# (-)-Epigallocatechin Gallate Reduces  Platelet-Derived Growth Factor-BB-Stimulated Interleukin-6  Synthesis in Osteoblasts: Suppression of SAPK/JNK

Authors: Shinji Takai; Rie Matsushima-Nishiwaki; Seiji Adachi; Hideo Natsume; Chiho Minamitani; Jun Mizutani; Takanobu Otsuka; Haruhiko Tokuda; Osamu Kozawa
Journal: Mediators of Inflammation
 (2008)

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## Abstract

We  previously showed that the mitogen-activated protein (MAP) kinase  superfamily, p44/p42 MAP kinase, p38 MAP kinase, and stress-activated protein kinase (SAPK)/c-JunN-terminal (JNK), positively plays a part in the platelet-derived growth factor-BB-
(PDGF-BB-) stimulated synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells while Akt and p70 S6 kinase negatively regulates the synthesis. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), one of the major green tea flavonoids, affects the synthesis of IL-6 in these cells and the mechanism. EGCG significantly reduced the IL-6 synthesis and IL-6 mRNA expression stimulated by PDGF-BB, EGCG reduced the PDGF-BB-stimulated IL-6 synthesis also in primary-cultured osteoblasts. EGCG had no effect on the levels of osteocalcin and osteoprotegerin in MC3T3-E1 cells. The PDGF-BB-induced autophosphorylation of PDGF receptor β was not suppressed by EGCG. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was not affected by EGCG. On the other hand, EGCG markedly suppressed the PDGF-BB-induced phosphorylation of SAPK/JNK. Finally, the PDGF-BB-induced phosphorylation of Akt and p70 S6 kinase was not affected by EGCG. These results strongly suggest that EGCG inhibits the PDGF-BB-stimulated synthesis of IL-6 via suppression of SAPK/JNK pathway in osteoblasts.


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## Body

## 1. Introduction

Interleukin-6 (IL-6) is a multifunctional cytokine that has important
physiological effects on a wide range of functions such as promoting B-cell
differentiation, T-cell activation, and inducing acute phase proteins [1–4]. It is generally
recognized that two functional cells, osteoblasts and osteoclasts, strictly
regulate bone metabolism, the former responsible for bone formation and the
latter for bone resorption [5]. The formation of bone structures and bone
remodeling results from the coupling process; bone resorption by activated osteoclasts with
subsequent deposition of new matrix by osteoblasts. In bone metabolism, it is
well recognized that IL-6 is one of the most potent osteoclastogenic factors [3, 4]. Bone resorption is mediated by the increased local production of
inflammatory cytokines such as tumor necrosis factor-α and IL-1. In osteoblasts [6–8], it has been
reported that bone resorptive agents such as tumor necrosis factor-α and IL-1 stimulate the synthesis of IL-6. As
for bone metabolism, IL-6 has been shown to stimulate bone resorption and
induce osteoclast formation [3, 4, 7, 9]. Thus, accumulating evidence indicates
that IL-6 secreted from osteoblasts plays a key role as a downstream effector
of bone resorptive agents. It has been shown that platelet-derived growth
factor-BB (PDGF-BB), a well-known mitogenic factor, increases proliferation and
inhibits the differentiation of osteoblasts [10]. PDGF-BB also enhances bone
resorption by increasing the number of osteoclasts, an effect that may be
secondary to an increase in the expression of IL-6 [10, 11]. Therefore,
modulation of PDGF-BB effect would be a possible therapeutic target of
osteoporosis. In our recent studies [12, 13], we have reported that PDGF-BB
stimulates IL-6 synthesis through p44/p42 MAP kinase, p38 MAP kinase, and
stress-activated protein kinase (SAPK)/c-Jun N-terminal (JNK), members of the MAP kinase superfamily [14], in
osteoblast-like MC3T3-E1 cells and that Akt and p70 S6 kinase limits the
synthesis. However, the exact mechanism of PDGF-BB underlying the IL-6
synthesis in osteoblasts has not yet been elucidated.Compounds
in foods such as vegetables and fruits have beneficial properties to human
being. Among them, it has been reported that flavonoids possess antioxidative,
antibacterial, and antitumor effects [15, 16]. Catechins are one of the major
flavonoids, which are present in various species of plants such as green tea [16].
In bone metabolism, it has been shown that catechin suppresses bone resorption
[17]. As for osteoclasts, it has been reported that (−)-epigallocatechin
gallate (EGCG) induces osteoclast apoptosis [18, 19] and suppresses the
differentiation [20]. However, the effects of EGCG on the expression of
cytokines or matrix-degrading enzyme from osteoclast-stimulated or treated
osteoblastic cells have not been reported as far as we know. As for
osteoblasts, it has been shown that catechin stimulates alkaline phosphatase
activity, a mature osteoblast phenotype [5], and reduces apoptosis in
osteoblast-like MC3T3-E1 cells [21]. However, the exact mechanism of catechin
in osteoblasts is not fully known.In the
present study, we investigated whether (−)-epigallocatechin gallate (EGCG), one
of the major green tea flavonoids, affects the PDGF-BB-stimulated IL-6
synthesis in osteoblast-like MC3T3-E1 cells and the mechanism behind it. We
here show that EGCG reduces the PDGF-BB-stimulated IL-6 synthesis via
attenuation of SAPK/JNK pathway in these cells.


## 2. Materials and Methods

### 2.1. Materials

Recombinant
PDGF-BB, IL-6 ELISA, osteocalcin ELISA, and osteoprotegerin
(OPG)
ELISA kit were
purchased from R&D Systems, Inc. (Minneapolis,
Minn, USA). EGCG was obtained from Calbiochem-Novabiochem
Corp. (La Jolla, Calif, USA). Phospho-specific PDGF receptorβ antibodies, PDGF receptor β antibodies, phospho-specific p44/p42 MAP
kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP
kinase antibodies, p38 MAP kinase antibodies, phospho-specific SAPK/JNK kinase
antibodies, SAPK/JNK antibodies, phospho-specific Akt antibodies, Akt
antibodies, phospho-specific p70 S6 kinase antibodies, and p70 S6 kinase
antibodies were purchased from Cell Signaling Technology (Beverly, Mass, USA). ECL Western
blotting detection system was purchased from Amersham Japan (Tokyo,
Japan). Other
materials and chemicals were obtained from commercial sources.


### 2.2. Cell Culture

The cloned
osteoblast-like MC3T3-E1 cells, which have been derived from newborn mouse
calvaria [22], were maintained as previously described [23]. Briefly, the cells
were cultured in α-minimum essential medium (α-MEM) containing 10% fetal calf serum (FCS) at
37°C in a humidified atmosphere of 5% CO2/95% air. The cells were
seeded into 35 mm diameter dishes (5×104/dish) or 90 mm diameter
dishes (5×105/dish) in α-MEM containing 10% FCS. After 5 days, the
medium was exchanged for α-MEM containing 0.3% FCS. The cells were used
for experiments after 48 hours.Primary-cultured
osteoblasts were obtained from the calvaria of newborn (1 or 2-day old) balb/c
mice as previously described [24]. They were seeded into 90 mm diameter dishes
(25×104 cells) in α-MEM containing 10% FCS. The medium was changed
every 3 days until the cells reached confluence at 5 days. Then, the medium was
exchanged for α-MEM containing 0.3% FCS. The cells were used
for experiments after 48 hours.


### 2.3. Assays for Osteocalcin, OPG, and IL-6

The
cultured cells were pretreated with vehicle or various doses of EGCG (1 to 30μM) for 24 hours, the levels of osteocalcin and OPG in the medium were measured by respective ELISA kit.
The cultured cells were stimulated by PDGF-BB in 1 mL of α-MEM containing 0.3% FCS, and then incubated
for the indicated periods. The conditioned medium was collected, and IL-6 in
the medium was then measured by IL-6 ELISA kit. When indicated, the cells were
pretreated with various doses of EGCG for 60 minutes.


### 2.4. Real-Time RT-PCR

The cultured cells were pretreated with 30μM EGCG or vehicle for 60 minutes, and then
stimulated by 50 ng/mL PDGF-BB for 60 minutes. Total RNA was isolated and
transcribed into complementary DNA using Trizol reagent and Omniscript Reverse
Transcriptase Kit. Real-time RT-PCR was performed using a Light Cycler system (Roche Diagnostics Basel, Switzerland) in capillaries and FastStart DNA Master SYBR Green I
provided with the kit. Sense and antisense primers were synthesized based on
the report of Simpson et al.for mouse GAPDH mRNA [25]. Sense and
antisense primers for mouse IL-6 mRNA were purchased from Takara Bio Inc. (Tokyo, Japan)
(primer set ID:MA039013). The amplified products were determined by melting
curve analysis and agarose electrophoresis. IL-6 mRNA levels were normalized
with those of GAPDH mRNA.


### 2.5. Analysis of Western Blotting

The
cultured cells were stimulated by PDGF-BB inα-MEM containing 0.3% FCS for the indicated
periods. The cells were washed twice with phosphate-buffered saline and then
lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM
Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, and
10% glycerol. The cytosolic fraction was collected as a supernatant after
centrifugation at 125,000×g for 10
minutes at 4°C. SDS-polyacrylamide gel electrophoresis (PAGE) was performed by
Laemmli [26] in 10% polyacrylamide gel. Western blotting analysis was performed
as described previously [27] by using phospho-specific PDGF receptor β antibodies, PDGF receptor β antibodies, phospho-specific p44/p42 MAP
kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP
kinase antibodies, p38 MAP kinase antibodies, phospho-specific SAPK/JNK kinase
antibodies, SAPK/JNK antibodies, phospho-specific Akt antibodies, Akt
antibodies, phospho-specific p70 S6 kinase antibodies, and p70 S6 kinase
antibodies with peroxidase-labeled antibodies raised in goat against rabbit IgG
being used as second antibodies. Peroxidase activity on PVDG membrane was
visualized on X-ray film by means of the ECL Western blotting detection system.
When indicated, the cells were pretreated with various doses of EGCG for 60 minutes.


### 2.6. Determinations

The
absorbance of enzyme immunoassay samples was measured at 450 nm with EL 340 Bio
Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, Vt, USA).
The densitometric analysis was performed using Molecular Analyst/Macintosh (Bio-Rad
Laboratories, Hercules, Calif, USA).


### 2.7. Statistical Analysis

The data
were analyzed by ANOVA followed by the Bonferroni method for multiple
comparisons between pairs, and aP<.05
was considered significant. All data are presented as the mean±SEM of triplicate determinations. Each experiment was repeated three times with
similar results.


## 2.1. Materials

Recombinant
PDGF-BB, IL-6 ELISA, osteocalcin ELISA, and osteoprotegerin
(OPG)
ELISA kit were
purchased from R&D Systems, Inc. (Minneapolis,
Minn, USA). EGCG was obtained from Calbiochem-Novabiochem
Corp. (La Jolla, Calif, USA). Phospho-specific PDGF receptorβ antibodies, PDGF receptor β antibodies, phospho-specific p44/p42 MAP
kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP
kinase antibodies, p38 MAP kinase antibodies, phospho-specific SAPK/JNK kinase
antibodies, SAPK/JNK antibodies, phospho-specific Akt antibodies, Akt
antibodies, phospho-specific p70 S6 kinase antibodies, and p70 S6 kinase
antibodies were purchased from Cell Signaling Technology (Beverly, Mass, USA). ECL Western
blotting detection system was purchased from Amersham Japan (Tokyo,
Japan). Other
materials and chemicals were obtained from commercial sources.


## 2.2. Cell Culture

The cloned
osteoblast-like MC3T3-E1 cells, which have been derived from newborn mouse
calvaria [22], were maintained as previously described [23]. Briefly, the cells
were cultured in α-minimum essential medium (α-MEM) containing 10% fetal calf serum (FCS) at
37°C in a humidified atmosphere of 5% CO2/95% air. The cells were
seeded into 35 mm diameter dishes (5×104/dish) or 90 mm diameter
dishes (5×105/dish) in α-MEM containing 10% FCS. After 5 days, the
medium was exchanged for α-MEM containing 0.3% FCS. The cells were used
for experiments after 48 hours.Primary-cultured
osteoblasts were obtained from the calvaria of newborn (1 or 2-day old) balb/c
mice as previously described [24]. They were seeded into 90 mm diameter dishes
(25×104 cells) in α-MEM containing 10% FCS. The medium was changed
every 3 days until the cells reached confluence at 5 days. Then, the medium was
exchanged for α-MEM containing 0.3% FCS. The cells were used
for experiments after 48 hours.


## 2.3. Assays for Osteocalcin, OPG, and IL-6

The
cultured cells were pretreated with vehicle or various doses of EGCG (1 to 30μM) for 24 hours, the levels of osteocalcin and OPG in the medium were measured by respective ELISA kit.
The cultured cells were stimulated by PDGF-BB in 1 mL of α-MEM containing 0.3% FCS, and then incubated
for the indicated periods. The conditioned medium was collected, and IL-6 in
the medium was then measured by IL-6 ELISA kit. When indicated, the cells were
pretreated with various doses of EGCG for 60 minutes.


## 2.4. Real-Time RT-PCR

The cultured cells were pretreated with 30μM EGCG or vehicle for 60 minutes, and then
stimulated by 50 ng/mL PDGF-BB for 60 minutes. Total RNA was isolated and
transcribed into complementary DNA using Trizol reagent and Omniscript Reverse
Transcriptase Kit. Real-time RT-PCR was performed using a Light Cycler system (Roche Diagnostics Basel, Switzerland) in capillaries and FastStart DNA Master SYBR Green I
provided with the kit. Sense and antisense primers were synthesized based on
the report of Simpson et al.for mouse GAPDH mRNA [25]. Sense and
antisense primers for mouse IL-6 mRNA were purchased from Takara Bio Inc. (Tokyo, Japan)
(primer set ID:MA039013). The amplified products were determined by melting
curve analysis and agarose electrophoresis. IL-6 mRNA levels were normalized
with those of GAPDH mRNA.


## 2.5. Analysis of Western Blotting

The
cultured cells were stimulated by PDGF-BB inα-MEM containing 0.3% FCS for the indicated
periods. The cells were washed twice with phosphate-buffered saline and then
lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM
Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, and
10% glycerol. The cytosolic fraction was collected as a supernatant after
centrifugation at 125,000×g for 10
minutes at 4°C. SDS-polyacrylamide gel electrophoresis (PAGE) was performed by
Laemmli [26] in 10% polyacrylamide gel. Western blotting analysis was performed
as described previously [27] by using phospho-specific PDGF receptor β antibodies, PDGF receptor β antibodies, phospho-specific p44/p42 MAP
kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP
kinase antibodies, p38 MAP kinase antibodies, phospho-specific SAPK/JNK kinase
antibodies, SAPK/JNK antibodies, phospho-specific Akt antibodies, Akt
antibodies, phospho-specific p70 S6 kinase antibodies, and p70 S6 kinase
antibodies with peroxidase-labeled antibodies raised in goat against rabbit IgG
being used as second antibodies. Peroxidase activity on PVDG membrane was
visualized on X-ray film by means of the ECL Western blotting detection system.
When indicated, the cells were pretreated with various doses of EGCG for 60 minutes.


## 2.6. Determinations

The
absorbance of enzyme immunoassay samples was measured at 450 nm with EL 340 Bio
Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, Vt, USA).
The densitometric analysis was performed using Molecular Analyst/Macintosh (Bio-Rad
Laboratories, Hercules, Calif, USA).


## 2.7. Statistical Analysis

The data
were analyzed by ANOVA followed by the Bonferroni method for multiple
comparisons between pairs, and aP<.05
was considered significant. All data are presented as the mean±SEM of triplicate determinations. Each experiment was repeated three times with
similar results.


## 3. Results

### 3.1. Effects of EGCG on the PDGF-BB-Stimulated IL-6 Synthesis in MC3T3-Cells and Primary-Cultured Mouse Osteoblasts

It has been
reported that PDGF-BB induces transcription of IL-6 in rat osteoblasts [11]. We
have previously found that PDGF-BB stimulates IL-6 synthesis in mouse
osteoblast-like MC3T3-E1 cells [12]. We first examined the effect of EGCG on
the PDGF-BB-stimulated IL-6 synthesis. EGCG, which alone had little effect on
the IL-6 levels, significantly reduced the PDGF-BB-stimulated synthesis of IL-6
(Figure 1(a)). EGCG (30 μM) caused about 50% reduction in the PDGF-BB effect.
In addition, we also examined the effect of EGCG in primary-cultured mouse
osteoblasts. PDGF-BB
significantly enhanced IL-6 synthesis in primary osteoblasts (Figure 1(b)). Furthermore, EGCG (30 μM) significantly reduced the PDBF-BB-stimulated
synthesis of IL-6 (Figure 1(b)). We next assessed the effect of EGCG on cell
viability by trypan blue dye exclusion test. We confirmed that the viability of
MC3T3-E1 cells incubated at 37°C for 24 hours in the presence of 30 μM EGCG was
more than 90% in comparison to that of the control cells. To
determine whether EGCG could affect the cell proliferation, we
counted the cell number before and after the 24-hour incubation with 30 μM EGCG.
We confirmed that EGCG did not affect the cell number at a dose
of 30 μM
(9.7±1.1×105 cells/mL for control; 9.1±1.6×105 cells/mL for 30 μM EGCG as measured during
the stimulation for 24 hours). Therefore, EGCG at 30 μM hardly affects the cell viability
or proliferation of osteoblast-like MC3T3-E1 cells up to 24 hours.Effect of EGCG on the PDGF-BB-stimulated
IL-6 synthesis and expression levels of IL-6 mRNA in osteoblasts. (a)
Osteoblast-like MC3T3-E1 cells were pretreated with various doses of EGCG for
60 minutes, and then stimulated by 50 ng/mL PDGF-BB or vehicle for 24 hours.
Each value represents the mean±SEM of triplicate determinations.
Similar results were obtained with two additional and different cell
preparations. P*<.05: compared to the value of
control. P**<.05: compared to the value of
PDGF-BB alone. (b) Primary cultures of osteoblast were pretreated with or
without 30 μM of EGCG for 60 minutes, and then stimulated
by 50 ng/mL PDGF-BB or vehicle for 24 hours. Each value represents the mean±SEM of triplicate determinations. Similar results were obtained with two
additional and different cell preparations. P*<.05: compared to value of the
PDGF-BB alone. (c) Osteoblast-like MC3T3-E1 cells were
pretreated with 30 μM EGCG or vehicle for 60 minutes, and then
stimulated by 50
ng/mL PDGF-BB for 60 minutes. Total RNA was isolated and
transcribed into complementary DNA. The expressions of IL-6 mRNA and GAPDH mRNA
were quantified by real-time RT-PCR. IL-6 mRNA levels were normalized with
those of GAPDH mRNA. Results were standardized for the value of control
(without EGCG and PDGF-BB). Each value represents the mean±SEM of triplicate
determinations. Similar results were
obtained with two additional and different cell preparations. P*<.05: compared to the value of
control. P**<.05: compared to the value of
PDGF-BB alone.
(a)(b)(c)


### 3.2. Effects of EGCG on the Levels of Osteocalcin and Osteoprotegerin in MC3T3-E1-Cells

Next,
to determine whether EGCG affects the differentiation of these
cells, we examined the effect of EGCG on the synthesis of osteocalcin, a
mature osteoblast phenotype [28], and osteoprotegerin (OPG), produced by
osteoblasts and inhibiting osteoclastic bone resorption [29], synthesis in
MC3T3-E1 cells. We
found that EGCG had no effect on the osteocalcin
(not detectable under the experimental condition at all; <1.56
ng/mL) and OPG (1967±34 pg/mL for vehicle;
1846±46 pg/mL with
30 μM of EGCG) synthesis.


### 3.3. Effect of EGCG on the PDGF-BB-Induced Expression Levels of IL-6 mRNA in MC3T3-E1 Cells

To clarify whether the suppressive effect by EGCG of
PDGF-BB-stimulated IL-6 synthesis is mediated through transcriptional event or
not, we examined the effect of EGCG on the PDGF-BB-induced IL-6 mRNA expression
by real-time RT-PCR. We found that EGCG (30μM) significantly downregulated the IL-6 mRNA
expression levels 60 minutes after the stimulation (Figure 1(c)),
suggesting that the suppressive effect of EGCG is mediated at least in part by the
reduction of IL-6 synthesis in osteoblast-like MC3T3-E1 cells.


### 3.4. Effect of EGCG on the PDGF-BB-Induced Autophosphorylation of PDGF Receptorβ in MC3T3-E1 Cells

In order to
clarify the inhibitory mechanism of EGCG behind the PDGF-BB-stimulated IL-6
synthesis in these cells, we next examined the effect of EGCG on the
PDGF-BB-induced autophosphorylation of PDGF receptorβ. EGCG failed to affect the PDGF-BB-induced
autophosphorylation of PDGF receptor β (Figure 2).
These results lead us to speculate that the mechanism of EGCG-effect on the
PDGF-BB-stimulated IL-6 synthesis may be mediated through downstream of PDGF
receptor activation.Figure 2
Effect of EGCG on the PDGF-BB-induced
autophosphorylation of PDGF receptorβ in
MC3T3-E1 cells. The cultured cells were pretreated with the indicated doses of
EGCG or vehicle for 60 minutes, and then stimulated by 50 ng/mL PDGF-BB or
vehicle for 3 minutes. The extracts of cells were subjected to SDS-PAGE with
subsequent Western blotting analysis with antibodies against phospho-specific
PDGF receptor β or PDGF
receptor β. The histogram shows quantitative
representations of the levels of PDGF-BB-induced autophosphorylation obtained
from laser densitometric analysis of three independent experiments. Each value
represents the mean±SEM of triplicate determinations. Similar results were
obtained with two additional and different cell preparations.


### 3.5. Effects of EGCG on the PDGF-BB-Induced Phosphorylation of p44/p42 Map Kinase, p38 MAP Kinase, or SAPK/JNK in MC3T3-E1 Cells

In our
previous study [13], we reported that PDGF-BB activates three major MAP
kinases, p44/p42 MAP kinase, p38 MAP kinase, and SAPK/JNK, resulting in the
synthesis of IL-6 in MC3T3-E1 cells. We next examined the effect of EGCG on the
PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or
SAPK/JNK. EGCG failed to affect the PDGF-BB-induced phosphorylation of p44/p42
MAP kinase or p38 MAP kinase (Figures 3(a) and 3(b)). On the contrary, EGCG,
which by itself had little effect on the phosphorylation levels of SAPK/JNK,
significantly suppressed the PDGF-BB-induced SAPK/JNK phosphorylation (Figure 4). According to the densitometric analysis, EGCG (30 μM) caused about 40% reduction in the
PDGF-BB-effect.Effect of EGCG on the phosphorylation of
p44/p42 MAP kinase and p38 MAP kinase induced by PDGF-BB in MC3T3-E1 cells. The
cultured cells were pretreated with the indicated doses of EGCG or vehicle for
60 minutes, and then stimulated by 50 ng/mL PDGF-BB or vehicle for 3 minutes. The
extracts of cells were subjected to SDS-PAGE with subsequent Western blotting
analysis with antibodies against (a) phospho-specific p44/p42 MAP kinase or
p44/p42 MAP kinase, and (b) phospho-specific p38 MAP kinase or p38 MAP kinase.
The histogram shows quantitative representations of the levels of
PDGF-BB-induced phosphorylation obtained from laser densitometric analysis of
three independent experiments. Each value represents the mean±SEM of
triplicate determinations. Similar results were obtained with two additional
and different cell preparations.
(a)(b)Figure 4
Effect of EGCG on the phosphorylation of
SAPK/JNK induced by PDGF-BB in MC3T3-E1 cells. The cultured cells were
pretreated with the indicated doses of EGCG or vehicle for 60 minutes, and then
stimulated by 50 ng/mL PDGF-BB or vehicle for 3 minutes. The extracts of cells
were subjected to SDS-PAGE with subsequent Western blotting analysis with
antibodies against phospho-specific SAPK/JNK or SAPK/JNK. The histogram shows quantitative
representations of the levels of PDGF-BB-induced phosphorylation obtained from
laser densitometric analysis of three independent experiments. Each value
represents the mean±SEM of triplicate determinations. Similar results were obtained with two
additional and different cell preparations. P*<.05: compared to the value of
PDGF-BB alone.


### 3.6. Effects of EGCG on the PDGF-BB-Induced Phosphorylation of Akt or p70 S6 Kinase Induced by PDGF-BB in MC3T3-E1 Cells

In our
previous studies [12, 13], we demonstrated that the PDGF-BB-activated Akt and
p70 S6 kinase limit
the synthesis of IL-6 in MC3T3-E1 cells. In order to investigate whether
EGCG-effect on the PDGF-BB-stimulated IL-6 synthesis is mediated via the
activation of Akt or
p70 S6 kinase in these cells, we examined the effect of EGCG on the
PDGF-BB-induced phosphorylation of Akt or p70 S6 kinase.
However, EGCG had little effect on the PDGF-BB-induced phosphorylation
of Akt (Figure 5(a)) or p70 S6 kinase (Figure 5(b)).Effect of EGCG on the
phosphorylation of Akt and p70 S6 kinase induced by PDGF-BB in MC3T3-E1 cells.
The cultured cells were pretreated with the indicated doses of EGCG or vehicle
for 60 minutes, and then stimulated by 50 ng/mL PDGF-BB or vehicle for 20 minutes.
The extracts of cells were subjected to SDS-PAGE with subsequent Western
blotting analysis with antibodies against (a) phospho-specific Akt or Akt, and
(b) phospho-specific p70 S6 kinase or p70 S6 kinase. The histogram shows
quantitative representations of the levels of PDGF-BB-induced phosphorylation
obtained from laser densitometric analysis of three independent experiments.
Each value represents the mean±SEM of triplicate determinations. Similar
results were obtained with two additional and different cell preparations.
(a)(b)


## 3.1. Effects of EGCG on the PDGF-BB-Stimulated IL-6 Synthesis in MC3T3-Cells and Primary-Cultured Mouse Osteoblasts

It has been
reported that PDGF-BB induces transcription of IL-6 in rat osteoblasts [11]. We
have previously found that PDGF-BB stimulates IL-6 synthesis in mouse
osteoblast-like MC3T3-E1 cells [12]. We first examined the effect of EGCG on
the PDGF-BB-stimulated IL-6 synthesis. EGCG, which alone had little effect on
the IL-6 levels, significantly reduced the PDGF-BB-stimulated synthesis of IL-6
(Figure 1(a)). EGCG (30 μM) caused about 50% reduction in the PDGF-BB effect.
In addition, we also examined the effect of EGCG in primary-cultured mouse
osteoblasts. PDGF-BB
significantly enhanced IL-6 synthesis in primary osteoblasts (Figure 1(b)). Furthermore, EGCG (30 μM) significantly reduced the PDBF-BB-stimulated
synthesis of IL-6 (Figure 1(b)). We next assessed the effect of EGCG on cell
viability by trypan blue dye exclusion test. We confirmed that the viability of
MC3T3-E1 cells incubated at 37°C for 24 hours in the presence of 30 μM EGCG was
more than 90% in comparison to that of the control cells. To
determine whether EGCG could affect the cell proliferation, we
counted the cell number before and after the 24-hour incubation with 30 μM EGCG.
We confirmed that EGCG did not affect the cell number at a dose
of 30 μM
(9.7±1.1×105 cells/mL for control; 9.1±1.6×105 cells/mL for 30 μM EGCG as measured during
the stimulation for 24 hours). Therefore, EGCG at 30 μM hardly affects the cell viability
or proliferation of osteoblast-like MC3T3-E1 cells up to 24 hours.Effect of EGCG on the PDGF-BB-stimulated
IL-6 synthesis and expression levels of IL-6 mRNA in osteoblasts. (a)
Osteoblast-like MC3T3-E1 cells were pretreated with various doses of EGCG for
60 minutes, and then stimulated by 50 ng/mL PDGF-BB or vehicle for 24 hours.
Each value represents the mean±SEM of triplicate determinations.
Similar results were obtained with two additional and different cell
preparations. P*<.05: compared to the value of
control. P**<.05: compared to the value of
PDGF-BB alone. (b) Primary cultures of osteoblast were pretreated with or
without 30 μM of EGCG for 60 minutes, and then stimulated
by 50 ng/mL PDGF-BB or vehicle for 24 hours. Each value represents the mean±SEM of triplicate determinations. Similar results were obtained with two
additional and different cell preparations. P*<.05: compared to value of the
PDGF-BB alone. (c) Osteoblast-like MC3T3-E1 cells were
pretreated with 30 μM EGCG or vehicle for 60 minutes, and then
stimulated by 50
ng/mL PDGF-BB for 60 minutes. Total RNA was isolated and
transcribed into complementary DNA. The expressions of IL-6 mRNA and GAPDH mRNA
were quantified by real-time RT-PCR. IL-6 mRNA levels were normalized with
those of GAPDH mRNA. Results were standardized for the value of control
(without EGCG and PDGF-BB). Each value represents the mean±SEM of triplicate
determinations. Similar results were
obtained with two additional and different cell preparations. P*<.05: compared to the value of
control. P**<.05: compared to the value of
PDGF-BB alone.
(a)(b)(c)


## 3.2. Effects of EGCG on the Levels of Osteocalcin and Osteoprotegerin in MC3T3-E1-Cells

Next,
to determine whether EGCG affects the differentiation of these
cells, we examined the effect of EGCG on the synthesis of osteocalcin, a
mature osteoblast phenotype [28], and osteoprotegerin (OPG), produced by
osteoblasts and inhibiting osteoclastic bone resorption [29], synthesis in
MC3T3-E1 cells. We
found that EGCG had no effect on the osteocalcin
(not detectable under the experimental condition at all; <1.56
ng/mL) and OPG (1967±34 pg/mL for vehicle;
1846±46 pg/mL with
30 μM of EGCG) synthesis.


## 3.3. Effect of EGCG on the PDGF-BB-Induced Expression Levels of IL-6 mRNA in MC3T3-E1 Cells

To clarify whether the suppressive effect by EGCG of
PDGF-BB-stimulated IL-6 synthesis is mediated through transcriptional event or
not, we examined the effect of EGCG on the PDGF-BB-induced IL-6 mRNA expression
by real-time RT-PCR. We found that EGCG (30μM) significantly downregulated the IL-6 mRNA
expression levels 60 minutes after the stimulation (Figure 1(c)),
suggesting that the suppressive effect of EGCG is mediated at least in part by the
reduction of IL-6 synthesis in osteoblast-like MC3T3-E1 cells.


## 3.4. Effect of EGCG on the PDGF-BB-Induced Autophosphorylation of PDGF Receptorβ in MC3T3-E1 Cells

In order to
clarify the inhibitory mechanism of EGCG behind the PDGF-BB-stimulated IL-6
synthesis in these cells, we next examined the effect of EGCG on the
PDGF-BB-induced autophosphorylation of PDGF receptorβ. EGCG failed to affect the PDGF-BB-induced
autophosphorylation of PDGF receptor β (Figure 2).
These results lead us to speculate that the mechanism of EGCG-effect on the
PDGF-BB-stimulated IL-6 synthesis may be mediated through downstream of PDGF
receptor activation.Figure 2
Effect of EGCG on the PDGF-BB-induced
autophosphorylation of PDGF receptorβ in
MC3T3-E1 cells. The cultured cells were pretreated with the indicated doses of
EGCG or vehicle for 60 minutes, and then stimulated by 50 ng/mL PDGF-BB or
vehicle for 3 minutes. The extracts of cells were subjected to SDS-PAGE with
subsequent Western blotting analysis with antibodies against phospho-specific
PDGF receptor β or PDGF
receptor β. The histogram shows quantitative
representations of the levels of PDGF-BB-induced autophosphorylation obtained
from laser densitometric analysis of three independent experiments. Each value
represents the mean±SEM of triplicate determinations. Similar results were
obtained with two additional and different cell preparations.


## 3.5. Effects of EGCG on the PDGF-BB-Induced Phosphorylation of p44/p42 Map Kinase, p38 MAP Kinase, or SAPK/JNK in MC3T3-E1 Cells

In our
previous study [13], we reported that PDGF-BB activates three major MAP
kinases, p44/p42 MAP kinase, p38 MAP kinase, and SAPK/JNK, resulting in the
synthesis of IL-6 in MC3T3-E1 cells. We next examined the effect of EGCG on the
PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or
SAPK/JNK. EGCG failed to affect the PDGF-BB-induced phosphorylation of p44/p42
MAP kinase or p38 MAP kinase (Figures 3(a) and 3(b)). On the contrary, EGCG,
which by itself had little effect on the phosphorylation levels of SAPK/JNK,
significantly suppressed the PDGF-BB-induced SAPK/JNK phosphorylation (Figure 4). According to the densitometric analysis, EGCG (30 μM) caused about 40% reduction in the
PDGF-BB-effect.Effect of EGCG on the phosphorylation of
p44/p42 MAP kinase and p38 MAP kinase induced by PDGF-BB in MC3T3-E1 cells. The
cultured cells were pretreated with the indicated doses of EGCG or vehicle for
60 minutes, and then stimulated by 50 ng/mL PDGF-BB or vehicle for 3 minutes. The
extracts of cells were subjected to SDS-PAGE with subsequent Western blotting
analysis with antibodies against (a) phospho-specific p44/p42 MAP kinase or
p44/p42 MAP kinase, and (b) phospho-specific p38 MAP kinase or p38 MAP kinase.
The histogram shows quantitative representations of the levels of
PDGF-BB-induced phosphorylation obtained from laser densitometric analysis of
three independent experiments. Each value represents the mean±SEM of
triplicate determinations. Similar results were obtained with two additional
and different cell preparations.
(a)(b)Figure 4
Effect of EGCG on the phosphorylation of
SAPK/JNK induced by PDGF-BB in MC3T3-E1 cells. The cultured cells were
pretreated with the indicated doses of EGCG or vehicle for 60 minutes, and then
stimulated by 50 ng/mL PDGF-BB or vehicle for 3 minutes. The extracts of cells
were subjected to SDS-PAGE with subsequent Western blotting analysis with
antibodies against phospho-specific SAPK/JNK or SAPK/JNK. The histogram shows quantitative
representations of the levels of PDGF-BB-induced phosphorylation obtained from
laser densitometric analysis of three independent experiments. Each value
represents the mean±SEM of triplicate determinations. Similar results were obtained with two
additional and different cell preparations. P*<.05: compared to the value of
PDGF-BB alone.


## 3.6. Effects of EGCG on the PDGF-BB-Induced Phosphorylation of Akt or p70 S6 Kinase Induced by PDGF-BB in MC3T3-E1 Cells

In our
previous studies [12, 13], we demonstrated that the PDGF-BB-activated Akt and
p70 S6 kinase limit
the synthesis of IL-6 in MC3T3-E1 cells. In order to investigate whether
EGCG-effect on the PDGF-BB-stimulated IL-6 synthesis is mediated via the
activation of Akt or
p70 S6 kinase in these cells, we examined the effect of EGCG on the
PDGF-BB-induced phosphorylation of Akt or p70 S6 kinase.
However, EGCG had little effect on the PDGF-BB-induced phosphorylation
of Akt (Figure 5(a)) or p70 S6 kinase (Figure 5(b)).Effect of EGCG on the
phosphorylation of Akt and p70 S6 kinase induced by PDGF-BB in MC3T3-E1 cells.
The cultured cells were pretreated with the indicated doses of EGCG or vehicle
for 60 minutes, and then stimulated by 50 ng/mL PDGF-BB or vehicle for 20 minutes.
The extracts of cells were subjected to SDS-PAGE with subsequent Western
blotting analysis with antibodies against (a) phospho-specific Akt or Akt, and
(b) phospho-specific p70 S6 kinase or p70 S6 kinase. The histogram shows
quantitative representations of the levels of PDGF-BB-induced phosphorylation
obtained from laser densitometric analysis of three independent experiments.
Each value represents the mean±SEM of triplicate determinations. Similar
results were obtained with two additional and different cell preparations.
(a)(b)


## 4. Discussion

In the
present study, we showed that EGCG significantly suppressed the PDGF-BB-stimulated
IL-6 synthesis and expression levels of IL-6 mRNA in osteoblast-like MC3T3-E1
cells. We found that EGCG reduced the PDGF-BB-stimulated IL-6 synthesis also in
primary-cultured mouse osteoblasts.
These findings suggest that the inhibitory effect of EGCG on the
PDGF-BB-stimulated IL-6 synthesis is not specific in a clonal osteoblast-like
MC3T3-E1 cells but it is common in osteoblasts. We confirmed that EGCG at 30μM
hardly affects the cell viability or proliferation of osteoblast-like MC3T3-E1
cells up to 24 hours. As for the effects of EGCG on the
differentiation of these cells, the results showed that EGCG had
no effect on the osteocalcin and OPG synthesis. However,
it has
been shown that catechin stimulates the alkaline phosphatase activity, a mature
osteoblast phenotype [5], and reduces bone-resorptive cytokine
production in osteoblast-like MC3T3-E1 cells [21]. Taking these
results into
account, it is suggested that EGCG can partially affect the
differentiation of osteoblast-like MC3T3-E1 cells. We next investigated the
mechanism of EGCG underlying the inhibitory effect on the IL-6 synthesis. It is
well known that the MAP kinase superfamily plays a crucial role in cellular
functions including proliferation, differentiation, and survival in a variety
of cells [14]. Three major MAP kinases, p44/p42 MAP kinase, p38 MAP kinase, and
SAPK/JNK, are known as central elements used by mammalian cells to transduce
the diverse messages [14]. We have previously reported that SAPK/JNK act as
positive regulators in PDGF-BB-induced IL-6 synthesis in MC3T3-E1 cells [13].
In the present study, we showed that EGCG did not affect the PDGF-BB-induced
phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. Therefore, it seems
unlikely that EGCG reduces the PDGF-BB-stimulated IL-6 synthesis through
downregulating activation of p44/p42 MAP kinase or p38 MAP kinase in
osteoblast-like MC3T3-E1 cells. On the other hand, we showed that the
PDGF-BB-induced phosphorylation of SAPK/JNK was markedly suppressed by EGCG.
These results suggest that EGCG downregulates the PDGF-BB-stimulated activation
of SAPK/JNK. Taking our findings into account, it is most likely that EGCG
inhibits PDGF-BB-stimulated IL-6 synthesis through downregulating the
activation of SAPK/JNK in osteoblast-like MC3T3-E1 cells. Further investigations are required to
clarify the precise mechanism of catechin underlying the suppression of IL-6
synthesis in osteoblasts.We
previously showed that Akt and p70 S6 kinase negatively regulates the
PDGF-BB-stimulated synthesis of IL-6 in osteoblast-like MC3T3-E1 cells [12, 13].
We additionally investigated the involvement of Akt and p70 S6 kinase in the
inhibitory effect of EGCG on the IL-6 synthesis. However, EGCG failed to affect
the PDGF-BB-induced phosphorylation of Akt or p70 S6 kinase. Therefore, it seems unlikely
that EGCG inhibits PDGF-BB-induced IL-6 synthesis via downregulating the
activation of Akt or
p70 S6 kinase in osteoblast-like MC3T3-E1 cells. Taking our findings into
account, it is most likely that EGCG inhibits PDGF-BB-stimulated IL-6 synthesis
through downregulating the activation of SAPK/JNK in osteoblast-like MC3T3-E1
cells.IL-6, which
is synthesized from osteoblasts, modulates a variety of bone cell function [3].
In bone metabolism, IL-6 secreted from osteoblasts acts as an
autocrine/paracrine factor, which induces osteoclast formation and stimulates
its activity to resorb bone [4, 7]. Based on our results, it is probable that
catechin-induced suppression of SAPJ/JNK activation by PDGF-BB has an
inhibitory effect on bone resorption via downregulating IL-6 synthesis in
osteoblasts. It has been reported that PDGF-BB is recognized as a potent
stimulator of osteoblast proliferation and collagen synthesis [30]. PDGF,
released during platelet aggregation, has a pivotal role in fracture healing as
a systemic factor, and that PDGF also regulates bone remodeling as a local
factor [30]. As for osteoporosis, one of the major problems in the health of
elderly persons in advanced countries, it is reported that administration of recombinant
PDGF-BB accelerates
fracture healing in the geriatric, osteoporotic rat [31]. However, the present
study indicates that PDBG-BB stimulates IL-6 synthesis, one of the potent bone resorptive
agents, not only by osteoblast-like MC3T3-E1 cells but also by cultured primary
osteoblast. In the present study, noteworthy, EGCG reduced PDGF-BB-stimulated
IL-6 synthesis in osteoblasts. Therefore, our present findings
led us to speculate that EGCG might enhance the fracture healing properties of
PDGF-BB by reducing the IL-6 synthesis in osteoblasts. Our present data would
provide a new insight in pharmacological effects of catechin on bone
metabolism.The
pharmacokinetics of EGCG in human volunteers taking single dosage of 1 600 mg/day showed a rapid absorption, with a maximum plasma concentration value of 11.08μM (=  3392 ng/mL); the time
to reach maximum plasma concentration was 2.2 hours, and the terminal
elimination half-life ranged between 1.9 and 4.6 hours [32]. Interestingly, 10-day repeated administration
of oral doses of EGCG of up to 800 mg/day was found to be safe and very well
tolerated [33]. In the present study, we showed that the significant
suppressive effect of EGCG on the PDGF-BB-stimulated IL-6 synthesis was clearly
observed at 10 μM. Therefore, it is most
likely that one could drink enough green tea to reach in vivo levels which are used
in our in vitro study. In addition, it has been shown that the plasma
concentration of EGCG required for cancer prevention or anti-inflammatory effects
is over 10 μM to 50 μM [34–36]. Our
results, regarding suppression of IL-6 synthesis, are consistent with these
previous findings. Little is known regarding the effective concentration of EGCG requiredto modulate intracellular signaling pathways. Further investigations using
primary-cultured osteoblasts in addition to MC3T3-E1 cells would be necessary
to elucidate the exact roles of catechin in the bone metabolism.In
conclusion, our present results strongly suggest that catechin inhibits the PDGF-BB-stimulated
synthesis of IL-6 via suppression of SAPK/JNK pathway in osteoblasts.



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*Source: 291808-2009-01-12.xml*