Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E2 and 15-deoxy-Δ12,14-PGJ2
14-PGJ2. PLoS ONE 6(11): e27630. doi:10.1371/journal.pone.0027630
Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin 12,14 (PG)E2 and 15-deoxy-D -PGJ2
Pu Wang 0
Fei Zhu 0
Konstantinos Konstantopoulos 0
Song Guo Zheng, University of Southern California, United States of America
0 1 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University , Baltimore , Maryland, United States of America, 2 Johns Hopkins Physical Sciences in Oncology Center and Institute for NanoBioTechnology, The Johns Hopkins University , Baltimore , Maryland, United States of America, 3 Institute for NanoBioTechnology, The Johns Hopkins University , Baltimore, Maryland , United States of America
Background: Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxyD12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE2 on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/Principal Findings: Using the T/C-28a2 chondrocyte cell line as a model system, we report that exogenous PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-kB p65 subunit leading to NF-kB activation. Binding of the activated NF-kB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-kB-dependent IL-6 synthesis in chondrocytes. Conclusions/Significance: We have delineated the signaling cascade by which PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in human chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.
Funding: This work was supported, in whole or in part, by the National Institutes of Health NIAMS Grant RO1 AR053358. http://www.nih.gov/. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Rheumatoid arthritis (RA) is characterized by systemic and local
inflammation, which results in cartilage and bone destruction.
Nonsteroidal anti-inflammatory drugs (NSAIDs), which are used
to treat RA, elicit their effects by inhibiting cyclooxygenase (COX)
activity . COX is known to exist in two isoforms: COX-1 and
COX-2. Despite their similar active site structures, products and
kinetics, only COX-2 is inducible and is primarily responsible for
the elevated production of prostanoids in chondrocytes .
COX2 catalyzes the rate-limiting step of prostaglandin (PG) synthesis.
PGE2 and PGD2 are the major PGs synthesized by chondrocytes.
PGD2 readily undergoes dehydration to yield the bioactive
cyclopentenone-type PGs of the J2-series such as
Accumulating evidence suggests that the effects of PGE2 on
chondrocyte function and cartilage tissue vary according to
concentration levels. At the nano- to micro-molar concentrations
produced by arthritic tissues [3,4], PGE2 has been associated with
catabolic effects because it suppresses the production of
proteoglycans and stimulates the degradation of extracellular matrix
[5,6,7]. In contrast, low (picomolar) concentrations of PGE2 exert
anabolic effects , as evidenced by stimulation of proteoglycan
(aggrecan) synthesis . Elevated levels of PGE2 have been
detected in the cartilage and synovial fluid from patients with RA
. It is believed that PGE2 plays a critical role in the generation
and maintenance of edema and erosion of cartilage and
juxtaarticular bone [1,10].
Elevated levels of 15d-PGJ2 have also been detected in joint
synovial fluids obtained from RA patients . However, the role
of 15d-PGJ2 in RA is still a matter of debate. 15d-PGJ2 has been
reported to induce chondrocyte apoptosis in a dose- and
timedependent manner through a peroxisome proliferator-activated
receptor-c (PPAR-c)-dependent pathway . Although
15dPGJ2 has also been shown to have a pro-apoptotic effect on other
cell types, such as endothelial cells , tumor cells  and
neurons , separate lines of evidence suggest that it may have
chondroprotective effects. For instance, 15d-PGJ2 and PGD2
counteract the induction of matrix metalloproteinases in
cytokineactivated chondrocytes [15,16], which have a key role in cartilage
degradation. 15d-PGJ2 have also been reported to block apoptosis
of human primary chondrocytes induced by the NF-kB inhibitor
Bay 11-7085 . Taken together, the contributions of PGD2 and
its metabolite 15d-PGJ2 to chondrocyte function remain
In addition to PGE2 and 15d-PGJ2, elevated levels of IL-6 have
been detected in synovial fluid from patients with RA . A
positive association between PGE2 and IL-6 production has been
suggested in many different cells, including astrocytes ,
macrophages , synovial  and gingival  fibroblasts,
osteoblasts , and chondrocytes . Moreover, we have
recently reported that PGE2 induces IL-6 expression in human
chondrocytes via cAMP/protein kinase A (PKA)- and
phosphatidylinositol 3 kinase (PI3-K)-dependent pathways . It is
believed that dysregulated overproduction of PGE2 is responsible
for inducing IL-6 synthesis in RA patients. In an animal model of
adjuvant-induced arthritis, the administration of a neutralizing
antibody against PGE2 to arthritic rats inhibited the edema,
hyperalgesia and IL-6 production at sites of inflammation . In
contrast, the role of PGD2 and 15d-PGJ2 in IL-6 regulation is still
a matter of debate. Although Thieringer, et al.  support the
notion that 15d-PGJ2 enhances IL-6 expression in LPS-treated
human peripheral blood monocytes, most previous studies showed
a negative relationship between 15d-PGJ2 and IL-6 production in
different cell lines, such as intestinal epithelial cells  and rat
pancreatic acinar AR42J cells . However, the potential effects
of 15d-PGJ2 in the presence and absence of PGE2 on IL-6
regulation in human chondrocytes have yet to be delineated.
Using the T/C-28a2 chondrocyte cell line as a model system,
we herein report that PGE2 and 15d-PGJ2 exert antagonistic
effects on IL-6 synthesis. Moreover, we delineate the signaling
pathway of IL-6 regulation in human chondrocytes primed with
exogenous PGE2 and/or 15d-PGJ2.
PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on
IL6 synthesis in human T/C-28a2 chondrocytes
Elevated levels of PGE2 , PGD2 (and its dehydration end
product 15d-PGJ2)  and IL-6  have been detected in joint
Figure 1. Dose-dependent regulation of TLR4, caveolin-1 and IL-6 synthesis by PGE2 or PGD2 or 15d-PGJ2 in human chondrocytes.
T/C-28a2 chondrocytes were treated with either PGD2 (A) or 15d-PGJ2 (B) for 48 h, or PGE2 (C) for 2 h. TLR4, caveolin-1 and IL-6 protein (upper) and
mRNA (lower) expression was determined by Western blotting or qRT-PCR, respectively. -actin and GAPDH served as internal controls in
immunoblotting and qRT-PCR, respectively. The Western blots are representative of three independent experiments, all revealing similar results. Data
represent the mean 6 S.E. of 3 independent qRT-PCR experiments. * and m, p,0.05 with respect to the corresponding vehicle control.
synovial fluids obtained from RA patients. We and others have
shown that PGE2 directly stimulates IL-6 production in human
articular chondrocytes [22,23,28]. Prior work has shown that
15dPGJ2 can positively or negatively regulate IL-6 synthesis in
different cell types. However, the effects of PGD2 and 15d-PGJ2
on IL-6 synthesis in human chondrocytes have yet to be
determined. It is believed that dysregulated overproduction of
IL-6 is responsible for the systemic inflammatory manifestions and
abnormal laboratory findings in RA patients. The human
T/C28a2 chondrocyte cell line was chosen as a model system, since T/
C-28a2 cells have been shown to behave much like primary
human chondrocytes when cultured under appropriate conditions
[23,29]. Our data reveal that prolonged (48 h) treatment of
human T/C-28a2 cells with exogenous PGD2 (Fig. 1A) or
15dPGJ2 (Fig. 1B) suppresses IL-6 mRNA and protein synthesis in a
dose-dependent manner. Moreover, marked downregulation of
IL-6 expression (data not shown) and spontaneous secretion (Fig.
S1A) is detected after 24 h to 48 h treatment of T/C-28a2 cells
with exogenously added 15d-PGJ2 (1 mM). 15d-PGJ2 (1 mM) also
repressed IL-6 secretion in human primary articular chondrocytes
(Fig. S1B). In marked contrast, and in agreement with previously
published data , PGE2 rapidly induces IL-6 mRNA and
protein synthesis in a dose-dependent fashion (Fig. 1C). Taken
together, these data illustrate that PGE2 and 15d-PGJ2 exert
opposing effects on IL-6 expression in human chondrocytes.
PGE2 and 15d-PGJ2 differentially regulate TLR4 and
caveolin-1 expression, which in turn modulate the IL-6
synthesis in human chondrocytes
Prior work showed that caveolin-1 diminishes the
lipopolysaccharide (LPS)-mediated nuclear translocation of NF-kB p65 and
IL-6 production in murine macrophage RAW264.7 cells via
binding and inactivating TLR4 . We therefore investigated the
effects of exogenous PGE2 and 15d-PGJ2 on TLR4 and caveolin-1
expression as well as their roles in IL-6 synthesis in human
T/C28a2 chondrocytes. Our data reveal that PGE2 induces TLR4
synthesis in a dose-dependent manner without affecting caveolin-1
expression (Fig. 1C). In contrast, PGD2 or 15d-PGJ2 concurrently
downregulates TLR4 and upregulates caveolin-1 mRNA and
protein synthesis in a dose-dependent fashion (Figs. 1A, B).
Interestingly, pre-treatment of T/C-28a2 cells with 15d-PGJ2
(1 mM) or PGD2 (8 mM) for 48 h abolishes the PGE2-dependent
IL-6 and TLR4 synthesis at both transcriptional and translation
levels (Figs. 2A, B). PGE2 does not alter the
PGD2/15d-PGJ2dependent upregulation of caveolin-1 (Fig. 2A).
In view of our data showing that PGE2 upregulates TLR4 while
leaving intact caveolin-1 expression, experiments were performed
using T/C-28a2 cells transfected with either a siRNA oligonucleotide
sequence specific for TLR4 or a plasmid containing the cDNA of
caveolin-1. The efficacy of these genetic interventions is demonstrated
at both the mRNA and protein levels (Fig. 2). Selective knockdown of
TLR4 significantly inhibits IL-6 upregulation without altering
caveolin-1 mRNA levels in PGE2-primed T/C-28a2 cells (Fig. 2C).
Ectopic expression of caveolin-1 is sufficient to suppress the levels of
IL-6 mRNA expression in control and PGE2-activated T/C-28a2
cells without impairing TLR4 synthesis (Fig. 2C).
In light of the effects of PGD2 or 15d-PGJ2 on TLR4 and
caveolin-1 expression, experiments were carried out using cells
transfected with a plasmid containing the cDNA of TLR4 or an
siRNA oligonucleotide specific for caveolin-1. As shown in Fig. 2D,
ectopic expression of TLR4 markedly increases IL-6 expression
compared with untreated control T/C-28a2 chondrocytes in the
absence of caveolin-1 regulation. Furthermore, TLR4
overexpression reverses the PGD2- or 15d-PGJ2-mediated IL-6
downregulation (Fig. 2D and Fig. S2). Similarly, caveolin-1 depletion
increases IL-6 synthesis in both untreated control and PGD2 or
15d-PGJ2-treated T/C-28a2 cells (Fig. 2D and Fig. S2). Taken
together, these data illustrate that PGE2 and 15d-PGJ2
differentially regulate TLR4 and caveolin-1 expression, which in turn
modulate IL-6 expression in human chondrocytes.
In view of the key role of cAMP production in PGE2-mediated
IL-6 synthesis , we examined the potential contribution of
TLR4 and caveolin-1 to the regulation of cAMP accumulation in
human T/C-28a2 chondrocytes. Our data reveal that selective
TLR4 knockdown attenuates the intracellular cAMP levels in
untreated control T/C-28a2 cells, as determined by the use of a
cAMP enzyme immunoassay kit (Fig. 3A). In contrast, ectopic
expression of TLR4 significantly augments cAMP levels (Fig. 3A).
However, caveolin-1 depletion or overexpression does not impair
the intracellular levels of cAMP (Fig. 3B). Cumulatively, these data
suggest that TLR4, but not caveolin-1, regulates cAMP
production in human chondrocytes.
TLR4 and caveolin-1 differentially regulate PI3-K, PKA and
ERK1/2 signaling pathways
We have recently reported that PGE2 induces IL-6 expression
in chondrocytes via a cAMP/PKA- and PI3-K-dependent
pathway . Moreover, prior work has implicated ERK1/2 as
a downstream target of PGE2 in myocytes . The functional
role of ERK1/2 in PGE2-mediated IL-6 synthesis in chondrocytes
was documented via the use of the MEK1/2 inhibitors PD98059
and U0126, which both significantly inhibited IL-6 expression at
both the transcriptional and translational levels (Fig. 3C).
Therefore, we sought to determine how prostaglandins and its
downstream effectors TLR4 and caveolin-1 regulate the activity of
PI3-K, PKA and ERK1/2. In agreement with our previous work
, exogenous PGE2 stimulates PI3-K and PKA activity at early
time points (2 h), as evidenced by increased phosphorylation of
Akt at Ser 473 and CREB at Ser-133, respectively, and returns to
basal levels after prolonged (48 h) stimulation (Fig. 4A). The same
temporal pattern is detected for the phosphorylation levels of
Figure 4. Exogenous PGE2 and 15d-PGJ2 differentially regulate TLR4 and caveolin-1, which are in turn responsible for the
phosphorylation of ERK1/2, Akt and CREB in T/C-28a2 chondrocytes. T/C-28a2 cells were incubated with either PGE2 (10 mM) for 2 h (A) or
15d-PGJ2 (1 mM) for 48 h (C, D). In other experiments, cells were pre-treated with 15d-PGJ2 (1 mM) for 48 h before incubation with PGE2 (10 mM) for
2 h (B). In select experiments, cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (D) or a plasmid
containing the cDNA of caveolin-1 (B) or TLR4 (D) before treatment with PGE2 or 15d-PGJ2. Phosphorylated ERK1/2 (Thr202/Tyr204), Akt (Ser 473) and
CREB (Ser 133) are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total ERK1/2, Akt,
CREB and -actin. TLR4 and caveolin-1 protein levels were also probed with an anti-TLR4 and an anti-caveolin-1 antibody, respectively. These western
blots are representative of three independent experiments, all revealing similar results.
ERK1/2 (Thr 202 and Tyr204) in PGE2-primed T/C-28a2 cells
(Fig. 4A). Of note, the total ERK1/2, Akt and CREB levels are not
impaired by PGE2 stimulation (Fig. 4A). Selective knockdown of
TLR4 or ectopic expression of caveolin-1 suppresses the
phosphorylation levels of Akt, CREB and ERK1/2 in
PGE2primed T/C-28a2 cells down to baseline controls (Fig. 4B).
15d-PGJ2 attenuates the levels of Akt, CREB and ERK1/2
phosphorylation below those of untreated controls in human
T/C28a2 chondrocytes (Fig. 4C). Maximal downregulation is detected
after 48 h of 15d-PGJ2 stimulation (Fig. 4C). Similarly, 15d-PGJ2
mitigates the enhanced phosphorylation levels of Akt, CREB and
ERK1/2 in caveolin-1-knockdown or TLR4 overexpressing
chondrocytes (Fig. 4D).
PGE2 and 15d-PGJ2 differentially regulate NF-kB
activation in human chondrocytes
NF-kB was identified as the key transcriptional factor
responsible for IL-6 synthesis in PGE2-primed chondrocytes
. Thus, we sought to determine the effects of 15d-PGJ2 on
NF-kB activation induced by exogenous PGE2. As shown in
Fig. 5A, 15d-PGJ2 blocked the PGE2-dependent transactivation of
NF-kB p65 subunit, as evidenced by the inhibition of
phosphorylation at Ser-276 and Ser-536. Moreover, exogenous 15d-PGJ2
exerted a pronounced inhibitory effect on IL-6 promoter activity
(Fig. 6A) and reduced the levels of the NF-kB gel shift (Fig. 7A)
and supershift (Fig. 7B) detected after T/C-28a2 chondrocyte
stimulation with PGE2.
TLR4 depletion or caveolin-1 overexpression blocked the
PGE2-dependent transactivation of NF-kB p65 subunit (Fig. 5A),
IL-6 promoter activity (Figs. 6B,C) as well as the levels of NF-kB
gel shift (Fig. 7A) and supershift (Fig. 7B). On the other hand,
exogenous 15d-PGJ2 represses the phosphorylation of p65 (Fig. 5B)
and its binding to the IL-6 promoter (Figs. 6D,E) as well as the
NFkB gel shift and supershift (Figs. 7C, D) induced by ectopic
expression of TLR4 or knockdown of caveolin-1 in human
T/C28a2 chondrocytes. These data were further validated using
chromatin immunoprecipitation assays (Fig. 8). Taken together,
our data disclose that PGE2 and 15d-PGJ2 exert antagonistic
effects on NF-kB activation, which are propagated via TLR4 and
caveolin-1. Our data also reveal the critical role of ERK1/2, in
addition to PI3-K and PKA , in the induction of IL-6
promoter activity (Fig. 6F) and increased levels of NF-kB gel shift
(Fig. 7E) and supershift (Fig. 7F) detected after T/C-28a2
chondrocyte stimulation with PGE2.
The synovial fluid of RA patients relative to normal controls
contains elevated levels of several soluble mediators including
PGE2, PGD2/15d-PGJ2 and IL-6, which contribute to the
systemic manifestations of the disease [9,17,27]. Although PGE2
has been reported to directly stimulate IL-6 production [22,23,28],
the potential role of PGD2/15d-PGJ2 in the modulation of IL-6
synthesis in human articular chondrocytes has yet to be
investigated. Here, we report that exogenous PGE2 and PGD2/
15d-PGJ2 exert opposing effects on IL-6 expression and secretion.
Specifically, PGE2 induces TLR4 synthesis, which is in turn
responsible for the activation of ERK1/2, PI3-K and PKA
pathways that act synergistically to activate NF-kB. Binding of the
NF-kB p65 subunit to IL-6 promoter elicits IL-6 synthesis (Fig. 9).
In contrast, exogenous PGD2/15d-PGJ2 concurrently
downregulates TLR4 and upregulates caveolin-1 expression, which in turn
suppress the PGE2-dependent activation of ERK1/2, PI3-K and
PKA pathways and NF-kB dependent IL-6 production (Fig. 9).
Prior work has shown that LPS binding to TLR4 induces
mPGES-1 synthesis and PGE2 production in mouse osteoblasts
. However, we herein show that PGE2 is necessary and
sufficient for induction of TLR4 at the transcriptional and
translational level in human T/C-28a2 chondrocytes. We
hypothesize that PGE2 induces TLR4 synthesis by an indirect
manner in the view of its function as an autocrine regulatory
factor. Our analysis also revealed that 15d-PGJ2 repressed TLR4
expression. In agreement with our data, Eun, et al.  reported
that treatment of human intestinal epithelial cells with 15d-PGJ2
attenuated LPS-induced TLR4 mRNA and protein expression,
thereby providing evidence that 15d-PGJ2 may downregulate
TLR4 expression. In contrast, Inoue, et al.  showed that
15dPGJ2 enhanced the expression of TLR4 in the LPS-induced acute
lung injury mice. However, this observation regarding the
potential stimulation of TLR4 expression by 15d-PGJ2 needs to
be interpreted with caution, since 15d-PGJ2 does not exhibit any
regulatory effect on TLR4 expression in the absence of LPS .
Of note, several lines of evidence suggest that 15d-PGJ2 is capable
of suppressing TLR4 expression in different cell lines, such as
mouse T lymphocytes , rat Schwann cells  and human
intestinal epithelial cells .
Caveolin-1 is upregulated in osteoarthritic cartilage .
Moreover, caveolin-1 binding to CD26 has been reported to play
a key role in T-cell-mediated antigen-specific response in RA .
Peroxisome proliferator-activated receptor c (PPARc) ligands,
such as 15d-PGJ2, upregulate caveolin-1 expression in human
carcinoma cells . In agreement with this prior observation, our
data reveal that 15d-PGJ2 induces caveolin-1 expression in human
T/C-28a2 chondrocytes. Interestingly, caveolin-1 was recently
shown to interact and inactivate TLR4-mediated IL-6 signaling in
murine RAW264.7 macrophages . Consistent with this
observation, we found that TLR4 and caveolin-1 exert
antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes.
The pro-inflammatory potential of TLR4 is associated with its
ability to induce IL-6 production in diverse cell types such as
human macrophages  and bladder epithelial cells [41,42].
TLR4 activation by LPS induces IL-6 expression in bladder
cancer cells via an ERK/p38/PI3-K-dependent pathway . In
line with our data, pharmacological inhibition of ERK attenuated
LPS-induced IL-6 synthesis in bladder cancer cells . In
contrast to our results, use of a PI3-K inhibitor (LY294002)
amplified IL-6 expression in LPS-primed bladder cancer cells .
Consistent with our observations, previous studies have shown that
TLR4 can activate PI3-K, ERK or PKA pathways either by direct
Figure 6. Regulation of the IL-6 promoter activity in human T/C-28a2 chondrocytes by exogenous PGE2 or 15d-PGJ2. T/C-28a2 cells
were pre-treated with 15d-PGJ2 (1 mM) for 48 h before incubation with PGE2 (10 mM) for 2 h (A). In other experiments, cells were incubated with
either PGE2 (10 mM) for 2 h (B, C, F) or 15d-PGJ2 (1 mM) for 48 h (D, E). In select experiments, T/C-28a2 cells were transfected with a plasmid
containing an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (E) or the cDNA of caveolin-1 (C) or TLR4 (D) before PG treatment. In
separate experiments, cells were incubated with PGE2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 mM) or U0126 (10 mM)
(F). Cells were transfected with the indicated siRNAs or cDNA constructs along with the IL-6 promoter reporter construct pIL-6-luc651 or pIL-6-luc651
DNF-kB before PG stimulation, as described under Experimental Procedures. Luciferase activities were measured by using the Dual-Luciferase
Reported Assay kit and normalized to sea pansy luciferase activity of co-transfected pRL-SV40. Data represent the mean 6 S.E. of at least 3
independent experiments. *, p,0.05 with respect to the pIL-6-luc651 DNF-kB and vehicle or mock transfected control. m, p,0.05 with respect to
significantly regulated (*) groups.
interaction with the PI3-K p85 regulatory subunit  or mitogen
activated protein kinase kinase kinase 3 (which is upstream of
ERK)  or by modulating intracellular cAMP level ,
respectively. Interestingly, caveolin-1 has been shown to suppress
PI3-K, PKA and ERK1/2 activity by direct interaction [46,47].
Cumulatively, our data along with previously published results
suggest that TLR4 and caveolin-1 modulate the intracellular
PI3K, ERK1/2 and PKA pathways in a reverse, antagonistic manner.
We have recently demonstrated the key role of the NF-kB p65
subunit in the induction of IL-6 synthesis in shear-activated or
PGE2 primed human T/C-28a2 chondrocytes via a
PI3-K/PKAdependent pathway [23,29]. We herein extend these observations
by showing the key role of ERK1/2 in the activation of the NF-kB
p65 subunit and its binding to the IL-6 promoter in
PGE2stimulated human chondrocytes. We further report that inhibitory
effects of 15d-PGJ2 in the activation of the PI3-K, PKA and
ERK1/2 pathways and NF-kB-dependent IL-6 synthesis, which
are mediated via the downregulation of TLR4 and upregulation of
caveolin-1. TLR4 was also reported to trigger a rapid IL-6
response in LPS-stimulated bladder epithelial cells , which
includes the sequential involvement of calcium, adenylyl cyclase
3generated cAMP and the transcription factor CREB. Even though
TLR4 stimulates intracellular cAMP production, which in turn
plays a key role in PGE2-dependent IL-6 synthesis, we have found
that CREB is not involved in the induction of IL-6 in human
chondrocytes stimulated with exogenous PGE2 .
In summary, we have elucidated the signaling pathway by which
PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in
human chondrocytes (Fig. 9), and demonstrated the key albeit
antagonistic actions of TLR4 and caveolin-1 in this process.
Understanding the signal transduction pathway of PG-regulated
IL6 synthesis in human chondrocytes will enable us to design therapeutic
strategies to reduce inflammation and pain in arthritic patients.
Materials and Methods
promoter reporter constructs pIL6-luc651 (-651/+1) and
pIL6luc651 DNF-kB (NF-kB site mutation) were gifts from Dr.
Eickelberg . pRL-SV40 vector encoded with renilla luciferase
gene was purchased from Promega (Madison, WI). The caveolin-1
and TLR4 cDNA plasmids were supplied from Origene
Technologies (Rockville, MD), and subcloned to the
pCMV6XL vector. The MEK1/2 inhibitors U0126 and PD98059 were
obtained from Sigma-Aldrich Corp. Antibodies specific for
actin, caveolin-1, Akt, p-Akt (Ser473), CREB, p-CREB (Ser133),
ERK1/2, p-ERK1/2 (Thr 202/Tyr 204), NF-kB p65, p-p65
(Ser276) and p-p65 (Ser536) were purchased from Cell Signaling
Technology, Inc. (Danvers, MA, USA). Antibody specific for
TLR4 was from Sigma-Aldrich Corp and monoclonal antibody
specific for IL-6 as well as TLR4 and caveolin-1 siRNAs were
from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). IL-6 and
Figure 9. Proposed cascade of signaling events regulating IL-6 synthesis in human chondrocytes treated with PGE2 and 15d-PGJ2.
PGE2 stimulates TLR4 synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the
NF-kB p65 subunit leading to NF-kB activation. Binding of the activated NF-kB p65 subunit to IL-6 promoter induces IL-6 synthesis in human
chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2,
PI3K and PKA activation, and ultimately with NF-kB-dependent IL-6 synthesis in chondrocytes.
cAMP EIA kits were from Cayman Chemical, All reagents for
qRT-PCR and SDS-PAGE experiments were purchased from
Bio-Rad Laboratories. Reagents for EMSA were obtained from
Pierce Chemical Company. The Dual-Luciferase Reporter Assay
kit was purchased from Promega (Madison, WI). The EZ-ChIP kit
was purchased from Upstate Biotechnology. All other reagents
were from Invitrogen (Carlsbad, CA), unless otherwise specified.
Cell culture and Treatment
Human primary articular chondrocytes (Cell Applications, Inc)
or T/C-28a2 chondrocytic cells (T/C-28a2 chondrocytic cells
were kindly provided by Dr. Goldring at Harvard Medical School,
Boston, MA, USA)  were seeded on 6-cm tissue culture dishes
(106 cells per dish) in human chondrocyte growth medium (Cell
Applications, Inc) or in DMEM/F12 medium supplemented with
10% FBS, respectively [23,29,50,51,52,53]. 24 h later, human
chondrocytic cells were grown in serum-free medium for another
24 h before being incubated with PGE2 (120 mM), 15d-PGJ2
(1 mM), PGD2 (0.58 mM) or vehicle (control) for prescribed
periods of time in the presence or absence of pharmacological
Transient Transfection and Reporter Gene Assays
For ectopic expression of caveolin-1 or TLR4, T/C-28a2
chondrocytes were transfected with 1.6 mg/slide of plasmid
containing cDNAs by using Lipofectamine 2000. In control
experiments, cells were transfected with 1.6 mg/slide of the
empty vector pCMV6-XL (OriGene Technologies). In select
experiments, T/C-28a2 cells were transfected with 1.6 mg/slide
of the IL-6 promoter reporter construct pIL-6-luc651 or
pIL6luc651 DNF-kB together with pRL-SV40 vector. In RNA
interference assays, T/C-28a2 cells were transfected with
100 nM of a siRNA oligonucleotide sequence specific for
caveolin-1 or TLR4. In control experiments, cells were
transfected with 100 nM of scramble siRNA. Transfected cells
were allowed to recover for at least 12 h in growth medium, and
then incubated overnight in medium containing 1%
NutridomaSP before their exposure to prostaglandins. In promoter activity
experiments, luciferase activities were measured by using the
Dual-Luciferase Reporter Assay kit (Promega), as previously
described described [23,29,54].
Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR assays were performed on the iCycler iQ detection
system (Bio-Rad) using total RNA, the iScript one-step RT-PCR
kit with SYBR green (Bio-Rad) and primers. The GenBank
accession numbers and forward (F-) and reverse (R-) primers are as
caveolin-1 (NM_001753), F-
TLR4 (NM_138554), F-
The GenBank accession numbers and forward (F-) and reverse
(R-) primers for IL-6 and GAPDH are provided in our previous
publications [23,29]. GAPDH was used as internal control.
Reaction mixtures were incubated at 50uC for 15 min followed
by 95uC for 5 min, and then 35 PCR cycles were performed with
the following temperature profile: 95uC 15 s, 58uC 30 s, 68uC
1 min, 77uC 20 s. Data were collected at the (77uC 20 s) step to
remove possible fluorescent contribution from dimer-primers
[23,29]. Gene expression values were normalized to GAPDH.
Western blot analysis
T/C-28a2 cells, from different treatment or transfection, were
lysed in RIPA buffer (25 mM TrisNHCl pH 7.6, 150 mM NaCl,
1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a
cocktail of proteinase inhibitors (Pierce Chemical Company). The
protein content of the cell lysates was determined using
bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical
Company). Total cell lysates (4 mg) were subjected to SDS-PAGE,
transferred to a membrane, and probed with a panel of specific
antibodies. Each membrane was only probed using one antibody.
-actin was used as loading control. All Western hybridizations
were performed at least in triplicate using a different cell
preparation each time.
Preparation of cytosolic and nuclear extracts
Cytosolic and nuclear extracts were isolated using the NE-PER
nuclear and cytoplasmic extraction kit (Pierce) following the
manufacturers instructions as previously described [23,29,54].
Gel-shift and supershift assay
A 59-biotinylated oligonucleotide probe
(59-GGGATTTTCC39) was synthesized containing the NF-kB cis-element present on
the IL-6 promoter. EMSAs were performed with a commercially
available nonradioisotopic EMSA kit (LightShift
Chemiluminescence EMSA kit; Pierce) as previous description [23,29,54].
Measurement of IL-6 and cAMP concentration in medium
The levels of IL-6 in medium and intracellular cAMP were
determined using the corresponding kits, following the
manufacturers instructions. The total protein concentration in the medium
was used as loading control, and the results were expressed as pg
IL-6 or pmol cAMP per mg of total protein.
Data represent the mean 6 S.E. of at least 3 independent
experiments. Statistical significance of differences between means
was determined by Students t-test or one-way ANOVA, wherever
appropriate. If means were shown to be significantly different,
multiple comparisons by pairs were performed by the Tukey test
Figure S1 Time-dependent regulation of IL-6 secretion
by 15d-PGJ2-treated human chondrocytes. T/C-28a2
chondrocytes (A) or human primary articular chondrocytes (B)
were treated with 15d-PGJ2 (1 mM) for the indicated time
intervals. IL-6 production was determined by an IL-6 enzyme
immunoassay kit. Data represent the mean 6 S.E. of at least 3
independent experiments. *, p,0.05 with respect to vehicle
Figure S2 The effects of PGD2 on TLR4, caveolin-1 and
IL-6 synthesis in T/C-28a2 chondrocytes. T/C-28a2 cells
were transfected with a siRNA oligonucleotide sequence specific
for caveolin-1 or a plasmid containing the cDNA of TLR4 before
incubated with PGD2 (8 mM) for 48 h. TLR4, caveolin-1 and IL-6
mRNA synthesis was determined by qRT-PCR. GAPDH served
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