Because these factors play important roles
during the terminal differentiation of keratinocytes associated with apoptotic processes
(40, 41, 42, 43), the control of the IL-22 function in OSCCs may therefore make it
possible to induce apoptosis in OSCC cells via differentiation.
The MMPs are a family of closely related proteolytic enzymes that are involved in
the degradation of various components of the basement membranes and extracellular
matrix. Each MMP plays a specific and important role in tumor invasion, spread and
metastasis through proteolytic potentials (44, 45, 46). MMP-2 and MMP-9 are known
as Gelatinase A and Geratibase B, respectively. They specifically cleave type IV
collagen that is one of the major structural components of the basement membrane. In
this study, IL-22 induced protein expression of MMP-2 in MISK81-5 cells, whereas
protein level of MMP-9 remained undetectable after IL-22 stimulation. In addition,
AG490 pretreatment reduced MMP-2 protein expression in MISK81-5 cells after IL-22
stimulation, probably through blocking of the STAT3 signaling pathway. Similar result
was seen in MISK81-5 cells after IL-6 stimulation. Recently, studies reported that
STAT3 signaling directly regulates MMP-2 expression, tumor invasion, and metastasis
in metastatic melanoma cells. MMP-2 is included in the target genes of STAT3 (47, 48).
The metastatic potential of tumor cells is thought to correlate with the activity of this
enzyme. Then, this result suggested that IL-22 may regulate migration and invasion of
OSCC cells via MMP-2 increased by STAT3 activation as well as IL-6. However,
further studies are required to clarify the MMP regulation in detail in the OSCC cells
48
affected by IL-22 and/or IL-6 because MMP-2 is also regulated differently from other
MMPs.
IL-22 also plays important roles in skin inflammatory process and wound healing. Of
note, it has partly become interesting to investigate the relationship between S100A7
and IL-22 in dermatoses (29). S100A7 is a member of the S100 Ca2+-binding protein
family consisting of at least 25 low molecular weight proteins (9–13 kDa). Genechip
microarray and proteomic analysis have demonstrated an aberrant S100A7 expression
in OSCCs (49, 50, 51). The S100A7 is a positive marker for oral carcinogenesis and
early tumor progression (52). The S100A7 gene is up-regulated in OSCC cells.
Transcription assay using chimera vectors suggested the putative SCC-specific
regulatory regions in the upstream of the S100A7 gene in our previous study (53).
However, the actual mechanism by which function of S100A7 and IL-22 promotes
tumorigenesis in the squamous cell carcinoma remains to be determined. Further studies
on the relationship between these factors in OSCC cells are required in the near future.
In summary, the current study showed that IL-22 affected a few important functions
of OSCC cells via the STAT3 pathways. In addition, these results suggested that IL-22
may play roles in carcinoma cell differentiation. Further studies are required to
understand the mechanisms by which IL-22 is involved in the biology of OSCC
carcinogenesis. Modifying the function of IL-22 and/or blockade of JAK/STAT signals
will lead to new perspectives and therapies with relatively few side effects in order to
improve the patient management in OSCC.
49
ACKNOWLEDGEMENTS
This research work was carried out under the supervision of Professor Hidetaka
Sakai, Department of Oral Pathology and Medicine, Division of Maxillofacial
Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University. I am
indebted to my supervisor for his unwavering support, encouragement, sincere guidance
and invaluable advice in accomplishing this research work.
My sincere thanks and gratitude also extend to Dr. Tamotsu Kiyoshima for
instructing me in all aspects about this research with unlimited patience, for his
guidance, valuable suggestions and fruitful discussions about this research project. I
also greatly appreciate the help of Dr. Ieyoshi Kobayashi, as well as to Dr. Kengo
Nagata for their kindness, guidance and support during my research work.
I wish to thank to all members of the laboratory of oral Pathology and medicine
especially Dr. Hiroaki Fujihara, Dr. Yukiko Ookuma for their support and
encouragement.
I would also like to extend my sincere homage and deepest gratitude to Professor
Seiji Nakamura, Department of Oral and Maxillofacial Oncology, Division of
Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu
University, for giving me the opportunity to conduct research at Kyushu University.
Without his kind support and guidance, it would have been impossible to finish this
work. I am also ever grateful to Emeritus Professor Masamichi Ohishi, Department of
50
Oral and Maxillofacial Surgery, Kyushu University, for his continued support and
guidance since beginning of my arrival at Kyushu University.
My sincere gratitude also extend to the rest of the member of Oral and Maxillofacial
Oncology department especially to Dr. Eiji Mitate for his kindness and unlimited
guidance in all aspects of my life in Japan.
I should also thank the international students in the faculty of Dental Science,
Kyushu University for their friendships and encouragements.
In the home front, heartiest appreciation and gratefulness for my father Mr. Moqbul
Ahmed and mother Mrs. Jahanara Begum chowdhurani as well as to my brothers for
their fountains of love, encouragement and support all along my life.
Lastly, how could I forget the name Dr. Md. Shoaib chowdhury, who is the uplifter
of my life.
Finally, I must thank my husband Dr. Rafiqul Islam and my dearest son Nahian
Ibrahim for taking care and supporting me greatly for completing my study.
51
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