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Original Articles |
-Induced Parathyroid Hormone-Related Peptide Production in Synovial Fibroblasts of Patients with Rheumatoid Arthritis
Department of Nutrition, Tokyo Metropolitan Institute of Gerontology (T.Y., A.S., Y.K.), Tokyo 173-0015, Japan; Sakamoto Clinic (H.S.), Kagoshima 899-73, Japan; Department of Internal Medicine (T.H.), Department of Orthopaedic Surgery (S.Y.), Tokyo Metropolitan Geriatric Hospital, Tokyo 173-0015, Japan; and Department of Orthopaedic Surgery, School of Medicine, University of Tokyo (H.O.), Tokyo 113-8655
Address all correspondence and requests for reprints to: Yasuko Koshihara, Ph.D., Department of Nutrition, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo, 173-0015, Japan. E-mail: ykoshi{at}tmig.or.jp
Abstract
Synovial fibroblasts, established in culture from patients with RA,
were treated with proinflammatory cytokines and prostaglandin
E2 (PGE2) for 24 h. These cells enhanced
the production and the messenger RNA expression of PTH-related peptide
(PTHrP) using proinflammatory cytokines, such as interleukin (IL)-1
,
tumor necrosis factor- without the coordination of other cytokines.
In addition, PGE2 which has been induced with IL-1, also
enhanced the production of PTHrP. The IL-1-induced PTHrP production
was inhibited by PG H synthetase (Cox) inhibitors, indomethacin, and
also by Cox-2 inhibitor, NS398. The synovial fibroblasts expressed
PGE2 receptor subtypes, EP2, EP3, EP4, but not EP1, as
detected by RT-PCR. Of the PGE2 receptor agonists, EP4
agonist showed the most marked induction of PTHrP, and EP2 agonist
partly induced the production. However, these PGE2
receptors were not induced by the treatment with IL-1 and
PGE2.
These results suggest that induction of PGE2 by IL-1 may
be an important component of the PTHrP production of the inflammatory
process in synovial tissues from patients with RA. These findings are
the first to demonstrate that PGE2 stimulates PTHrP
production, which is mediated mostly by EP2 and EP4 receptors.
THE PTH-RELATED peptide (PTHrP) was first
identified as a factor causing malignant humoral hypercalcemia
(1, 2). It was suggested that this peptide plays important
roles in the development (3) and pathophysiology (4, 5) of numerous diseases. In contrast to PTH, the expression of
PTHrP is observed in various tissues (6, 7, 8, 9, 10), as have a
variety of actions, such as osteoclastic bone resorption
(11), keratinocyte differentiation (12),
cartilage formation (13), and calcium homeostasis
(14). Recently, Okano et al. (15)
and Kohno et al. (16) demonstrated that
synovial fluids from rheumatoid arthritis (RA) patients contain high
levels of PTHrP in comparison with osteoarthritis (OA) patients. PTHrP
expression in articular tissue has been reported in chondrocytes and
synovial cells using immunohistochemistry and in situ
hybridization (15). Therefore, it was suggested that PTHrP
might contribute to bone destruction in RA joints. Synovial-lining
cells are constituted from three types (type A, B, and D). Funk
et al. (17) recently demonstrated that PTHrP
was produced by synovial fibroblasts isolated from rheumatoid synovium.
The production was enhanced by proinflammatory cytokines as IL-1ß and
tumor necrosis factor (TNF)-. In the present studies, we also found
that prostaglandin E2
(PGE2) enhanced PTHrP production in synovial
fibroblasts similar to IL-1. The two forms, IL-1 and IL-1ß, are
products of adjacent but highly divergent genes. Both forms bind with
high affinity and signal only through the type I IL-1 receptor
(18, 19). IL-1, as well as TNF-, is one of the most
important cytokines for developing synovial inflammation and plays a
dominant role in the etiopathology of RA. It was also reported that
IL-1 activated fibroblasts, isolated from inflamed synovia of patients
with RA, to synthesize and release large amounts of
PGE2 (20). Then, whether
PGE2 is involved in IL-1-induced PTHrP
production was investigated using PGH synthetase (cyclooxgenase, Cox)
inhibitors.
Prostaglandins are important mediators of inflammation in RA (21, 22). The nonsteroidal antiinflammatory drugs, which are inhibitors of Cox, are used extensively in the treatment of RA. Of the various prostanoids, PGE2 is produced by rheumatoid synovial tissues and probably plays key role in the erosion of cartilage and juxta-articular bone (23, 24).
The actions of PGE2 are mediated through the
binding event with specific membrane-bound G protein-coupled prostanoid
EP receptors (25). There are at least four subtypes of the
EP receptor, termed EP1, EP2, EP3, and EP4, which have been defined on
the basis of their different pharmacological profiles and signal
transduction pathways. The activation of EP1, EP2/EP4, and EP3
receptors results in elevation of intracellular
Ca2+, stimulation of adenylate cyclase, or
inhibition of adenylate cyclase, respectively. Then, which
PGE2 subtype receptors were mediated on
PGE2-induced PTHrP production in synovial cells
was investigated using specific EP agonists. In the study, we
demonstrated that IL-1-induced PTHrP production may be produced
through PGE2, and mostly mediated by EP2/EP4
receptors in synovial fibroblasts with RA.
Materials and Methods
Patients
RA was diagnosed according to the criteria of the American Rheumatism Association. Synovial tissues were obtained from a male patient aged 51 yr during total knee joint replacement surgery for severe inflammatory or destructive lesions. Informed consent was obtained from the patient.
Isolation and culture of synovial fibroblasts
The synovial cells were isolated as described previously
(26). The cells isolated from synovial tissues were
cultured with -MEM containing 20% heat-inactivated horse serum (HS,
Morgate, Australia) in a 5% CO2/95%
air-incubator. The culture medium was replaced twice each week. When
cells reached confluence, these cells that dispersed after agitating
with 0.05% pronase E and 0.05% EDTA in Ca2+ and
Mg2+-free PBS for 5 min were transferred to new
plastic dishes in a split ratio of 1:2 or 1:4. The cells at more than 7
population doubling levels (PDL) were used for subsequent experiments.
These cells used here consisted of fibroblasts alone, without dendritic
or monocytic cells.
Treatment with cytokines and prostaglandin
The synovial fibroblasts at more than 7 PDL were treated with
pronase/EDTA solution for 5 min to detach them from culture dishes and
then collected by centrifugation at 250 x g for 5 min.
The collected cells were plated onto 12-well multiple dishes at
105 cells/well with 1.0 mL of -MEM containing
20% HS. When cells reached confluence, they were incubated with 1 or
10 ng/mL of recombinant human IL-1 (Genzyme, Cambridge,
MA), and 10-5
M or
10-6
M of PGE2 (Funakoshi,
Tokyo, Japan) for 24 h after replacement with -MEM containing
with 5% HS. PGE2 was dissolved in ethanol and
added to be 0.1% in culture medium. The PTHrP levels in the
conditioned medium were measured directly by immunoradiometric assay
(IRMA). Because -MEM interferes with the assay, the medium was
changed to DMEM.
For the addition of prostaglandin synthetase inhibitors such as
indomethacin and NS398, these inhibitors were added simultaneously or
2 h before the addition of IL-1 in the culture. The
PGE2 receptor agonists, EP1, EP2, EP3, and EP4
agonists, which were kindly supplied by Ono Pharmaceutical Company
(Osaka, Japan), were also added as cytokines. The EP1, EP2, EP3, and
EP4 agonists were generated, and the specificity of the respective EP
agonists was analyzed by measuring the binding affinity of the agonists
to respective EPs expressed in CHO cells (Table 1
) (27, 28). The 1,000-fold
concentrated inhibitors and PGE2 receptor
agonists were dissolved in DMSO and added to be 0.1% in culture
medium. DMSO vehicle instead of compounds was added in untreated
control cells. The amount of DNA in each well was measured by the
method of Burton (29) after extraction with hot 5%
perchloric acid. Each well contained 30–40 µg DNA.
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PTHrP levels in the conditioned medium were measured by the two-site IRMA method (Nichols Institute Diagnostics, San Juan Capistrano, CA) using two kinds of polyclonal antibodies directed against the synthetic 125I-labeled N-terminal region of human PTHrP (1–40) and biotin-coated human PTHrP (60–72) by the addition of avidin-coated beads. PTHrP (1–86) standards were diluted in the same medium used for samples. The sensitivity of this assay was 0.2 fmol/mL. The interassay coefficient of variation was 7.67–16.89%. Human intact PTH (1–84), the PTH region (1–34), the N-terminal region of PTHrP (1–40), and the C-terminal region of PTHrP (109–141) were not detected. Details have been described previously (30). Determination of human PGE2 was measured using enzyme-linked immunosorbent assay (ELISA) (Cayman Chemical, Ann Arbor, MI). The sensitivity of this assay was 7.8 pg/mL. The intraassay coefficient of variation was <10%. The specificity for PGs except PGE was less than 0.01%.
RT-PCR
The RT reaction was performed using an RNA LA PCR kit (Ver.1.1,
Takara Biochemicals, Osaka, Japan). First, 1 µg of total RNA from
synovial fibroblasts (T-26 cells) at 7 PDL treated with 10 ng/mL of
inflammatory cytokines such as IL-1, IL-6, and TNF-, and
10-5 M of
PGE2 for 24 h, was hybridized to oligo
dT-adaptor primer, and the RT reaction was carried out using AMV
reverse transcriptase XL (Life Sciences, St. Petersburg, FL) for
1 h at 42 C. The PCR reaction of PTHrP was
performed using HotStarTaq (QIAGEN Inc., Valencia, CA).
The synthetic forward primer (5'-CTGGT TCAGC ATGGG AGGGTC-3') and the
reverse primer (5'-GTTAG GGGAC CACCT CCGAGGT-3') were designed to
amplify a 231-bp fragment. The PCR profile followed the method of Li
et al. (31) with slight modification;
denaturation at 95 C for 15 min followed by 44
cycles of denaturation at 94 C for 1 min,
reannealing at 60 C for 30 sec, extension of 72
C for 1 min. Semiquantitative PCR reaction of
PTHrP was performed by the method of Alipov et al.
(32) with slight modification. The synthetic forward
primer (5'-AGACTGGTTCAGCAGTGGAG-3') and the reverse primer
(5'-ATCGAGCTCCAGCGACGTTG-3') were designed to amplify a 510-bp
fragment. The PCR profile followed as denaturation at 95
C for 15 min followed by 31, 33, and 35 cycles of
denaturation at 94 C for 1 min, reannealing at 57
C for 1 min, extension of 72
C for 1 min. PCR products were separated by
electrophoresis on a 2.0% agarose gel. The PCR reaction of
PGE2 receptors was performed as follows. The
synthetic forward primers designed from the published sequence for EP1
(5'-CTCGCCGCCCTGGTGTGCAACACGC-3'), EP2 (5'-TTCATCCGGCACGGGCGGACCGC3'),
EP3 (5'-TGTGTCGCGCAGTACCGGCG-3'), EP4 (5'-CCTCCTGAGAAAGACAGTGCT-3') and
reverse primers for EP1 (5'-GGCCTCCCAGGCGCTCGGTGTTAGGCC-3'), EP2
(5'-GTCAGCCTGTTTACTGGCATCTG-3'), EP3 (5'-CGGGCCACTGGACGGTGTACT-3'), EP4
(5'-AAGACACTCTCTGAGTCCT-3') were used. The PCR profile followed the
method of Zeng et al. (33).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used to
control cDNA samples.
Results
Cytokine-induced PTHrP production
The synovial fibroblast strain (T-26 cells) was established from
the synovial tissues of a patient with RA. The cells produced
detectable amounts of IL-6 and IL-8 without any stimulants (data not
shown), but not IL-1, IL-1ß, or TNF-. Long termed cultured
cells used here did not contain monocytes/macrophages or dendritic
cells, due to the characteristics of cytokine production.
Proinflammatory cytokines including TNF- and IL-1 and ß mediate
the joint destruction that characterizes RA.
NH2-terminal PTHrP, a potent bone resorbing
agent, could also be a member of the synovial cascade of
tissue-destructive cytokines whose expression is induced in RA. Whether
proinflammatory cytokines such as IL-1, IL-6 or TNF- induce the
expression of PTHrP messenger RNA (mRNA) in synovial fibroblasts was
investigated by RT-PCR.
As shown in Fig. 1, untreated cells
mildly expressed the mRNA, but treatment with IL-1 and
PGE2 markedly enhanced the expression. IL-6 did
not increase the expression of PTHrP. The level of GAPDH expression did
not vary among the samples. These findings suggested that synovial
fibroblasts from patients with RA produce PTHrP during the inflammation
process. Proinflammatory cytokines- and
PGE2-induced PTHrP production in synovial
fibroblasts was confirmed by IRMA, and obtained the similar results as
that of mRNA expression (data not shown).
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and PGE2 linearly increased
PTHrP production up to 24 h and 32 h, respectively, and
decreased, thereafter (Fig. 2A). An
increase of the mRNA level by IL-1- and PGE2-treatment appeared
earlier than that of protein level (Fig. 2B). The high expression at
1 h may be affected by the medium change containing serum, because
of no differences among 3 samples.
|
-induced PTHrP
production
To clarify the PGE2 contribution in
IL-1-induced PTHrP production, firstly, PGE2
production in IL-1-treated cells was determined by ELISA. The
synovial fibroblastic cells produced PGE2 by the
addition of IL-1 (1 or 10 ng/mL) (Table 2).
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-induced PTHrP production was mediated through
PGE2 production was examined using PG H
synthetase (Cox) inhibitors. These enzymes have been identified in two
forms, a constitutive form; Cox-1, and an inducible form; Cox-2. Cox-2
has recently been identified as an isoform of Cox-1, which is expressed
in synovial tissues of patients with RA. De novo synthesis
of Cox-2 polypeptide in cultured rheumatoid synovial fibroblasts-like
cells (synoviocytes) was enhanced by IL-1ß or PMA. In sharp contrast,
Cox-1 transcripts were not modulated by IL-1ß (34).
These Cox-1 and Cox-2 inhibitors were added simultaneously or 2 h
before the addition of IL-1. As shown in Fig. 3, indomethacin inhibited 90% of PTHrP
production from 10-7 to
10-5
M in both treatment conditions, such as
pretreatment and simultaneous treatment. NS398, a selective inhibitor
of Cox-2, also inhibited 90% of PTHrP production at the above doses
and conditions. Because PGE2 also enhanced PTHrP
production, and IL-1-induced PTHrP production was inhibited by PG
synthetase inhibitors, PG production mediates a significant portion of
the effect of IL-1 on PTHrP production. The concentration of the
inhibitors used was adequate to inhibit PGE2
production, because they were reduced to a basal level by the addition
of inhibitors in IL-1-treated cells (Table 3).
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PGE2 acts through its receptors, as EP1,
EP2, EP3, and EP4 receptors. To detect the receptor subtypes that acted
in the PGE2-induced PTHrP production, receptor
agonists were added to the culture. Specific binding affinities (Ki) of
receptor agonists to PGE2 receptor are summarized
in Table 1
. EP2, EP3, and EP4 agonists showed extremely specific
binding affinity to their EPs expressed in CHO cells, but EP1 agonist
showed less specificity to EP1 receptor in comparison with other
agonists. EP1 and EP3 agonists did not have any effects on
intracellular cAMP elevation but elevated the intracellular
Ca2+ level with lower potency than
PGE2. High concentrations of EP4 agonist also
elevated the Ca2+ level as EP3 agonist. EP2 and
EP4 agonists elevated the intracellular cAMP level through their own
receptors (data not shown). Using these agonists, the EP1 agonist did
not induce PTHrP production. However, high concentrations of EP2 and
EP4 agonists enhanced PTHrP production. The EP4 agonist significantly
enhanced it in a dose-dependent manner. EP3 did not statistically
enhance the production (Fig. 4). These
findings suggest that EP2 and EP4 appear to be involved in the effects
of PGE2 on PTHrP production.
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and PGE2, but EP1
and EP3 agonists did not (Fig. 5). The
level of GAPDH expression did not vary among those samples. In
addition, the RT-PCR findings showed that EP2 and EP4 receptors existed
in synovial fibroblasts, but their mRNA expression was not enhanced by
the IL-1 and PGE2 treatments (Fig. 6A). These things were confirmed by
semiquantitative PCR method as shown in Fig. 7. EP3 receptor was also detected in
another PCR-condition as a mild and a long-term period reaction (Fig. 6B). Even in gentle PCR conditions, EP1 receptor expression was not
detected (data not shown).
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IL-1 and TNF- protein were readily detected in synovial fluid,
and IL-1, TNF-, IL-6, IFN-
, GM-CSF, M-CSF, and LIF were detected
in RA synovial tissue. These data have been reviewed by Feldman
et al. (35). Incubation of synovial fibroblasts
with inflammatory cytokines as IL-1 and IL-ß, and
PGE2 for 24 h showed an increase in PTHrP
production, but IL-6 and TNF- scarcely induced PTHrP production
(data not shown), as shown by the expression of PTHrP mRNA (Fig. 1).
Funk et al. (17) also reported that high
concentrations of IL-6, 200 ng/mL, did not enhance PTHrP release, but
TNF- at 20 ng/mL clearly induced the production. If high
concentrations of TNF- had been used in the present study, TNF-
might have induced PTHrP release. They also found that IL-1ß enhanced
PTHrP production in synoviocytes of RA, but they did not investigate
the mechanism (17).
We found that IL-1 and PGE2 induced PTHrP
production in three synovial fibroblast strains established from three
patients with RA. The PGE2-induced PTHrP
production was increased to a similar level as IL-1 treatment. In
the present study, we confirmed that PTHrP production by IL-1 in
synovial fibroblasts was mediated through PGE2.
IL-1 treatment enhanced PGE2 production, which
is similar mediation of PGE2 by IL-1 as has
been reported in other parameters of other cells (20).
IL-1-induced PTHrP production was blocked by Cox inhibitors, both
Cox-1 and/or Cox-2 inhibitors. Although, de novo synthesis
of Cox-2 in synoviocytes was induced by IL-1ß, but Cox-1 transcripts
were not modulated by IL-1ß (34). These findings
suggested that modulation of Cox-2 expression by IL-1ß might be an
important component of the PTHrP production in synovial tissues from
patients with RA. In IL-1-induced PTHrP production, Cox-2 transcripts
were induced to contribute to the PGE2
production. These findings suggested that IL-1 first induced
PGE2 production, and then extracellular
PGE2 induced PTHrP production through the EP2/EP4
receptor. The intracellular signaling in the production of PTHrP by EP4
receptor agonists, is not yet clarified. However, it has been reported
that EP4 receptor activates adenylate cyclase and stimulates the
intracellular concentration of cAMP (36).
Little is known about the regulation of PTHrP synthesis and release in nontumoral cells. We reported that the production of PTHrP was remarkably stimulated by the protein kinase C (PKC) activator phorbol-12-myristate-13 acetate (PMA) not by forskolin in OA synovial fibroblasts (26). Forskolin can elevate the intracellular cAMP level by direct activation of the catalytic unit of the enzyme without requiring GTP. However, PGE2 receptor coupled with G protein to elevate cAMP (30). Therefore, intracellular cAMP elevation may not be necessary to promote PTHrP, but stimulation of the G protein is required. In RA synovial fibroblasts, PMA remarkably enhance PTHrP production in comparison with inflammatory cytokines, but forskolin barely enhanced it (data not shown). In lung squamous carcinoma, cAMP stimulation have been shown to promote PTHrP secretion (37). The extracellular calcium concentration was recently shown to influence PTHrP production in human keratinocytes and rat Leydig tumor cells (26).
This is the first study to demonstrate that induction of
PGE2 by IL-1 may be an important component of
the PTHrP production of the inflammatory process in synovial tissues
from patients with RA, and PGE2 stimulates PTHrP
production, which is mediated mostly by EP2 and EP4 receptors.
Acknowledgments
We thank T. Maruyama (Ono Pharmaceutical Company) for generous gifts of PGE2 receptor agonists.
Received July 19, 2000.
Revised December 28, 2000.
Revised February 21, 2001.
Accepted March 13, 2001.
References
and interleukin 1-ß bind to the same receptor. J Immunol. 136:4509–4514.[Abstract]
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) for 1, 3, 8, 24,
32, or 48 h. Control cells (
) were incubated with vehicle.
PTHrP released in the medium was measured by IRMA (A). ***,
P < 0.001; **, P < 0.01; and
*, P < 0.05 vs. untreated control
cells at each incubation time. PTHrP expression by RT-PCR was
represented in (B). Numbers at the bottom of each
photograph show 1, untreated control cells: 2, IL-1
