Interleukin-17 Induces Expression of Chemokines and Cytokines in Prostatic Epithelial Cells but Does Not Stimulate Cell Growth In Vitro Zongbing You 1,* , Dongxia Ge 1 , Sen Liu 1 , Qiuyang Zhang 1 , Alexander D. Borowsky 2 , and Jonathan Melamed 3 1 Department of Structural & Cellular Biology, Department of Orthopaedic Surgery, Tulane Cancer Center, Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research and Regenerative Medicine, Tulane Center for Aging, Tulane University School of Medicine, New Orleans, Louisiana 70112 2 Department of Pathology & Laboratory Medicine and Center for Comparative Medicine, University of California Davis, Davis, California 95616 3 Department of Pathology, New York University School of Medicine, New York, New York 10016 Abstract BACKGROUND—Interleukin-17 (IL-17A) expression is increased in prostate cancer. This study investigated the expression of IL-17A receptor C (IL-17RC) in prostatic intraepithelial neoplasia (PIN) lesions and the effects of IL-17A on prostatic epithelial cells in in-vitro studies. METHODS—IL-17RC expression in human and rodent prostate tissues was detected by immunohistochemistry. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were used to determine mRNA and protein expression in human and mouse prostatic epithelial cell lines. RESULTS—IL-17RC protein was increased in human and rodent PIN lesions, compared to the normal human and rodent prostatic epithelium. IL-17A treatment activated the Nuclear Factor-κB (NF-κB) and/or Extracellular signal-Regulated Kinase (ERK) pathways in human PIN and LNCaP cell lines as well as mouse prostate cancer cell line TRAMP-C1. IL-17A treatment did not affect cell growth of the cell lines studied. However, IL-17A induced expression of CXCL1, CXCL2, CCL2, CCL5, and IL-6 in human and mouse prostatic epithelial cell lines. When the full-length IL-17RC was over-expressed in human PIN and LNCaP cell lines, activation of NF-κB and/or ERK pathways and expression of CXCL1, CXCL2, and CCL5 chemokines were significantly enhanced upon IL-17A treatment. CONCLUSION—These findings suggest that the prostatic epithelial cells in PIN lesions may respond to IL-17A stimuli with augmented synthesis of chemokines, due to increased IL-17RC expression. * Correspondence to: Zongbing You, MD, PhD, Department of Structural & Cellular Biology, Tulane University School of Medicine, 1430 Tulane Ave SL 49, New Orleans, LA 70112. Fax: 504-988-1687; Tel: 504-988-0467; [email protected]. Conflicts of Interest The authors have no conflicts of interest in publication of this chapter. NIH Public Access Author Manuscript Int J Med Biol Front. Author manuscript; available in PMC 2014 October 01. Published in final edited form as: Int J Med Biol Front. 2012 ; 18(8): 629–644. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
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Interleukin-17 Induces Expression of Chemokines and Cytokines in Prostatic Epithelial Cells but Does Not Stimulate Cell Growth In Vitro
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Interleukin-17 Induces Expression of Chemokines and Cytokinesin Prostatic Epithelial Cells but Does Not Stimulate Cell GrowthIn Vitro
Zongbing You1,*, Dongxia Ge1, Sen Liu1, Qiuyang Zhang1, Alexander D. Borowsky2, andJonathan Melamed3
1Department of Structural & Cellular Biology, Department of Orthopaedic Surgery, Tulane CancerCenter, Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research andRegenerative Medicine, Tulane Center for Aging, Tulane University School of Medicine, NewOrleans, Louisiana 70112
2Department of Pathology & Laboratory Medicine and Center for Comparative Medicine,University of California Davis, Davis, California 95616
3Department of Pathology, New York University School of Medicine, New York, New York 10016
Abstract
BACKGROUND—Interleukin-17 (IL-17A) expression is increased in prostate cancer. This study
investigated the expression of IL-17A receptor C (IL-17RC) in prostatic intraepithelial neoplasia
(PIN) lesions and the effects of IL-17A on prostatic epithelial cells in in-vitro studies.
METHODS—IL-17RC expression in human and rodent prostate tissues was detected by
(qRT-PCR) and Western blot analyses were used to determine mRNA and protein expression in
human and mouse prostatic epithelial cell lines.
RESULTS—IL-17RC protein was increased in human and rodent PIN lesions, compared to the
normal human and rodent prostatic epithelium. IL-17A treatment activated the Nuclear Factor-κB
(NF-κB) and/or Extracellular signal-Regulated Kinase (ERK) pathways in human PIN and LNCaP
cell lines as well as mouse prostate cancer cell line TRAMP-C1. IL-17A treatment did not affect
cell growth of the cell lines studied. However, IL-17A induced expression of CXCL1, CXCL2,
CCL2, CCL5, and IL-6 in human and mouse prostatic epithelial cell lines. When the full-length
IL-17RC was over-expressed in human PIN and LNCaP cell lines, activation of NF-κB and/or
ERK pathways and expression of CXCL1, CXCL2, and CCL5 chemokines were significantly
enhanced upon IL-17A treatment.
CONCLUSION—These findings suggest that the prostatic epithelial cells in PIN lesions may
respond to IL-17A stimuli with augmented synthesis of chemokines, due to increased IL-17RC
expression.
*Correspondence to: Zongbing You, MD, PhD, Department of Structural & Cellular Biology, Tulane University School of Medicine,1430 Tulane Ave SL 49, New Orleans, LA 70112. Fax: 504-988-1687; Tel: 504-988-0467; [email protected].
Conflicts of InterestThe authors have no conflicts of interest in publication of this chapter.
NIH Public AccessAuthor ManuscriptInt J Med Biol Front. Author manuscript; available in PMC 2014 October 01.
Published in final edited form as:Int J Med Biol Front. 2012 ; 18(8): 629–644.
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Keywords
IL-17; PIN; prostate cancer
INTRODUCTION
Almost all surgical prostate specimens contain evidence of inflammation [1–3]. Chronic
inflammation invokes proliferative inflammatory atrophy (PIA) of prostate – a potential
precursor lesion to prostatic intraepithelial neoplasia (PIN) and/or carcinoma [4,5]. The
cause of prostate inflammation includes infection, urine reflux, diet, estrogen, and physical
trauma [6,7]. Normal prostate may contain a small number of T cells, B cells, macrophages,
and mast cells that increase in number with aging [8].
In animal models of prostate cancer, chronic inflammation with T cells was noted in a
significant proportion of the mice with high-grade PIN [9]. When TRAMP mice were fed
with the COX-2 inhibitor celecoxib, only 25% mice developed prostate cancer, compared to
a rate of 100% in control mice without celecoxib treatment. None of the celecoxib-treated
mice had any metastatic tumor, whereas the control mice showed metastasis to the lymph
nodes (65%), lungs (35%) and liver (20%) [10]. This study highlights the role of
inflammation in cancer initiation and progression in this model. Inflammatory cells were
also noted in the PB-Cre4 × Ptenloxp/loxp model at 26–29 weeks of age [11]. At this stage,
more than 80% of the prostate tissue was composed of microinvasive cancer cells and PIN,
with less than 20% of stroma and inflammatory cells. The cancer-dominant mouse prostate
tissues had 3- to 8-fold increase of some cytokines and chemokines such as CXC ligand 1
(CXCL1), C-C ligand 2 (CCL2) and CCL20, compared to the age-matched normal mouse
prostate tissues [11]. In a 2-amino-1-methyl-6-phenylimidazo (4,5-b) pyridine (PhIP)-treated
rat model [12], significantly more inflammation occurred in the PhIP-treated rat prostate
glands than in the controls and inflammation preceded proliferative atrophy and PIN.
In human PIA lesions, over 80% of inflammatory cells are CD3+ T cells, of which most are
CD4+ [4,13,14]. The CD4+ T helper cells are classified into TH1, TH2, and TH17 subtypes.
TH1 cells produce interferon (IFN)-γ and interleukin (IL)-2, while TH2 cells produce IL-4,
IL-5, and IL-13. TH17 is a new subtype that produces IL-17A and IL-17F [15,16]. The
inflammatory cells and the inflammation-stimulated prostatic epithelial and stromal cells
produce a variety of cytokines, chemokines, and growth factors [13,14,17]. It is known that
sequence variants of IL-1β, IL-8, IL-10, tumor necrosis factor (TNF) α and vascular
epithelial growth factor are associated with human prostate cancer risk [18].
IL-17A is a pivotal cytokine that stimulates expression of other cytokines and chemokines
such as TNFα, IL-6, and IL-8 [19–22]. The percentage of TH17 cells in prostate-infiltrating
lymphocytes was higher than that in the peripheral blood, though this TH17 skewing was
inversely correlated with Gleason grade [23]. IL-17A expression is increased in 79% of
benign prostate hyperplasia (BPH) and 58% of prostate cancer specimens [14]. IL-17RA and
IL-17RC are the receptors for IL-17A and IL-17F [24–27]. Both receptors have been found
in prostate cancer [14,28,29]. The full-length IL-17RC protein inhibited TNFα-induced
apoptosis in a human prostate cancer cell line LNCaP [30]. IL-17RC protein expression as
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This study demonstrates that IL-17RC expression is increased in human and rodent PIN
lesions. Over-expression of IL-17RC enhances IL-17A-induced signalling activity and
expression of chemokines.
Acknowledgments
The authors thank Dr. Prescott L. Deininger (Director of Tulane Cancer Center) and Tulane Cancer Center CoreFacilities for research support; Dr. Johng S. Rhim who was the original source of RWPE-1 and pRNS-1-1 cell lines;Dr. Mark Stearns who was the original source of the PIN cell line and Dr. Alice C. Levine who provided the PINcell line. Grant sponsors: National Institutes of Health’s Centers of Biomedical Research Excellence (COBRE)grant (2P20RR020152-06), Department of Defense grants (W81XWH-05-1-0567 and W81XWH-10-1-0937), thedevelopmental funds of the Tulane Cancer Center, Louisiana Cancer Research Consortium, and Tulane Frameworkfor Global Health Seed Grant.
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Fig. 1. IL-17RC expression was increased in human and rodent PIN lesions as shown byimmunohistochemical stainingA. Human high-grade PIN lesions (in the three circles); original magnification, ×100. B.
High magnification (×400) of a representative PIN lesion; ar rows indicate PIN; open arrow
indicates normal epithelium; and arrowhead indicates a blood vessel. C. Rat normal prostatic
epithelium (open arrow) and a blood vessel (arrowhead). D. Rat normal atrophic prostatic
epithelium (open arrow) and epithelial hyperplasia (arrow). E. Rat normal prostatic
epithelium (open arrow) and PIN (arrow). F. TRAMP mouse PIN lesions (arrows) and
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adjacent normal epithelium (open arrows). G. Pten wild-type mouse normal prostatic
epithelium (open arrow). H. Pten conditional knockout mouse PIN (arrows). Original
magnification, × 400.
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Fig. 2. Effects of IL-17A on signaling pathways and cell growth in the prostati c epithelial celllinesA. Western blot analysis of phosphorylated ERK1/2 (pERK1/2) and regular ERK1/2 in
human PIN -C and PINRC cells. B. Western blot analysis of pERK1/2 and ERK1/2 in
mouse TRAMP -C1 cells. C. Western blot analysis of pIκBα, IκBα, pERK1/2, ERK1/2,
pAKT, and pSTAT3 in human LNCaP-C and LNCaP-RC cells. For loading control, the
membranes were stripped and probed for GAPDH. D. Cell growth assay. The cells were
cultured in the complete culture medium in 12-well plates. Triplicate wells per group were
treated with or without 20 ng/ml of IL-17A for 4 days. The medium was replaced by serum-
free DMEM containing 5 mg/ml of MTT. After 4 hours of incubation, medium was removed
and the formazan dye was dissolved by dimethylsulfoxide. Absorbance at 595 nm was read.
The difference between the groups in comparison was not statistically significant (P > 0.05).
You et al. Page 15
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Table I
Sequences of PCR primers used for qRT-PCR
Gene Sequence (5’ to 3’)
hCXCL1 Forward AACCGAAGTCATAGCCACAC
Reverse GTTGGATTTGTCACTGTTCAGC
hCXCL2 Forward CTGCGCTGCCAGTGCTT
Reverse CCTTCACACTTTGGATGTTCTTGA
hCCL2 Forward CAAGCAGAAGTG GGTTCAGGAT
Reverse TCTTCGGAGTTTGGGTTTGC
hCCL5 Forward CCTCGCTGTCATCCTCATTG
Reverse GGGTTGGCACACACTTGG
hIL6 Forward GGTACATCCTCGACGGCATCT
Reverse GTGCCTCTTTGCTGCTTTCAC
mCxcl1 Forward CACCCAAACCGAAGTCATAG
Reverse AAGCCAGCGTTCACCAGA
mCxcl2 Forward CGCCCAGACAGAAGTCATAG
Reverse TCCTCCTTTCCAGGTCAGTTA
mCcl2 Forward GCCTGCTGTTCACAGTTGC
Reverse TGTATGTCTGGACCCATTCCT
mCcl5 Forward CACCACTCCCTGCTGCTT
Reverse ACACTTGGCGGTTCCTTC
mIl6 Forward CTACCCCAATTTCCAATGCT
Reverse ACCACAGTGAGGAATGTCCA
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Tab
le II
Che
mok
ines
and
cyt
okin
es in
duce
d by
IL
-17A
in h
uman
and
mou
se p
rost
atic
epi
thel
ial c
ell l
ines
Gen
eH
uman
nor
mal
cel
lsH
uman
PIN
cel
lsH
uman
can
cer
cells
Mou
se c
ance
r ce
lls
RW
PE
-1pR
NS-
1-1
PIN
-CP
IN-R
CL
NC
aP-C
LN
CaP
-RC
TR
AM
P-C
1
CX
CL
12.
7±1.
82.
0±1.
71.
9±0.
63.
1±0.
5*2.
8±1.
615
.0±
3.3*
32.7
±1.
1
CX
CL
22.
3±1.
11.
9±1.
32.
0±0.
43.
0±0.
7*2.
0±1.
12.
4±1.
59.
3±1.
5
CC
L2
1.0±
0.5
1.2±
0.3
1.2±
0.4
1.8±
1.1
2.4±
1.9
2.8±
1.3
4.5±
1.0
CC
L5
0.4±
0.5
1.3±
0.3
1.3±
1.2
1.0±
0.5
1.5±
1.2
9.2±
2.8*
2.8±
1.1
IL-6
1.3±
0.3
1.5±
0.4
1.7±
0.3
1.3±
0.3
2.3±
1.4
1.8±
0.6
5.6±
1.0
The
cel
l lin
es w
ere
trea
ted
with
or
with
out 2
0 ng
/ml o
f IL
-17A
for
2 h
. The
mR
NA
exp
ress
ion
was
ana
lyze
d by
qR
T-P
CR
, whi
ch w
as n
orm
aliz
ed b
y G
APD
H a
nd p
rese
nted
as
mea
n ±
sta
ndar
d de
viat
ion
offo
ld in
crea
se o
f th
ree
expe
rim
ents
usi
ng th
e un
trea
ted
cont
rol c
ells
as
base
line.
Ast
eris
k in
dica
tes
that
the
fold
incr
ease
was
of
stat
istic
al s
igni
fica
nce
(P <
0.0
5), c
ompa
red
to th
e PI
N-C
or
LN
CaP
-C c
ells
,re
spec
tivel
y.
Int J Med Biol Front. Author manuscript; available in PMC 2014 October 01.