Lee et al. Cell Death and Disease (2018)9:401
DOI 10.1038/s41419-018-0433-0
Cell Death & Disease
ARTICLE
Open Access
Kaempferol targeting on the fibroblast
growth factor receptor 3-ribosomal S6
kinase 2 signaling axis prevents the
development of rheumatoid arthritis
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Cheol-Jung Lee1, Su-Jin Moon2,3, Jeong-Hee Jeong3, Sangbae Lee4, Mee-Hyun Lee1,5, Sun-Mi Yoo1, Hye Suk Lee1,
Han Chang Kang 1, Joo Young Lee1, Weon Sun Lee6, Hee-Jin Lee6, Eun-Kyung Kim3, Joo-Yeon Jhun3, Mi-La Cho3,
Jun-Ki Min2,3 and Yong-Yeon Cho1
Abstract
Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although
involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in
RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF)
concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and
migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6
kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity
by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3
backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGFFGFR3–RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of
activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated
phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and
sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high
frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol
administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced
osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of
osteoclast markers, such as tartrate-resistant acid phosphatase, integrin β3, and MMP9. Conclusively, our data suggest
that bFGF-induced FGFR3–RSK2 signaling may play a critical role during the initiation and progression of RA in terms
of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for
RA.
Correspondence: J-K. Min (min6403@catholic.ac.kr) or Y-Y. Cho
(yongyeon@catholic.ac.kr)
1
Integrated Research Institute of Pharmaceutical Sciences & BK21 PLUS Team
for Creative Leader Program for Pharmacomics-based Future Pharmacy,
College of Pharmacy, The Catholic University of Korea, 43, Jibong-ro, Wonmigu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea
2
Department of Internal Medicine, College of Medicine, Division for
Rheumatology, The Catholic University of Korea, 505, Banpo-dong, Seocho-gu,
Seoul 137-701, Republic of Korea
Full list of author information is available at the end of the article
These authors contributed equally: Cheol-Jung Lee, Su-Jin Moon
These authors contributed equally: Jun-Ki Min and Yong-Yeon Cho
Edited by A. Stephanou.
Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory
disease characterized by infiltration of immune cells into
the synovium and hyperplasia of the synovial lining.
Synovial lining cells in RA joints increase to 10–15 cell
layers1–3 due to the influx and proliferation of
© The Author(s) 2018
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Official journal of the Cell Death Differentiation Association
Lee et al. Cell Death and Disease (2018)9:401
inflammatory cells, which eventually manifest as pannus
formation, which grows in a tumor-like fashion and is a
pathognomic finding of RA4. Since the angiogenesis and
proliferation of fibroblast-like synoviocytes (FLSs) play
pivotal roles in mechanisms involved in RA pathogenesis5,
altered activities of angiogenic and growth factors in RA
synovium or synovial fluids (SF) have been considered as
treatment targets for the disease5–7.
Fibroblast growth factor (FGF) is a family of heparinbinding growth factors that shows increased concentration in RA SF compared with that in osteoarthritis (OA)6.
In a previous study, basic FGF (bFGF) concentration in
RA SF better reflected the severity of joint destruction
compared with other cytokines, such as tumor necrosis
factor α (TNF-α), interleukin (IL)-1, or IL-66. In addition,
bFGF overexpression in experimental arthritis mice
resulted in worsened arthritis severity, and it depended on
enhanced angiogenesis and osteoclastogenesis. Previous
studies have shown the anti-apoptotic effects of bFGF in
RA FLSs8 and its RANKL-inducing properties on RA
FLSs9, which are findings that predict the activation of
osteoclasts and structural damage to the affected joints. In
terms of angiogenesis, bFGF activity in endothelial cells
stimulates angiogenic events partly by upregulating vascular endothelial growth factor10. However, the pathophysiological roles of bFGF in RA and its signaling in
immune cells or FLSs have not been well understood.
Proinflammatory cytokines such as TNF-α, IL-1, and
IL-6 induce inflammatory reaction and chemokine production in FLSs, resulting in the increased influx of
additional proinflammatory cells, including macrophages,
into the synovium11. It has become clear that these
proinflammatory cytokines work together with other
mediators, such as IL-17 in an additive or synergistic
way12. Traditionally, the imbalance between type 1 helper
T (Th1) and type 2 helper T (Th2) subsets has been
suggested to lie at the center of RA pathogenesis13.
However, in the past decade, the key paradigm has
changed because numerous studies have identified the
pivotal roles of IL-17 and IL-17-expressing CD4+ T-cells,
known as Th17 cells, in RA development and progression14. Prostaglandin E2 also plays a key role in FLS
activation induced by proinflammatory cytokines and
epidermal
growth
factors
(EGFs)
in
RA15.
Cyclooxygenase-2 (COX-2) is highly expressed in the
synovial lining of RA joints because of the persistent
activities of proinflammatory cytokines, such as TNF-α,
IL-1β, and IL-616, 17. Ribosomal S6 kinase 2 (RSK2) is an
important kinase that modulates the transactivation
activities of AP-1 and NF-κB, which regulate Cox-2 gene
expression in cells where growth factors and/or environmental stresses are present18–20, indicating the potential
role of RSK2 in inflammatory diseases, such as RA. FGF
receptor 3 (FGFR3) is one of four receptor tyrosine
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kinases that respond to FGF. Interestingly, FGFR3 activates RSK2 through tyrosine phosphorylation21, and its
effect is associated with an enhanced MEK/ERK pathway22, 23. We discovered that kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), a
flavonoid found abundantly in edible plants, inhibited
RSK2 N-terminal kinase activity by binding to the active
pocket24, resulting in the inhibition of cell proliferation
induced by EGF25. Recently, it was found that kaempferol
inhibits RSK2, MSK1, and Src kinase activities26, 27. These
results indicate that kaempferol might have other target
proteins. The possibility of this hypothesis was suggested
by the fact that the kaempferol-mediated direct inhibition
of FGFR3–RSK2 signaling suppresses cell proliferation in
JB6 Cl41 cells28. One recent study showed that the inhibition of sonic hedgehog signaling inhibits the proliferation of RA FLSs, which is associated with decreased
expression of FGFR1 and FGFR329.
Although a few studies have suggested that bFGF may
be implicated in RA pathogenesis, its downstream signals
and intracellular effects have not been well elucidated.
Therefore, we investigated whether bFGF and its downstream signals contribute to the pathogenesis of RA, and
how it affects RA FLS proliferation and osteoclastogenesis. To elucidate this, we studied a proliferation assay in
human RA FLSs and signaling activity in RA synovium.
Furthermore, an in vivo effect was confirmed through
collagen-induced arthritis (CIA) mice, an experimental
murine model of RA. Here, we found that kaempferol was
identified as a compound that inhibits FGFR3 kinase
activity in RA FLSs, resulting in significant inhibition of
FLS proliferation and migration. Furthermore, kaempferol
treatment in arthritis mice attenuated arthritis severity
and osteoclastogenesis.
Results
bFGF stimulates proliferation of FLSs in humans
To determine whether bFGF plays a pivotal role in RA
pathogenesis, we measured and compared bFGF concentrations in the SF of RA (n F 79) and OA (n = 31)
patients. The mean ages of the patients with RA and OA
who participated in our study were 58 and 64 years,
respectively. The median duration of disease in RA
patients who participated in our study was 5.8 (interquartile range [IQR], 1.5–10) years. The concentration of
bFGF measured in the SF of RA patients was higher than
that in the SF of OA patients (Fig. 1a). The median values
of bFGF in RA SF and OA SF were 9.1 (IQR, 7.9–11.8)
and 7.7 (IQR, 7.3–8.5) pg/ml, respectively. Interestingly,
the bFGF level in the SF of RA patients positively correlated with SF white blood cell (WBC) and neutrophil
counts (Supplementary Figure S1a and b). However, neither the Disease Activity Score 28, which is the most
widely used RA activity index, nor the serum level of C-
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Fig. 1 bFGF stimulates proliferation of FLSs in humans. a bFGF contents in SF obtained from OA patients (n = 31) and RA patients (n = 79) were
measured by sandwich ELISA using specific antibodies. Each dot represents the concentration of bFGF from each patient. The median values of bFGF
in the RA SF and OA SF denotes the center of interquartile range. *p < 0.01. b, c bFGF induces proliferation of human FLSs (b) and human MH7A (c)
by G1/S cell-cycle transition in a dose-dependent manner compared with untreated controls. Data were obtained from three independent
experiments. d, e Synovial tissues from OA patients (n = 5) and RA patients (n = 5) were analyzed to measure cell proliferation potential using Ki-67
(d) and the activation of RSK2 and FGFR3 using p-RSK2 (T577) and p-FGFR3 (Y724) antibodies (e). Scale bars, 50 μm. The photograph is a
representative confocal image obtained from an immunohistofluorescence assay; the fluorescence intensities of Ki-67, p-RSK, and p-FGFR3 were
normalized by DAPI intensity. The average fold change of the intensity presented in graphs was obtained from five synovial tissues from OA and RA
patients. *p < 0.05; **p < 0.01
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Lee et al. Cell Death and Disease (2018)9:401
reactive protein showed any correlation with bFGF levels
in SF (data not shown). Based on these findings, we
examined bFGF effects on cell proliferation using both
human FLSs isolated from RA patients and MH7A, a
human RA synovial cell line. The results showed that
bFGF-induced cell proliferation (Fig. 1b, c, left graphs) by
stimulating G1/S cell-cycle transition (Fig. 1b, c, right
graphs). The stimulatory effect of FLS proliferation was
confirmed by immunofluorescence assay, showing that
Ki-67 expression was elevated about three-fold in RA
synovium compared with that in OA (Fig. 1d). Based on
previous reports indicating that RSK2 is directly regulated
by FGFR322, 28, we examined the activation levels of RSK2
and FGFR3 in human RA and OA synovium. The results
showed that the fluorescence intensities for the phosphoRSK2 (Thr577) and -FGFR3 (Tyr724) were increased in
RA synovium compared with that in OA tissues (Fig. 1e).
Involvement of FGFR3 and RSK2 in RA in humans
Since the RSK2 signal was found to be higher in the RA
synovium compared with that in OA, we determined
which cells mainly express RSK2 in RA synovium. An
immunohistochemical study showed that active RSK2 was
abundantly detected in the hyperplastic lining layer
(indicated by the blue dotted line) and sublining layer
(indicated by the green dotted line) of RA synovium
(Fig. 2a, first panels). Moreover, double immunohistochemistry using antibodies against RSK2 (brown), CD68
(as macrophage markers, red), CD3 (as T-cell marker,
red), and CD20 (as B-cell marker, red) macrophages were
present in the hyperplastic lining layer and sublining area
at a fairly high frequency (Fig. 2a, second panel), and Tcells were present in the sublining layer of lymphoid
aggregates (Fig. 2a, third panel), similar to previous
reports30, 31. CD20+ B cells were abundantly present in
ectopic lymphoid structures with germinal center-like
characteristics (Fig. 2a, fourth panel). The results further
showed that some T-cells and most macrophages had
activated RSK signals, but B cells did not (Fig. 2a, second
to fourth panels). Since the activated RSK2 signal was
highly detected in the hyperplastic lining of RA synovium,
we performed RSK2 knockdown using the lentiviral
shRNA expression vector. We found that RSK2 knockdown attenuated cell proliferation and migration of
human RA FLSs (Fig. 2b, c). Interestingly, compared with
OA tissue, RA synovium harbored higher levels of activated RSK2 (phospho-RSK2-Thr577) and FGFR3 (phospho-FGFR3-Tyr724) (Fig. 2d). Moreover, the cells
expressing activated forms of RSK2 and FGFR3 were
highly co-stained with CD68, and moderately with CD4
(Fig. 2d). Taken together with Figs. 1 and 2, these results
imply that bFGF and its downstream FGFR3/
RSK2 signaling axis might play pivotal roles in the proliferation and migration of RA FLSs and persistent
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inflammation in RA joints, which suggests the rationale
for FGFR3/RSK2 signaling targeting in RA to inhibit the
development and progression of RA.
Kaempferol targets the kinase domain of FGFR3
Based on the ability of kaempferol to inhibit both
FGFR3 and RSK2 activity28, we investigated the therapeutic efficacy of kaempferol for RA. Before proving this,
we needed to confirm the mechanisms of FGFR3 inhibition by kaempferol. First, we conducted computational
reverse docking with 17 plasma membrane residential
kinase structures downloaded from the Protein Data Bank
(PDB; http://www.rcsb.org/pdb/home/home.do), and
found that kaempferol showed the lowest stand-free
energy value, with about –9.083 kcal/mol against the
ATP-binding site of the FGFR3 kinase (PDB code: 4K33)
(Fig. 3a). A structural analysis proposed that kaempferol
can form hydrogen bonds with the backbone oxygen of
Glu555 and Ala558, as well as the side chain of Lys508,
with distances of about 2.7, 3.0, and 3.7 Å, respectively
(Fig. 3b). This proposal was proven by a competition assay
using ATP-agarose beads and an active FGFR3 kinase
domain, indicating that FGFR3 kinase domain binding to
ATP was decreased by the addition of kaempferol in a
dose-dependent manner (Fig. 3c). Notably, the binding of
kaempferol to the FGFR3 kinase domain was confirmed
by a pull-down assay with cyanogen bromide (CNBr)activated sepharose beads and a commercially active
FGFR3 kinase domain (Fig. 3d), or a membrane-bound
FGFR3 extracted from the membrane fraction of MH7A
cells (Fig. 3e). Moreover, western blotting (Fig. 3f) and an
immunocytofluorescence assay (Fig. 3g) showed that
bFGF-induced FGFR3 phosphorylation was inhibited by
about 50% at 0.4 μM of kaempferol treatment and almost
abrogated at 2 μM of kaempferol in MH7A cells. To sum
up, we discovered that kaempferol can selectively inhibit
FGFR3 activity by targeting its kinase domain.
bFGF-induced cell migration is mediated through the
RSK2 signaling pathway
Since kaempferol targets RSK2 N-terminal kinase24 and
the FGFR3 kinase domain (Fig. 3), we analyzed the effects
of kaempferol on the proliferation and migration of
human RA FLSs. Kaempferol inhibited bFGF-induced FLS
proliferation in a dose-dependent manner (Fig. 4a). No
cytotoxicity was observed in RA FLSs up to a concentration of 80 μM of kaempferol (Supplementary Figure S2a), similar to our previous observation25. Since
RSK2 plays an important role in cell proliferation and
migration19, 32, we determined the effects of kaempferol
on the transactivation activities of AP-1 and NF-κB and
Cox-2 promoter activity in RSK2+/+ and RSK2−/− mouse
embryonic fibroblasts (MEFs). The results showed that
bFGF-induced AP-1 transactivation activity and Cox-2
Lee et al. Cell Death and Disease (2018)9:401
Fig. 2 (See legend on next page.)
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(see figure on previous page)
Fig. 2 Involvement of FGFR3 and RSK2 in RA in humans. a Representative double immunohistochemical analysis of human RA synovial tissues
(×100, n = 3). The tissues were hybridized with a combination of phospho-RSK2 (Thr577) antibody (shown in brown), CD68 (a macrophage marker,
shown in red), CD3 (a T-cell marker, shown in red), and CD20 (a B-cell marker, shown in red), as indicated. Boxed area was magnified (×400). The area
marked by the blue dotted line indicates the hyperplastic lining layer, and the area marked by the green dotted line indicates the sublining layer in
synovium. b, c The knockdown effects of RSK2 on cell proliferation (b) and cell migration (c) in human RA FLSs. Cell proliferation was measured by
MTS assay (b), and the migrated area (graph) was quantified by measuring the uncovered area of the wound using Image J (Ver. 1.6). Scale bars, 100
μm (c). Data were obtained from three independent experiments, and values are represented as means ± SEM. *p < 0.001 by Student’s t-test. d
Representative photographs (×40) of triple immunohistofluorescence analysis of tissues obtained from OA (n = 5) and RA (n = 5) patients. p-RSK2
and p-FGFR3 were co-stained with CD4- (a T-cell marker; upper panels) or CD68- (a macrophage marker; bottom panels) specific antibodies as
indicated and analyzed by confocal microscopy. Yellow color in the merged photographs indicates the coexistence of CD4+ T-cells or CD68+
macrophages in the synovial tissues of OA and RA patients, respectively. Scale bars, 40 μm
promoter activity were decreased by kaempferol in
RSK2+/+ MEFs, but not in RSK2−/− MEFs, in a dosedependent manner (Fig. 4b). Similar results were obtained
from RSK2+/+ and RSK2−/− MEFs in naive cell culture
conditions (Supplementary Figure S2b), suggesting that
the genetic depletion of RSK2 or kaempferol treatment
might suppress cell migration of FLSs. As expected, the
bFGF-induced cell migration shown in RSK2+/+ MEFs
was attenuated in RSK2−/− MEFs (Fig. 4c and Supplementary Figure S2c). The inhibitory effects of cell
migration induced by bFGF were similarly observed by
kaempferol treatment in MH7A and human RA FLSs in a
dose-dependent manner (Fig. 4d). Zymography using the
culture supernatant of FLSs indicated that FLS migration
inhibition by kaempferol was mediated through the
inhibition of MMP-9 and MMP-2 activities (Fig. 4e). Since
RSK2 mediated bFGF signaling in FLS proliferation and
migration (Fig. 4a–d), we needed to confirm the specificity of kaempferol on FGFR3. We found that bFGFinduced FGFR3 phosphorylation was inhibited by
PKC412, a FGFR3 inhibitor, and kaempferol, but not by
U0126, an MEK inhibitor (Fig. 4f). Taken together, our
results suggest that the FGFR3–RSK2 signaling axis might
play an important role in the proliferation and migration
of FLSs, which is the most fundamental treatment target
to prevent progressive joint destruction in RA.
Kaempferol inhibits RA development in a collage-induced
arthritis mouse model
We investigated whether kaempferol can affect Th17
lineage differentiation (Fig. 5a, b). To investigate the
effects of kaempferol under Th17 cell-polarizing conditions, isolated murine CD4+ T-cells were cultured in the
presence of anti-CD3, anti-CD28, TGFβ, IFNγ, IL-4, and
IL-6 with or without kaempferol for 72 h. We found that
kaempferol decreased not only the number of IL-17expressing CD4+ T-cells (Fig. 5a), but also the level of IL17, IL-21, and TNF-α in the culture supernatant (Fig. 5b)
in a dose-dependent manner. The MTT assay to investigate the cytotoxicity of kaempferol resulted in no
Official journal of the Cell Death Differentiation Association
cytotoxicity in murine CD4+ T-cells up to 25 μM of
kaempferol (data not shown). Next, we investigated
whether kaempferol suppressed inflammation and joint
destruction in an experimental RA murine model (CIA).
One group of mice was intraperitoneally injected with 2
mg/kg of kaempferol three times a week after type II
collagen (CII) boosting immunization, and the other
group was only injected with the vehicle. The results
showed that kaempferol treatment in CIA mice ameliorated arthritis severity and incidence compared with
vehicle-treated mice (Fig. 5c). Histological sections of
hind paw joints showed that kaempferol treatment in CIA
mice attenuated the severity of inflammation, cartilage
damage, and bone erosion, which were investigated by
hematoxylin-eosin (H&E) staining (Fig. 5d, top panels)33.
Additionally, cartilage loss assessed by safranin O was
prevented by kaempferol treatment in CIA mice (Fig. 5d,
middle panels). The reduction of tartrate-resistant acid
phosphate-positive (TRAP+, indicated by black arrow)
cells in the joints of CIA+kaempferol mice indicated that
osteoclastogenic activity was suppressed (Fig. 5d, bottom
panels and right graph) compared with control group
mice. To validate whether kaempferol treatment negatively regulated Th17 lineage differentiation in vivo, the
mRNA levels of genes involved in Th17 differentiation,
such as IL-17, Ahr, CCL20, and RORγt, were measured in
draining lymph node cells isolated from each group of
mice. The mRNA levels of the genes were lower in CIA
+kaempferol mice than in vehicle-treated mice (Fig. 5e).
Since the signal transducer and activator of transcription3 (STAT3) is a pivotal transcriptional factor during the
differentiation of Th17 cells from naïve CD4+ T-cells
through IL17a and IL17f gene expression34, and Src is
known to be capable of activating STAT3 activity by
direct tyrosine phosphorylation35, 36, cell populations in
the spleen tissue of CIA + kaempferol mice showing
CD4+/IL-17+ (mainly Th17), CD4+/pSTAT3-S727+,
CD4+/pSTAT3-Y705+, and CD4+/Src+ were analyzed by
confocal microscope. The number of IL-17+/CD4+ cells
was decreased by kaempferol treatment in CIA mice
Lee et al. Cell Death and Disease (2018)9:401
Fig. 3 (See legend on next page.)
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(see figure on previous page)
Fig. 3 Kaempferol targets the kinase domain of FGFR3. a Docking scores calculated by Protein Preparation Wizard in Maestro v9.2 using
kaempferol 3D structures (CID 5282102) and crystal structures of indicated RTKs obtained from the Protein Data Bank. b The lowest docking score of
kaempferol to the active pocket of FGFR3 represents the formation of hydrogen bonds with Lys508, Glu555, and Ala558, as indicated. c Kaempferol
competition assay. Binding of FGFR3 kinase domain to ATP-conjugated agarose beads was competed with the indicated doses of kaempferol. The
graph indicates fold change of band intensity from three independent western blots of the FGFR3 pull-down assay. Data were obtained from three
independent experiments, and values are represented as means ± SEM. *p < 0.01; **p < 0.001. d, e Kaempferol binding to FGFR3 was confirmed by a
CNBr-kaempferol pull-down assay using a commercial FGFR3 kinase domain (c) and a membrane fraction of MH7A cells (d). The western blot is
representative of three independent experiments. f, g The effects of kaempferol on bFGF-induced FGFR3 phosphorylation at Tyr724 was analyzed by
western blotting (f) and immunocytofluorescence (g) using indicated p-FGFR3 specific antibodies in MH7A cells. The band intensity of western blot
(f) was measured using Image J (Ver. 1.6), and each indicated area (g) in the immunocytofluorescence confocal image (×400) was magnified (×630).
Scale bars, 20 μm
(Fig. 5f and Supplementary Figure S3a). Moreover, the
numbers of CD4+/pSTAT3+ (both Ser727 and Tyr705)
and CD4+/Src+ splenic T-cells were significantly
decreased by kaempferol treatment compared with
vehicle-treated arthritis mice (Fig. 5f and Supplementary
Figure S3b). These results demonstrated that kaempferol
inhibited autoimmune arthritis by suppressing Th17 cell
differentiation from naïve T-cells.
Kaempferol inhibits osteoclast differentiation
Next, we examined the effects of kaempferol on osteoclast differentiation. We found that ex vivo M-CSF/
RANKL-induced differentiation of bone marrow-derived
monocytes/macrophages (BMMs) isolated from CIA
+kaempferol mice significantly inhibited the formation of
multinucleated TRAP+ giant cells compared with those of
CIA+vehicle mice (Fig. 6a). An in vitro study confirmed
that kaempferol treatment in naïve murine BMMs significantly inhibited M-CSF- and RANKL-induced osteoclastogenesis (Fig. 6b). We further characterized the
effects of kaempferol on the molecular mechanisms for
osteoclast differentiation by an analysis of mRNA levels
for osteoclast-specific genes, including TRAP, calcitonin
receptor, cathepsin K, c-Jun, and p50 (a member of NF-κB)
(Fig. 6c). These results strongly supported the hypothesis
that kaempferol inhibits osteoclast differentiation
(Fig. 6c). Since osteoclasts phenotypically characterized by
high TRAP activity are multinucleated cells formed by the
fusion of hematopoietic lineage-derived monocytes/macrophages, we next investigated the effects of kaempferol
on M-CSF/RANKL-stimulated BMM morphology and
nuclear numbers in an osteoclast. The total nuclear
number of multinucleated (≥3 nuclei) giant cells with the
phenotypic features of osteoclasts was significantly
reduced by kaempferol in a dose-dependent manner,
whereas the number of cells with a single nucleus were
increased (Fig. 6e). These results indicated that kaempferol inhibits the differentiation of multinucleated osteoclasts from undifferentiated BMMs with a single nucleus.
Taken together, our findings indicate that bFGF/FGFR3mediated RSK2 activation induces FLS proliferation,
Official journal of the Cell Death Differentiation Association
migration, and inflammatory responses, resulting in the
induction of osteoclastogenesis. Thus, blockage of the
bFGF/FGFR3/RSK2 signaling axis by kaempferol may
inhibit the progressive structural damage of RA joints that
is induced by overwhelming osteoclast activity.
Discussion
In this study, the kaempferol-antagonizing bFGF/
FGFR3/RSK2 axis effectively reduced clinical and histologic scores in CIA mice. The main mechanism by which
kaempferol exerted its antiarthritic efficacy was the inhibition of RA FLS proliferation and migration and the
significant suppression of Th17 differentiation and
osteoclastogenesis. In joints, FLSs produce lubricating SF
in the joint cavity, and is involved in the production of
matrix components and matrix-degrading enzymes during matrix remodeling37. Proinflammatory factors produced by FLSs and immune cells such as activated T-cells
induce the secretion of matrix-degrading enzymes and
inflammatory factors that contribute to joint erosion and
enhance the inflammatory cycle in RA1–3, 38. Since the
RSK2 signaling pathway induces the transactivation
activities of AP-1, and NF-κB regulates cell proliferation,
cell migration, and inflammation19, 39 by regulation of
activated T-cell 3 (NFAT3) nuclear factors and RSK2/
NFAT3-mediated IL-2 promoter activity39, these results
suggest that RSK2 plays a critical role in T-cell activation
in vitro and in vivo40, and provide us with an opportunity
to establish the hypothesis that RSK2 might be involved in
RA development.
On the other hand, typical hallmarks of RA are thickening of the synovial lining and synovial hyperplasia by
the enhancing fibroblast proliferation and immune cell
infiltration41. This phenomenon eventually contributes to
joint damage. Chronologically, the expression of bFGF in
synovial tissues from patients with RA was detected by
immunohistological staining42. Later, bFGF concentrations were measured in two different groups (one group
with less joint damage [Larsen grade 1–3], and another
with severe joint damage [Larsen grade 4–5]) of RA
patients and a group of OA patients. The results indicated
Lee et al. Cell Death and Disease (2018)9:401
Fig. 4 (See legend on next page.)
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(see figure on previous page)
Fig. 4 bFGF-induced cell migration is mediated through the RSK2 signaling pathway. a The efficacy of kaempferol on bFGF-induced human
FLS proliferation was measured by MTS assay. b AP-1 transactivation and Cox-2 promoter activities were measured in RSK2+/+ and RSK2−/− MEF by
transfection of pAP-1-luciferase (top graph) and pCox-2 promoter-luciferase (bottom graph) reporter plasmids as indicated. c Effects of bFGF-induced
cell migration in RSK2+/+ and RSK2−/− MEFs. The migrated area was quantified by measuring the uncovered area of the wound using Image J (Ver.
1.6). d Efficacy of kaempferol on the cell migration of MH7A and human FLSs. The migrated area (graphs) was quantified by measuring the
uncovered area of the wound using Image J (Ver. 1.6). e Efficacy of kaempferol on MMP-9 and MMP-2 activity was analyzed by gelatin zymography
using the indicated culture supernatants of human FLSs. f Representative photographs for kaempferol specificity on FGFR3 phosphorylation at Tyr724
induced by bFGF stimulation in MH7A cells. PKC412, an FGFR3 inhibitor; U0126, an MEK inhibitor. Data were obtained from three independent
experiments. Each indicated area in the immunocytofluorescence confocal image (×400) was magnified (×630). Scale bars, 20 μm. a–d Data were
obtained from three independent experiments, and values are represented as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t-test
that the bFGF concentrations in the SF of RA patients
with severe joint damage (70–73 pg/ml) were 5.5- and
20.6-fold higher than that in the SF of RA patients with
less joint damage (8.4–18.9 pg/ml) and OA patients (3.6
pg/ml), respectively6. Based on the positive correlation
between bFGF in RA SF and WBC and neutrophil counts,
and the significant differences in SF bFGF concentrations
according to the severity of radiographic damage, it is
suggested that bFGF in RA joints can play a pivotal role in
progressive joint destruction and may represent local
inflammatory status in affected joints.
Osteoclast differentiation of BMMs is a complicated
process governed by a variety of signaling pathways.
Osteoclasts are multinucleated bone resorbing cells
formed by the cytoplasmic fusion of mononuclear precursors such as macrophages. Previous reports have
indicated that the chemical inhibition of the MEKs/ERKs
signaling pathway using PD98059 or U0126 suppresses
RANKL-induced osteoclast differentiation40, 41. Moreover, c-Fos, a component of AP-1, and NF-κB are wellknown regulatory molecules that induce osteoclast differentiation43, 44. Since the inhibition of the ERKs/
RSK2 signaling axis abrogated AP-1 and NF-κB transactivation activity32, we established our initial hypothesis
that dual targeting of kaempferol on the kinase activities
of FGFR3 and RSK2 might suppress osteoclastogenesis.
Indeed, recent studies have shown that bFGF increases
osteoclast activity in vitro and that this increasing activity
is inhibited by FGFR3-deficient osteoclasts, which
revealed the directly positive effect of bFGF-FGFR3 signaling on osteoclast regulation45. However, bFGF signaling is also known to affect osteoblast activity46. bFGF is
expressed in osteoblasts and induces osteogenesis in a
stage-specific manner in osteoblastic cells47. Despite these
concomitant effects of bFGF on osteoblasts and osteoclasts, kaempferol showed significant anti-arthritis and
anti-osteoclastic effects in vivo and in vitro.
Conclusively, our study indicated that bFGF-FGFR3
interaction stimulates FLS proliferation and cell migration
mediated through both direct and indirect signaling
pathways to RSK2 in the synovial tissues, resulting in the
Official journal of the Cell Death Differentiation Association
provocation of inflammatory response-macrophage infiltration. The macrophages in the synovial tissue differentiated to multinucleated osteoclasts by the stimulation
of M-CSF and RANKL, resulting in RA development.
Thus, the dual targeting of kaempferol on both FGFR3
and RSK2 may prevent RA development through the
inhibition of osteoclast differentiation (Fig. 6f).
The efficacy of kaempferol was previously suggested by
the fact that kaempferol harbored about 7 μM, 15 μM, and
25 μM of IC50 against RSK2, Src kinase and MSK1,
respectively24, 26, 27. In the present study, we found that
kaempferol targeted the kinase domain of FGFR3 with
about 400 nM of IC50 in a cell culture system (Fig. 2e, f).
The 400 nM IC50 value of kaempferol is the lowest
effective concentration against known target kinases such
as RSK2, MSK1, and Src. Interestingly, the kaempferol
concentration in human plasma was maximally reached at
100–150 nM at 5.8 h by serving a bowl of thick endive
soup (300 g; 8.65 mg kaempferol equivalent) with a slice of
white bread and a glass of water after fasting from
flavonoid-rich foods such as fruits, juices, green or leafy
vegetables, onions, tomatoes, tea, and red wine for 48 h
before the examination48. These results suggest that longterm daily consumption of a kaempferol-rich diet can
help prevent RA development.
Materials and methods
Chemicals and antibodies
Chemicals utilized for molecular and cellular biology
and buffer preparation were purchased from SigmaAldrich (St. Louis, MO, USA). Cell culture medium
including Dulbecco’s Modified Eagle’s Medium (DMEM;
Cat#: 10-013-CVR, Corning, New York, NY, USA),
modified Eagle’s Medium (MEM; Cat#: 10-010-CVR,
Corning), and supplements including penicillin and
streptomycin (Cat#: 15140-122, Gibco, Waltham, MA,
USA) were purchased from Life Science Technologies
(Rockville, MD, USA). Antibodies for phospho-FGFR3
(Cat#: SC-33041), FGFR3 (Cat#: SC-13121), p-RSK2 T577
(Cat#: SC-16407, SC-377501), RSK2 (Cat#: SC-9986), and
β-actin (Cat#: SC-69879) were purchased from Santa
Lee et al. Cell Death and Disease (2018)9:401
Fig. 5 (See legend on next page.)
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Lee et al. Cell Death and Disease (2018)9:401
Page 12 of 21
(see figure on previous page)
Fig. 5 Kaempferol inhibits RA development in the collage-induced arthritis mouse model. a, b Inhibitory effects of kaempferol on Th17 lineage
differentiation and inflammatory cytokine production were analyzed using mouse splenocytes. The inhibitory effects of kaempferol on Th17polarized T-cell differentiation (a), secretion of IL-17 (b, left graph), IL-21 (b, middle graph), and TNF-α (b, right graph) were determined by counting
IL-17+/CD4+-expressing T-cell cells using flow cytometry (a) and sandwich ELISA (b), respectively. Data were obtained from three independent
experiments, and values are represented as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 vs. untreated cells. c Kaempferol effects on RA
development in CIA mice were analyzed as described in the Materials and Methods. RA incidence (top graph) and clinical arthritis severity (bottom
graph) were obtained from CIA + vehicle control group (n = 10) and CIA + kaempferol group (n = 10). Values are represented as means ± SD. Each
point of the CIA + kaempferol groups was compared with the corresponding CIA + vehicle control group. *p < 0.05. d Representative photographs
of the effects of kaempferol on RA development in CIA mice. Tissue specimens obtained from the hind paw joints of each group of mice (each n = 5)
(c) at the end point of the experiment were analyzed by staining with H&E, safranin O, and TRAP, respectively. Inflammation, cartilage, bone damage,
and TRAP+ osteoclasts were quantified as described in the Materials and Methods. Data were obtained from each group (CIA + vehicle, n = 5; CIA +
kaempferol, n = 5), and values are represented as means ± SEM. **p < 0.001. e Inhibitory effects of kaempferol on the mRNA expression of genes
involved in Th17 differentiation by real-time PCR using draining lymph node cells of CIA + vehicle and CIA + kaempferol mice, as indicated. f
Inhibitory effects of kaempferol on the activated signaling proteins involved in the differentiation of Th17 cells were measured by
immunohistofluorescence assay using the spleens of CIA + vehicle and CIA + kaempferol mice, as indicated. The spleen tissue specimens were costained as indicated, and the positive cells were counted using randomly photographed confocal images obtained from three different areas. e, f
Data were obtained from each mouse group (CIA + vehicle, n = 3; CIA + kaempferol, n = 3), and values are represented as means ± SEM. *p < 0.05;
**p < 0.01; ***p < 0.001
Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies
for phospho-RSK T359/S363 (Cat#: 9344), phosphoERK1/2 (Cat#: 9106), ERK1/2 (Cat#: 9102), and Ki-67
(Cat#: 9027) were purchased from Cell Signaling Technology (Beverly, MA, USA) for the western blot analysis,
immunohistochemistry, and immunocytofluorescence.
Fetal bovine serum (Cat#: 26140-079, Gibco) was purchased from Life Science Technologies and heat inactivated before utilization. Human recombinant bFGF (Cat#:
SRP4037, Sigma-Aldrich), dimethylsulfoxide (DMSO,
Cat#: D8418, Sigma-Aldrich), and kaempferol (Cat#:
ALX-385-005, Enzo, Farmingdale, NY, USA) were purchased from Sigma-Aldrich.
Computational docking of kaempferol
Computational docking was performed to investigate
the binding mode for the crucial functional groups of
kaempferol and receptor tyrosine kinases, including the
FGFR family. All crystal structures of the kinase domains
of the receptor tyrosine kinases were obtained from the
Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). The crystal structure was prepared using the
Protein Preparation Wizard in Maestro v9.2. Hydrogen
was added consistent with a pH of 7.0., and all water
molecules were removed. The structure was then minimized with a root-mean-square deviation cutoff value of
0.3 Å. The program Glide v5.7, which approximated a
complete systematic search of the conformational,
orientational, and positional space of the docked ligand,
was used for ligand docking. The receptor grid was created with the centroid of the crystal ligand as the center of
the grid. Docked ligands were treated flexibly while the
kinase domain of receptor tyrosine kinase was held rigidly
in the docking procedure. Flexible docking was performed
using the standard precision mode. The number of poses
Official journal of the Cell Death Differentiation Association
per ligand was set to 10 in the post-docking minimization,
and the best pose (with the lowest energy) was the output.
The other parameters were kept at the default values.
Cell culture
RSK2+/+ and RSK2−/− MEFs (generously gifted from
Dr. J.C. Brunung, Institute for Genetics, Center for
Molecular Medicine Cologne, Cologne, Germany) were
cultured in DMEM supplemented with 10% fetal bovine
serum (FBS) and antibiotics at 37 °C in a 5% CO2 incubator. MH7A, a human RA synovial cell line obtained
from the Riken cell bank (Ibaraki, Japan) through Dr. EunHee Moon, Department of Bioscience and Biotechnology,
Sejong University (Seoul Korea), were cultured in RPMI
1640 (Cat#: 10-040-CVR, Corning) supplemented with
10% FBS, penicillin (final concentration, 100 U/ml),
streptomycin (P/S, final concentration, 0.1 mg/ml), and Lglutamine at 37 °C in a 5% CO2 incubator. The primary
FLS cells obtained from Bucheon St. Mary’s Hospital were
treated with 2 μg/ml of puromycin for 3 days to eliminate
the non-infected cells.
RSK2 knockdown in primary human FLSs
To establish the RSK2 knockdown primary human
FLSs, we first produced Lenti-sh-RSK2 viral particles in
HEK 293 T-cells purchased from the American Type
Culture Collection. The HEK293T cells were transfected
with pLenti-sh-RSK2 (Dharmacon, Lafayette, CO, USA)
and packing plasmids (psPAX2 and pMD2.G from
Addgene, Cambridge, MA, USA) according to the manufacturer’s suggested protocols. The medium containing
viral particles was collected at 24 h and 48 h after transfection, filtered with 0.45 μM filters (Cat#723-2545,
Thermo Fisher Scientific, Waltham, MA, USA), and used
to infect human FLSs with 4 μg/ml of polybrene. After 48
Lee et al. Cell Death and Disease (2018)9:401
Fig. 6 (See legend on next page.)
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Lee et al. Cell Death and Disease (2018)9:401
Fig. 6 (See legend on next page.)
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Lee et al. Cell Death and Disease (2018)9:401
Page 15 of 21
Fig. 6 Kaempferol inhibits osteoclast differentiation. a Inhibitory effects of kaempferol on ex vivo osteoclast formation. The BMMs obtained from
CIA + vehicle and CIA + kaempferol mice were analyzed in terms of the osteoclast formation induced by M-CSF or M-CSF + RANKL. TRAP+
osteoclasts (≥3 nuclei/TRAP+ cell) were counted. Photographs (×100) are representative of TRAP staining obtained from each mouse group (CIA +
vehicle, n = 3; CIA + kaempferol, n = 3), and values obtained from the whole well of a 48-well plate are presented as means ± SEM. *p < 0.05. b
Inhibitory effects of kaempferol on in vitro osteoclast formation. Naïve murine BMMs were subjected to osteoclast differentiation by combinational
stimulation of kaempferol, M-CSF, and RANKL as indicated. TRAP+ osteoclasts ( ≥ 3 nuclei/TRAP+ cell) were counted. Photographs (×100) are
representative of TRAP staining obtained from three independent experiments, and values obtained from the whole well of a 48-well plate are
represented as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. c Inhibitory effects of kaempferol on osteoclast-specific genes. Naïve murine BMMs
stimulated with M-CSF/RANKL and indicated doses of kaempferol for 4 days, and mRNA levels of indicated osteoclast-specific genes were measured
by real-time PCR. Data were obtained from three independent experiments, and values are represented as means ± SEM. **p < 0.01; ***p < 0.001. d
Representative photographs of morphological osteoclast analysis. The indicated area shows a multinucleated giant osteoclast cell body after
treatment with kaempferol and M-CSF/RANL, as indicated. Scale bars, 40 μm. e Inhibitory effects of kaempferol on osteoclast differentiation. The total
nuclear number of multinucleated (≥3 nuclei) giant cells with the phenotypic features of osteoclasts and the number of cells with a single nucleus
were counted. Data were obtained from three independent experiments using a four-chamber slide, and values are represented as means ± SEM. *p
< 0.05; **p < 0.01; ***p < 0.001. f Schematic of the signaling pathway targeted by kaempferol for the inhibition of osteoclast differentiation. bFGFFGFR3 interaction transduces activation signaling to RSK2, resulting in hyperplasia by the induction of inflammation, FLS proliferation, and cell
migration through NF-κB and AP-1. Eventually, the macrophages in synovium differentiate to bone absorbing osteoclasts. Thus, the dual targeting of
kaempferol on both FGFR3 and RSK2 may prevent RA in humans
h incubation, non-infected primary FLSs were eliminated
by treatment of 2 μg/ml of puromycin (Cat#A111308,
Thermo Fisher Scientific) for 3 days. The survived cells
were immediately utilized to the cell proliferation and cell
migration assays.
nm. The inhibition of cell proliferation by kaempferol was
evaluated by comparing the absorbance of the samples to
a vehicle (DMSO)-treated control group over 96 h at 24 h
intervals.
Th17 cell differentiation and flow cytometry analysis
Western blotting
Each equal amount of protein was resolved by 8–10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene
difluoride (PVDF, Merck Millipore Ltd, Burlington, MA,
USA) membranes. The membranes were then incubated
in a blocking buffer containing 5% skim milk/1 × PBS
and probed with specific antibodies as indicated,
respectively. The proteins were visualized by an
enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using a Chemidoc XRS imager system (Bio-Rad Laboratories,
Hercules, CA, USA).
MTS assay
To measure cell proliferation, MH7A (9 × 103 cells/
cm2), primary FLSs (9 × 103 cells/cm2), or RSK2 knockdown FLSs (9 × 103 cells/cm2) were seeded into 96-well
plates in 100 μl of cell culture medium and incubated for
2 h at 37 °C in a 5% CO2 incubator. At 0 h, the absorbance
was measured at optical densities of 492 nm and 690 nm
using
the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS)-based CellTiter 96® Aqueous One Solution
according to the manufacturer’s instructions (Promega,
Madison, WI, USA). Briefly, 20 μl of the MTS solution
was added to the well, followed by incubation for 1 h at 37
°C in a 5% CO2 incubator. The reaction was stopped by
adding 25 μl of 10% SDS solution to each well, and the
absorbance was measured immediately at 492 and 690
Official journal of the Cell Death Differentiation Association
To measure the cell-cycle, FLS (2.5 × 103 cells/cm2)
were seeded into 60-mm-diameter dishes and cultured
overnight at 37 °C in a 5% CO2 incubator. To examine the
cell-cycle transition by bFGF stimulation, primary FLS
obtained from RA patients were treated with the indicated
doses of bFGF for 24 h in complete cell culture medium.
The cells were trypsinized, fixed, and then stained with
propidium iodide (20 μg/ml) for 15 min at 4 °C. The cellcycle distribution was measured by flow cytometry (BD
FACSCalibur™ flow cytometer, Franklin Lakes, NJ, USA).
To measure the effects of kaempferol on Th17 differentiation, splenic CD4 + T-cells were stimulated with
plate-bound anti-CD3 mAb (0.5 μg/ml; BD Biosciences,
San Jose, CA), soluble anti-CD28 mAb (1 μg/ml; BD
Biosciences), anti-IFN-γ Ab (2 μg/ml; R&D Systems,
Minneapolis, MN), anti-IL-4 Ab (2 μg/ml; R&D Systems),
recombinant TGF-β (2 ng/ml; R&D Systems), and
recombinant IL-6 (20 ng/ml; R&D Systems) for 3 days.
The cells were treated with an indicated dose of kaempferol for 3 days. Cells were stained with various combinations of fluorescent antibodies against CD4 and IL-17
(eBioscience, San Diego, CA, USA). Prior to intracellular
staining, the cells were restimulated for 4 h with phorbol
myristate12 acetate (25 ng/ml) and ionomycin (250 ng/ml)
in the presence of GolgiStop (BD Bioscience). The cells
were permeabilized and fixed Cytofix/Cytoperm (BD
Bioscience), as per the manufacturer’s instructions and
further stained with anti-IL-17 or anti-Foxp3 Flow cytometry was conducted on FACSCalibur flow cytometer
(BD Biosciences).
Lee et al. Cell Death and Disease (2018)9:401
Wound healing assay
MH7A (3 × 105 cells/cm2), RSK2+/+ (3 × 105 cells/cm2),
RSK2−/− MEFs (3 × 105 cells/cm2), and FLSs (3 × 105
cells/cm2) were seeded into culture inserts (Cat#: 80209,
Ibidi GmbH, Martinsried, Germany), cultured, and
starved for 24 h. Next, the culture inserts were removed
after 2 h of mitomycin-C treatment to stop cell proliferation. The cells were pretreated with the indicated
doses of kaempferol for 30 min and co-treated with 1 or
10 ng/ml of bFGF in addition to the indicated doses of
kaempferol. Representative images (×40) of cell migration
for wound healing were captured at 0, 6, and 24 h using an
ECLIPSE Ti inverted fluorescence microscope (NIKON
Instruments Korea, Gangnam, Seoul, Korea). The migrated area was measured using Image J (NIH Image J Ver.
1.6, Bethesda, MD, USA).
Reporter gene assay
RSK2+/+ (5 × 103 cells/cm2) and RSK2−/− (5 × 103 cells/
cm2) MEFs were transiently transfected with 400 ng each
of a pAP-1-luciferase, pNF-κB-luciferase, or pCOX-2promoter luciferase reporter plasmid with 20 pg of the
phRL-SV40 Renilla luciferase reporter plasmid in 24-well
plates. The cells were cultured for 24 h and then starved in
serum-free medium for 16 h. The cells were pretreated
with the indicated doses of kaempferol for 30 min, and
then co-treated with bFGF (10 ng/ml) and the indicated
doses of magnolin for 24 h. The cells were disrupted, and
firefly luciferase activity was measured using a VICTOR
X3 (PerkinElmer, Waltham, MA, USA). Firefly luciferase
activity was normalized by Renilla luciferase activity to
equalize the transfection efficiency.
Gelatin zymography
MMP-2 and MMP-9 activities were evaluated by gelatin
zymography using the cell culture supernatants. Briefly,
FLSs (2 × 104cells/cm2) were seeded into 60-mm-dishes,
cultured, and treated with the indicated doses of bFGF
and kaempferol for 24 h. The culture supernatants were
then harvested, and 20 μg of protein from each sample
was loaded on a polyacrylamide gel containing 0.2%
gelatin. The gel was washed by 2.5% Triton X-100 buffer
for 20 min, and then incubated for 24 h at 37 °C in a
renaturing buffer (50 mM Tris-HCl, pH 7.5, 10 mM
CaCl2, 1 μM ZnCl2, 0.01% NaN3). The gels were stained
with Coommassie Brilliant Blue and destained in methanol/acetic acid.
Pull-down assay
CNBr-activated sepharose 4B beads (Cat#: 71-7086-00
AF, GE Healthcare, Little Chalfont, 9NA, UK) were activated according to the manufacturer’s suggested protocol.
Briefly, CNBr-sepharose 4B beads (0.3 g) were suspended
in a final concentration of 1 mM HCl (30 ml) for 5 min by
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Page 16 of 21
rotation, and then washed with 30 ml of 1 mM HCl three
times. Kaempferol (40 mM) dissolved in 100% DMSO was
combined with coupling buffer (0.1 M of NaHCO3, pH
8.3, containing 0.5 M of NaCl) to achieve the final concentration of 10 μM. The activated CNBr-sepharose beads
and kaempferol were mixed for 1 h at room temperature,
and excess kaempferol was washed with at least five
volumes of coupling buffer. The remaining active groups
of CNBr-beads were blocked by adding the blocking
buffer (0.1 M of Tris-HCl, pH 8.0) for 2 h standing. The
CNBr-kaempferol beads (50% slurry) were aliquoted and
stored at 4 °C until utilized. The binding between FGFR3
and kaempferol was examined by affinity chromatography. The kinase domain of active FGFR3 (100 ng) or
the total membrane fraction protein (500 μg) was incubated with 30 μL of CNBr-kaempferol beads (50% slurry)
for 2 h or overnight at 4 °C. The beads were washed three
times and suspended in 20 μL of 1 × SDS sample buffer.
Bound proteins were resolved by 10% SDS-PAGE and
visualized by western blot using total-FGFR specific
antibodies and horseradish peroxidase (HRP)-conjugated
secondary antibodies.
Kaempferol/ATP competition assay
Active FGFR3 (100 ng) and each indicated dose of
kaempferol were combined and preincubated in binding
buffer (10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 2
mM DTT, 0.01% NP-40) overnight at 4 °C, and 25 μl of
ATP-agarose or control beads (50% slurry, Innova
Bioscience, Cambridge, UK) was added and incubated for
2 h at 4 °C. The beads were then washed with binding
buffer and mixed with 20 μl of SDS sample buffer, and
FGFR3 bound ATP-agarose beads were then visualized by
western blotting using total-FGFR3-specific antibodies
and HRP-conjugated secondary antibodies. Band intensity
was measured using a densitometry computer program
(NIH Image J).
Immunocytofluorescence assay
MH7A cells (1 × 104 cells/cm2) were seeded into
four-chamber culture slides and cultured for 12 h. The
cells were then starved with FBS-free MEM for 16 h,
pretreated with the indicated doses of kaempferol,
U0126, and PKC412 for 30 min, and then co-treated
with bFGF (10 ng/ml). The cells were fixed with 4%
formalin, permeabilized with 0.5% Triton X-100/1 ×
PBS, blocked with 1% of BSA, and hybridized with antirabbit phospho-FGFR3 Tyr724-specific antibodies
overnight at 4 °C in a humidified chamber. The FGFR3
proteins were visualized by hybridization with secondary antibodies conjugated with anti-rabbit-Alexa-568
(Cat#: A11036, Thermo Fisher Scientific) under an
ECLIPSE Ti inverted fluorescence microscope (NIKON
Instruments Korea).
Lee et al. Cell Death and Disease (2018)9:401
Page 17 of 21
Table 1 Primer list to be utilized for quantitative real-time PCR
IL-17
sense
5′-ACC TCA CAC GAG GCA CAA GT-3′
antisense
5′-CCC AAC AGC TGG AAT AGA GC-3′
sense
5′- AGC AGC TGT GTC AGA TGG TG-3′
antisense
5′-CTG AGC AGT CCC CTG TAA GC-3′
sense
5′-CAG CTG TTG CCT CTC GTA CA-3′
antisense
5′-CAC CCA GTT CTG CTT TGG AT-3′
sense
5′-TGT CCT GGG CTA CCC TAC TG-3′
antisense
5′-GTG CAG GAG TAG GCC ACA TT-3′
TRAP
sense
5′-CTG TGG GCT TTA AGG ACA GC-3′
antisense
5′-ACA TAG CCC ACA CCG TTC TC-3′
Integrin β3
sense
5′-CTG TGG GCT TTA AGG ACA GC-3′
antisense
5′-GAG GGT CGG TAA TCC TC-3′
sense
5′-CTG TCC AGA CCA AGG GTA CAG CCT-3′
antisense
5′-GAG GTA TAG TGG GAC ACA TAG TGG-3′
sense
5′-CGG ACT TTG ACA CAG CAG AA-3′
antisense
5′-AGC AGC AAT CGA CAA GGA GT-3′
sense
5′-CAG CAG AGG TGT GTA CTA TG-3′
antisense
5′-GCG TTG TTC TTA CGA GC-3′
sense
5′-GCA GAA AGT CAT GAA CCA CG-3′
antisense
5′-TCG CAA CCA GTC AAG TTC TC-3′
Ahr
CCL20
ROR-yt
MMP9
Calcitonin receptor
Cathepsin K
c-Jun
p50 (a component of NF-κB)
β-actin
sense
5′-GTC TCT GGG GGT ACC ATC AAA G-3′
antisense
5′-AGG ATG TCT CCA CAC CAC TGT-3′
sense
5′-GAA ATC GTG CGT GAC ATC AAA G-3′
antisense
5′-TGT AGT TTC ATG GAT GCC ACA G-3′
Approval statements of human subjects and animal
studies
Human subject experiments using synovial fluids were
obtaind from 31 OA patients (27 women and four men
met the ACR criteria)35 and 79 RA patients (63 women
and 16 men met the ACR criteria)36 who visited the
outpatient department at the Division of Rheumatology,
Bucheon St. Mary’s Hospital. WBC and neutrophil counts
in the SF of OA and RA patients were calculated using a
Neubauer hemocytometer and Wright’s stain, respectively. The amount of bFGF in SF was measured using an
ELISA kit (R&D Systems, Minneapolis, MN, USA)
according to the manufacturer’s instructions. SF samples
were stored at –80 °C until analysis. RA and OA synovial
tissues were obtained from patients with RA or OA
undergoing total joint replacement surgery. RA FLSs were
isolated by enzymatic digestion of synovial tissues
obtained from RA patients undergoing total knee replacement surgery. Human experiments were approved by
the Institutional Review Board (IRB) of human subjects at
Official journal of the Cell Death Differentiation Association
Bucheon St. Mary’s Hospital (approval number:
HC14TISI0070), The Catholic University of Korea, and
conducted in accordance with IRB guidelines and regulations. All patients were informed and gave their written
consent, and the study was performed in accordance with
the Helsinki II Declaration. Synovium samples were fixed
in 4% paraformaldehyde solution overnight at 4 °C,
dehydrated with alcohol, washed, embedded in paraffin,
and sectioned into thick slices. On the other hand, all of
the animal experiments, including CIA induction and
kaempferol administration, were approved by the Institutional Animal Care and Use Committee (IACUC) at the
Catholic University of Korea (approval number: 20130020-01) under specific pathogen-free conditions in
accordance with IACUC guidelines and regulations.
Measurement of bFGF in synovial fluids of OA and RA
patients
The concentration of bFGF in the synovial fluid of each
RA and OA patients were measured using a commercial
Lee et al. Cell Death and Disease (2018)9:401
sandwich ELISA kit (Cat#: DFB50, R&D Systems). To
eliminate the influence of rheumatoid factor on the
cytokine level in ELISA, all synovial fluids were precleared
using a commercial reagent to block heterophilic antibodies (Cat#: 500-11-001, HeteroBlock; Omega Biologicals Inc., Bozeman, MT, USA).
Culture of human FLSs
Synoviocytes were isolated by enzymatic digestion of
synovial tissue specimens. The tissue samples were
minced into 2–3 mm pieces and treated for 4 h with 4 mg/
ml type II collagenase (Worthington, Freehold, NJ) in
DMEM at 37 °C in 5% CO2. Dissociated cells were centrifuged at 500× g, resuspended in DMEM supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 100 units/
ml penicillin, and 100 ng/ml streptomycin, plated in 75cm2 flasks, and incubated overnight. The non-adherent
cells were then removed, and the adherent cells were
cultivated in DMEM supplemented with 10% fetal calf
serum. Synoviocytes from passages 4–8 were used in each
experiment. The cells were morphologically homogeneous and exhibited the appearance of synovial fibroblasts, with typical bipolar configuration under inverse
microscopy. The cells were seeded in six- or 24-well
plates, eight-well chamber slides, or 100-mm culture
dishes supplemented with 10% fetal bovine serum.
Immunohistofluorescence assays
To determine the expression of phospho-RSK2 and
phopsho-FGFR3 in synovial tissues from RA and OA
patients, the paraffin-invaded human synovial slices (5μm) were deparaffinized by incubation at 60 °C for 2 h.
The deparaffinized slides were rehydrated, unmasked by
soaking in boiling 10 mM sodium citrate buffer (pH 6.0)
for 10 min, and allowed to cool to room temperature
gradually or treated with pepsin for 30 min. The slides
were then blocked with 5% goat serum in 1 × PBX/0.5%
Triton X-100 for 1 h at RT, and then hybridized with the
indicated antibodies, phospho-FGFR3 (Y724) (1:50),
phospho-RSK2 (T577) (1:50), CD68 (1:50) and CD4
(1:50), in 1 × PBS/0.5% Triton X-100 buffer overnight at 4
°C. The slides were washed and hybridized with secondary
antibodies conjugated with Alexa-488 (Cat#: A11055, BioRad Laboratories), −568 (Cat#: A11036, Bio-Rad
Laboratories), or −647 (Cat#: A21235, Bio-Rad Laboratories) for 2 h at RT in the dark as indicated. Image stacks
were captured using laser scanning confocal microscopy
(LSM 710, Carl Zeiss Korea Co. Ltd., Seoul, Korea). To
analyze the effects of kaempferol on signaling pathways
for immune responsiveness in CIA-kaempferol treatment,
the spleen tissue was snap-frozen in liquid nitrogen and
stored at −70 °C. The spleen tissue sections (5-μm) were
fixed in acetone and co-hybridized with a specific anti-
Official journal of the Cell Death Differentiation Association
Page 18 of 21
body using Fluorescein isothiocyanate-labeled anti-CD4
(Cat#: 553046, BD Biosciences, San Jose, CA, USA) and
phycoerythrin (PE)-labeled anti-IL-17 (Cat#: 559502, BD
Biosciences), PE-labeled anti-Src antibodies (Cat#:
ab47405, Abcam, Cambridge, UK), PE-labeled antiphospho-STAT3-Y705 (Cat#: 612569, BD Biosciences),
or -S727 antibodies (Cat#: 558557, BD Biosciences).
Image stacks were captured using laser scanning confocal
microscopy (LSM 710, Carl Zeiss Korea Co. Ltd.). Positive
cells were counted manually at a higher magnification
(projected on a screen) by four individuals, and the results
were expressed as means ± standard deviation.
Double immunohistochemistry of human RA tissues
To determine cell types encountering phospho-RSK2 in
RA tissues in RA patients, the RA tissues were fixed in
formalin and embedded in paraffin, and then slices with 3μm paraffin sections were prepared. The sections were
deparaffinized, and then pretreated with cell conditioning
solution (CC1, Ventana, Tucson, AZ, USA) and UV
irradiation to abrogate the endogenous hydroperoxidase
activity. First, the sections were hybridized with primary
antibodies, including CD3 (Cat#: A0452, 1:100 dilution,
DAKO Korea, Sonpa-gu, Seoul, Korea), CD20 (Cat#:
M0755, 1:100 dilution, DAKO), and CD68 (Cat#: M0814,
1:100 dilution, DAKO), as indicated for 32 min and with
AP-conjugated secondary antibody for 8 min. The proteins were visualized using an Ultra View Universal Red
detection kit (Cat#: 760-501, Ventana). Second, the sections were hybridized with phospho-RSK2-Thr577 (Cat#:
sc16407, 1:100 dilution, Santa Cruz Biotechnology) for 32
min with HRP-conjugated secondary antibody for 8 min.
RSK2 proteins were visualized by colorimetric detection
using 3,3-diaminobenzidine with H2O2. Finally, the sections were counterstained with Hematoxylin II (Ventana)
for 4 min and Bluing Reagent (Ventana) for 4 min. The
sections were observed under light microscope (BX50,
Olympus, Japan).
Kaempferol effects on the IL-17, IL-21 and TNF-α
production in differentiated Th17 cells
The examine the effect of kaempferol on the IL-17, IL21, and TNF-α production in differentiated T-cells, culture supernatants were obtained from Th17-polarized
differentiated T-cells by the treatment of a indicated dose
of kaempferol. The levels of IL-17, IL-21, and TNF-α in
the supernatants of murine splenocyte cultures were
measured sandwich enzyme-linked immunosorbent assay
(Cat#:MAB721, Cat#:841338, AF-410-NA, R&D Systems,
respectively). Horseradish peroxidase-avidin (R&D Systems) was used for color development Absorbance was
measured at 405 nm on an ELISA microplate reader
(Molecular Devices, Sunnyvale, CA, USA).
Lee et al. Cell Death and Disease (2018)9:401
Kaempferol effects on arthritis inhibition in CIA mouse
model
DBA/J1 mice (20 male, 6 weeks old) purchased from
Orient Bio Inc. (Guro-gu, Seoul, Korea) were acclimated
for 1 week with on a 12-h dark/light cycle and allowed
food and water ad libitum. To induce arthritis, DBA/1 J
mice were intradermally injected at the base of the tail
with 100 μg of chicken CII emulsified in complete
Freund’s adjuvant (1:1 w/v; Chondrex, Redmond, WA,
USA) and boosted intradermally 14 days later. Arthritic
score measurements were performed as follows: 0 = no
joint swelling; 1 = slight edema and erythema limited to
the foot or ankle; 2 = slight edema and erythema from the
ankle to the tarsal bone; 3 = moderate edema and erythema from the ankle to the tarsal bone; and 4 = edema
and erythema extending from the ankle to the entire leg,
with severe swelling of the wrist or ankle. The final
arthritis score was calculated as the sum of scores from all
four legs, which were assessed by three independent
observers with no knowledge of the experimental groups.
The mice were randomly divided two groups (vehicle and
kaempferol injection groups). Kaempferol (2 mg/kg) dissolved in 10% DMSO was administered through i.p.
injection three times a week after induction of arthritis for
next 70 days. The onset and severity of arthritis were
determined by three independent observers, based on the
previously described scoring system49. The mice were
observed twice a week for the onset and severity of joint
inflammation for up to end point (day 84) after the initial
immunization. Before being sacrificed at 84 days after CIA
induction by cervical dislocation, the mice were anesthetized using 2–3% isoflurane. At end point, the mice were
scarified and hind joint tissues, spleen, and tibias and
femurs were harvested for the further studies. The hind
joint tissues from CIA-vehicle and CIA-kaempferol
groups were subject to histopathological examination
such as scoring of inflammation, destruction of cartilage,
and bone damage according to published criteria50, 51 by
Hematoxylin-Eosin (H&E) staining and safranin O
staining.
Histopathological analysis of mouse arthritic joint tissues
The ankle joint specimens (5 μm) obtained from CIA
and CIA-kaempferol administered mice were fixed with
4% paraformaldehyde, decalcified in a histological decalcifying agent (Calci-Clear Rapid, Cat#: HS105, National
Diagnostics-Chayon Laboratories, Gangnam-gu, Seoul,
Korea), and embedded in paraffin. The tissue slices were
then stained with H&E and safranin O to detect proteoglycans in the joint tissues. The H&E-stained tissue
slices were scored for inflammation and bone erosion as
previously reported33, 51, and cartilage damage was
determined using safranin O staining. The extent of cartilage damage was scored as described previously33, 51.
Official journal of the Cell Death Differentiation Association
Page 19 of 21
Mouse joint tissue was fixed in 10% formalin and decalcified in EDTA bone decalcifier, and ankle joints were
processed for paraffin embedding, from which 7-μm-thick
tissue sections were prepared. Sections were stained for
TRAP using the Leukocyte Acid Phosphatase kit (SigmaAldrich) according to the manufacturer’s protocol. TRAP
+ multinucleated cells with ≥3 nuclei were counted as
osteoclasts. All histological assessments were performed
by two independent blinded observers.
Ex vivo and in vitro osteoclastogenesis
For the ex vivo osteoclastogenesis experiment, BMMs
obtained from CIA-vehicle and CIA-kaempferol mice
were isolated from the tibias and femurs of the mice by
flushing the bone marrow cavity with α-minimum
essential medium (α-MEM, Cat#: LM008-01, Wel gene,
Gyeongsangbuk-do, Gyeongsna-si, Korea). The cells were
centrifuged, red blood cells were removed using the ACK
buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM
Na2EDTA), and the cells were then plated in six-well
plates (Cat#: 140675, Thermo Scientific Inc., MA, USA) in
α-MEM for 12 h. The floating cells were collected, seeded
in 48-well plates (1 × 105 cells/cm2) or four-chamber
slides (1 × 105 cells/cm2, Cat#: T460-27, Waltham, MA,
USA), and cultured in α-MEM supplemented with 10 ng/
ml of M-CSF (R&D Systems) for 3 days for differentiation
into macrophage-like osteoclast precursor cells. The nonadherent cells were washed out, and the remaining
osteoclast precursor cells were cultured in α-MEM supplemented with 10 ng/ml of M-CSF and 50 ng/ml of
RANKL (PeproTech, Rocky Hill, NJ, USA) for 4 days to
generate osteoclasts. The differentiated osteoclasts were
visualized by TRAP staining. For in vitro osteoclastogenesis, non-treated DBA/J1 mice were utilized to isolate
and induce the osteoclastogenesis as described above. The
osteoclast precursor cells were cultured in α-MEM supplemented with 10 ng/ml of M-CSF and 50 ng/ml of
RANKL either with or without kaempferol (10 μM) for
4 days to generate osteoclasts. The differentiated osteoclasts were visualized by TRAP staining. The tibias and
femurs were examined to determine the ex vivo osteoclastogenesis induced by the macrophage-colony stimulating factor (M-CSF; Cat#: 300-25, PeproTech)/receptor
activator of the NF-κB ligand (RANKL; Cat#: 310-01 C,
PeproTech).
Quantitative real-time polymerase chain reaction (PCR)
Total RNA was extracted using TRIzol Reagent
(Molecular Research Center, Cincinnati, OH, USA)
according to the manufacturer’s suggested protocol.
Complementary DNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied
Science, Cat# 04 896 866 001, Mannheim, Germany)) and
random hexamer primers. A Light-Cycler 2.0 instrument
Lee et al. Cell Death and Disease (2018)9:401
(software version 4.0; Roche Diagnostics) was used for
PCR amplification. All reactions were performed using
LightCycler FastStart DNA Master SYBR Green I mix
(Takara) following the manufacturer's instructions. The
primers for the quantitative real-time PCR were summarized in Table 1.
Statistical analysis
Data are presented as the mean ± SEM. The
Mann–Whitney U test or Student's t-test was used for
comparing values between two groups. One-way analysis
of variance followed by Bonferroni’s post hoc test was
used to compare the differences between three or more
groups. To assess the Gaussian distribution and the
equality of variance, the Shapiro–Wilk test and Levene
test were used, respectively. Differences between arthritis
incidences at a given time point were analyzed by the χ2
contingency analysis. The program used for the statistical
analysis was the SPSS statistical software package, standard version 16.0 (SPSS, Chicago, IL, USA). P-values <
0.05 (two-tailed) were considered significant.
Acknowledgements
This study was supported by the Research Fund of The Catholic University of
Korea (M-2017-B0002-00127), the Ministry of Science, ICT and Future Planning
(NRF-2017R1A2B2002012, -2017M3A9F5028608, and -2017R1A4A1015036), the
Ministry of Education (BK21PLUS grant NRF-22A20130012250) and by the
Institute of Clinical Medicine Research, Bucheon St. Mary’s Hospital Research
Fund (BCMC13IA01).
Author details
Integrated Research Institute of Pharmaceutical Sciences & BK21 PLUS Team
for Creative Leader Program for Pharmacomics-based Future Pharmacy,
College of Pharmacy, The Catholic University of Korea, 43, Jibong-ro, Wonmigu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea. 2Department of
Internal Medicine, College of Medicine, Division for Rheumatology, The
Catholic University of Korea, 505, Banpo-dong, Seocho-gu, Seoul 137-701,
Republic of Korea. 3The Rheumatism Research Center, Catholic Research
Institute of Medical Science, The Catholic University of Korea, 505, Banpo-dong,
Seocho-gu, Seoul 137-701, Republic of Korea. 4Division of Immunology,
Beckman Research Institute of the City of Hope, 1500, E. Duarte Rd, Duarte, CA
91010, USA. 5China-US(Henan) Hormel Cancer Institute, No. 127, Dongming
Road, Jinshui District, Zhengzhou 450008 Henan, China. 6Clinical Medicine
Research Institute of Bucheon St. Mary’s Hospital, The Catholic University of
Korea, 327, Sosa-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-717, Republic of
Korea
1
Author contributions
C-J.L., S-J.M., J-H.J., M-H.L., H.S.L., and S-M.Y. performed experiments of FLS
culture, cell proliferation assay, knockdown of RSK2, western blotting,
immunohistochemistry, and immunohistofluorescence. W.S.L.. and H-J.L.
collected clinical samples and analyzed bFGF levels. S.L. conducted
computational docking of kaempferol. J-H.J., E-K.K., J.Y.J., and M-L.C. performed
CIA-induced RA experiments. H.C.K. and J.Y.L. performed BMM collection and
wound healing. S-J.M., J-K.M., and Y-Y.C. performed experimental design,
analyzed data, and wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Official journal of the Cell Death Differentiation Association
Page 20 of 21
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41419-018-0433-0).
Received: 11 October 2017 Revised: 22 February 2018 Accepted: 22
February 2018
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