In this study, we identified a novel suppression mechanism, operating in the early phase of adipogenesis, that increased the production of anti-adipogenic PGF2a and PGE2by enhancing cyclo
Trang 1adipogenesis by positive feedback loop for enhancement
Toshiyuki Ueno and Ko Fujimori
Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, Japan
Keywords
3T3-L1, PGF2a; adipocytes; COX-2; CREB;
MEK ⁄ ERK
Correspondence
K Fujimori, Laboratory of Biodefense
and Regulation, Osaka University of
Pharmaceutical Sciences, 4-20-1 Nasahara,
Takatsuki, Osaka 569-1094, Japan
Fax: +81 72 690 1215
Tel: +81 72 690 1215
E-mail: fujimori@gly.oups.ac.jp
(Received 6 April 2011, revised 31 May
2011, accepted 8 June 2011)
doi:10.1111/j.1742-4658.2011.08213.x
Prostaglandin (PG) F2asuppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor c In this study, we identified a novel suppression mechanism, operating in the early phase of adipogenesis, that increased the production of anti-adipogenic PGF2a and PGE2by enhancing cyclooxygenase (COX) 2 expression through the PGF2a -activated FP receptor⁄ extracellular-signal-regulated kinase (ERK) ⁄ cyclic AMP response element binding protein (CREB) cascade COX-2 expression was enhanced with a peak at 1 h for the mRNA level and at 3 h for the pro-tein level after the addition of Fluprostenol, an FP receptor agonist The Fluprostenol-derived elevation of COX-2 expression was suppressed by the co-treatment with an FP receptor antagonist, AL8810, with a mitogen-acti-vated protein kinase (MEK; ERK kinase) inhibitor, PD98059 ERK was phosphorylated within 10 min after the addition of Fluprostenol, and its phosphorylation was inhibited by the co-treatment with AL8810 or PD98059 Moreover, FP receptor mediated activation of the MEK⁄ ERK cascade and COX-2 expression increased the production of PGF2a and PGE2 An FP receptor antagonist and each inhibitor for MEK and COX-2 suppressed the PGF2a-derived induction of synthesis of these PGs Further-more, promoter-luciferase and chromatin immunoprecipitation assays dem-onstrated that PGF2a-derived COX-2 expression was activated through binding of CREB to the promoter region of the COX-2 gene in 3T3-L1 cells These results indicate that PGF2a suppresses the progression of the early phase of adipogenesis by enhancing the binding of CREB to the COX-2 pro-moter via FP receptor activated MEK⁄ ERK cascade Thus, PGF2aforms a positive feedback loop that coordinately suppresses the early phase of adipo-genesis through the increased production of anti-adipogenic PGF2aand PGE2
Introduction
Adipose tissue plays a critical role as a site for energy
storage [1,2] and has been identified as an endocrine
organ that secretes various biologically active molecules
called adipocytokines [3] However, excessive lipid accumulation or enlarged size of adipocytes is asso-ciated with diseases such as obesity and diabetes [4]
Abbreviations
AKR, aldo-keto reductase; ChIP, chromatin immunoprecipitation; COX, cyclooxygenase; CRE, cyclic AMP responsive element; CREB, CRE-binding protein; EIA, enzyme immunoassay; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein
kinase⁄ extracellular signal-regulated kinase kinase; pAb, polyclonal antibody; PG, prostaglandin; PLA 2 , phospholipase A 2 ; PPAR, peroxisome proliferator-activated receptor.
Trang 2Adipocyte differentiation (adipogenesis) is a complex
process including the coordinated changes in hormone
sensitivity and gene expression in response to various
stimuli including lipid mediators
Prostaglandins (PGs) are a group of lipid mediators
with numerous functions, and they are synthesized
from membrane lipids by three enzymatic steps First,
arachidonic acid is liberated by phospholipase A2s
(PLA2s) Second, it is converted to PGH2by either
cy-clooxygenase (COX) 1 or COX-2 Third, PGH2, which
is a common precursor of all prostanoids, is
metabo-lized to various PGs by the action of specific PG
syn-thases [5,6] PGs are involved in the regulation of
adipocyte differentiation Lipocalin-type
PGD-syn-thase-produced PGD2 enhances adipocyte
differentia-tion [7], whereas PGE2 and PGF2a suppress the
differentiation of adipocytes through EP4 [8] and FP
[9–12] receptors, respectively, indicating that PGE2and
PGF2aare anti-adipogenic factors PGF2aplays a
vari-ety of physiological roles in the body and is
synthe-sized by the reduction of either the 9,11-endoperoxide
moiety of PGH2 or the 9-keto group of PGE2 in an
NAD(P)H-dependent manner, both of which reactions
are catalyzed by aldo-keto reductases (AKRs) [13] We
recently showed that AKR1B3 acts as the PGF
syn-thase in adipocytes and that AKR1B3-derived PGF2a
suppresses the early phase of adipogenesis [14,15]
through a specific cell surface G-protein-coupled
recep-tor, the FP receptor [16,17], which binding leads to the
activation of various kinases [16,18–20]
COX consists of two isozymes, COX-1 and COX-2,
and is the rate-limiting enzyme catalyzing the
conver-sion of arachidonic acid into endoperoxide
intermedi-ates that are ultimately converted by specific synthases
to prostanoids [5,21,22] COX-1 is constitutively
expressed in most cells including the adipocytes,
whereas COX-2 expression is induced by various
stim-uli and is transiently activated in the early phase of
adipogenesis [15] The expression of antisense COX-2
mRNA results in the upregulation of adipogenesis,
thus indicating that COX-2 is involved in the
produc-tion of anti-adipogenic prostanoids [23] In contrast,
selective COX-2 inhibitors impair adipocyte
differenti-ation through inhibition of the clonal expansion phase
[24] Moreover, COX-2-deficient mice exhibit
sup-pressed adipocyte differentiation [25] Thus,
arachi-donic acid metabolism during adipogenesis is a process
governed at multiple levels, suggesting a complex role
for PGs during fat cell development [26] The effects of
COX in adipogenesis are controversial, because
COX-2-derived PGs play suppressive roles in the early phase
of adipogenesis [14,15], while COX-2 and
lipocalin-type PGD-synthase-derived PGD2 activates the late
phase of adipogenesis [7] The regulation of adipogene-sis by PGs is thus not yet fully understood
In this study, we demonstrate the effect of PGF2a–
FP receptor signaling on the expression of COX-2 and suppression of the early phase of adipogenesis in mouse adipocytic 3T3-L1 cells PGF2a promoted the produc-tion of anti-adipogenic PGF2aand PGE2by enhancing the expression of COX-2 through FP-receptor-activated mitogen-activated protein kinase⁄ extracellular signal-regulated kinase kinase (MEK)⁄ extracellular signal-regulated kinase (ERK) cascade and the binding of cyclic AMP response element (CRE) binding protein (CREB) to the COX-2 promoter Therefore, PGF2a suppresses the early phase of adipogenesis by de novo synthesis of anti-adipogenic PGF2a and PGE2 through
a positive feedback FP receptor-MEK⁄ ERK-CREB-COX-2 loop
Results
Activation of FP receptor increases PGF2a production with enhanced expression of COX-2 During the adipogenesis of mouse 3T3-L1 cells, PGF2a
is produced in the early phase of adipogenesis with a peak at 3 h after the initiation of adipogenesis [15]
In this study, we found that when 3T3-L1 cells were incubated with Fluprostenol, an FP receptor agonist, for 1 h, PGF2a production was significantly increased (Fig 1A), indicating that PGF2a may enhance PGF2a production To identify the molecular mechanism of this Fluprostenol-mediated increase in PGF2a produc-tion in adipocytes, at first we investigated the expres-sion of genes involved in the biosynthesis of PGF2a
and FP receptor in Fluprostenol-treated 3T3-L1 cells
by performing quantitative PCR analysis The tran-scription level of the COX-2 gene was increased 3.7-fold, whereas that of the FP receptor gene was decreased about 28%, compared with the levels of the vehicle control (Fig 1B) The expression levels of cyto-solic PLA2 (cPLA2), COX-1 and AKR1B3 genes were not altered by the treatment of the cells with Flupros-tenol (Fig 1B) Moreover, we confirmed that PGF2a could activate COX-2 expression in 3T3-L1 cells about 3.1-fold compared with the vehicle control (Fig 1C) Next, we examined the time course change in the COX-2 expression level in the Fluprostenol-treated 3T3-L1 cells The transcription of COX-2 mRNA was increased at 1 h after the addition of Fluprostenol and then quickly decreased to a level lower than that detected in the vehicle-treated cells (Fig 1D) The COX-2 protein level was increased with a peak at 3 h after the addition of Fluprostenol and then decreased
Trang 3A B COX-2
0.1
*
FP 0.75
1.6
*
0.06
0.04
0.02
0.25
**
1.2
0.8
0.4
Fluprostenol
Fluprostenol
0
COX-1 10
Fluprostenol
0 0
C
0.06
7.5
5
7.5
5
7.5
5
2.5
0.04
0.02
*
Fluprostenol
0 2.5
Fluprostenol
0
2.5
0 Fluprostenol
0
COX-2mRNA level (/TBP mRNA
COX-2mRNA level (/TBP mRNA
1
1.5 0.1
0.2
COX-2 Actin
0 0.5
Time after addition of Fluprostenol (h)
0
0.05
*
Time after addition of Fluprostenol (h)
Fig 1 Enhancement of COX-2 expression by Fluprostenol, an FP receptor agonist (A) Induction of PGF 2a production by treatment with Fluprostenol Pre-adipocytic 3T3-L1 cells were incubated with Fluprostenol (0.5 n M ; Cayman Chemical) or vehicle for 1 h, followed by treat-ment with A23187 (5 l M ) for 10 min at 37 C, after which the medium was collected to measure the PGF 2a level by EIA *P < 0.01, com-pared with vehicle-treated cells (B) Expression levels of COX-2, FP receptor, cPLA 2 , COX-1 and AKR1B3 genes in the Fluprostenol-treated 3T3-L1 cells Cells were incubated with Fluprostenol (0.5 n M ) for 1 h, and the expression level of each gene was measured by quantitative PCR The data are presented as the mean ± SD of three independent experiments *P < 0.01, **P < 0.05, compared with vehicle control (C) Transcription level of COX-2 gene in the PGF2a-treated 3T3-L1 cells Cells were cultured with PGF2a(100 n M ) for 1 h, and the mRNA level
of COX-2 gene was then measured by quantitative PCR The data are presented as the mean ± SD of three independent experiments.
*P < 0.01, compared with vehicle control (D) Expression level of COX-2 gene in Fluprostenol-treated 3T3-L1 cells Cells were incubated with Fluprostenol (0.5 n M ) for various times Expression levels of COX-2 gene were measured by quantitative PCR The data are presented as the mean ± SD of three independent experiments *P < 0.01, compared with vehicle-treated cells (E) Protein level of COX-2 was detected by western blot analysis Cells were treated as described in (D) Crude cell extracts (20 lg) were loaded in each lane *P < 0.01, compared with vehicle-treated cells.
Trang 4to almost the same level as detected in the
vehicle-trea-ted cells, although the actin levels were not altered in
each sample (Fig 1E) These results, all taken
together, reveal that Fluprostenol-activated FP
recep-tors increased PGF2aproduction and enhanced COX-2
expression in 3T3-L1 cells
Involvement of MEK signaling in the PGF2a
FP receptor-activated COX-2 expression
To elucidate the regulatory mechanism of the PGF2a
FP receptor activated COX-2 expression in 3T3-L1
cells, we cultured the cells with AL8810, which is
an FP receptor antagonist, or with an MEK
inhibi-tor, PD98059; a PLC inhibiinhibi-tor, U73122; a PKC
inhibitor, bisindolylmaleimide I; and a general COX
inhibitor, indomethacin, or a selective COX-2
inhibi-tor, NS-398, in the presence of Fluprostenol for 1 h
COX-2 expression was enhanced by the treatment with
Fluprostenol compared with that obtained with the
vehicle control (Fig 2A) Fluprostenol FP
receptor-activated COX-2 expression was abolished by the
co-treatment with AL8810 or PD98059 (Fig 2A) In
contrast, U73122, bisindolylmaleimide I, indomethacin
and NS-398 had no effect on the expression level of
Fluprostenol FP receptor-activated COX-2 expression
(Fig 2A) Moreover, the Fluprostenol-derived
eleva-tion of COX-2 mRNA expression was suppressed by
AL8810 or PD98059 in a concentration-dependent
manner (Fig 2B), results which agree well with the
COX-2 protein level demonstrated by western blot
analysis, although the actin levels were not changed in
each sample (Fig 2C) These results indicate that
Fluprostenol-derived enhancement of COX-2
expres-sion occurred through FP receptor and MEK signaling
in 3T3-L1 cells
Phosphorylation of ERK1⁄ 2 by activated PGF2a–FP
receptor signaling
The MEK⁄ ERK signaling pathway is critical in the
determination of the specificity of cellular responses
such as cell proliferation and cell differentiation [27]
Next, we examined whether ERK1⁄ 2 was activated by
MEK in the Fluprostenol-treated 3T3-L1 cells ERK1⁄ 2
was expressed constitutively even in the absence of
Fluprostenol, and slightly phosphorylated in the
vehi-cle-treated cells (Fig 3A) When the cells were cultured
in DMEM containing Fluprostenol, phosphorylation of
ERK1⁄ 2 was enhanced with a peak 10 min after the
addition of Fluprostenol and then decreased (Fig 3A)
To confirm the activation of ERK1⁄ 2 via
Fluproste-nol-activated FP receptors, we cultured the cells in the
presence of AL8810 or PD98059 and Fluprostenol, and assessed the level of phosphorylated ERK1⁄ 2 pro-tein by western blot analysis Fluprostenol-mediated phosphorylation of ERK1⁄ 2 was diminished by AL8810 (Fig 3B) Moreover, PD98059 also decreased
it (Fig 3B) These results indicate that Fluprostenol activated the MEK⁄ ERK signaling pathway in 3T3-L1 cells within 10 min through the FP receptor
Enhanced de novo synthesis of PGF2aand PGE2
by activation of FP receptor MEK⁄ ERK cascade Next we examined whether FP receptor-mediated acti-vation of the MEK⁄ ERK cascade would enhance PGF2a production in 3T3-L1 cells PGF2a production was increased by the treatment with Fluprostenol (Figs 1 and 4, left panel) Fluprostenol-derived eleva-tion of PGF2a production was decreased by the treat-ment with AL8810, PD98059, indomethacin or NS-398 (Fig 4, left panel), whereas U73122 and bisindolylma-leimide I had no effect on the production (Fig 4, left panel) Moreover, when the cells were incubated with PD98059 or NS-398, PGF2a production decreased to the basal level, indicating that basal and induced PGF2a production are dependent upon COX-2 and MEK⁄ ERK cascade Furthermore, we found that PGE2 production was also enhanced by the activation
of the FP receptor MEK⁄ ERK cascade (Fig 4, right panel) The co-treatment with AL8810, PD98059, indomethacin or NS-398 decreased the Fluprostenol-induced PGE2production (Fig 4, right panel) In con-trast, U73122 and bisindolylmaleimide I had no effect
on it (Fig 4, right panel) Moreover, the levels of other PGs, PGD2, prostacyclin and thromboxane, were not altered when the cells were incubated with Flupro-stenol (data not shown) These results reveal that Fluprostenol-derived activation of the FP receptor MEK⁄ ERK cascade enhanced de novo synthesis of anti-adipogenic PGF2aand PGE2in 3T3-L1 cells
Fluprostenol-activated COX-2 expression through binding of CREB to COX-2 promoter
To examine the transcriptional activation mechanism
of the COX-2 gene triggered by activation of the FP receptor in 3T3-L1 cells, we conducted luciferase repor-ter assays using various deleted or mutated promorepor-ter- promoter-reporter plasmids (Fig 5A) The transcription initiation site of mouse COX-2 gene was determined previously [28] When the construct carrying the promoter region from )500 to +124, named )500 ⁄ +124, was used for the transfection, efficient reporter activity was detected (Fig 5A) Moreover, when the )500 ⁄ +124 construct
Trang 5transfected cells were treated with Fluprostenol,
lucifer-ase reporter activity was 138% (black column) of
that of the vehicle (white column); and this
Fluproste-nol-activated COX-2 promoter activity was suppressed
by the co-treatment with AL8810 (gray column)
Further sequential deletion analysis of the region from )500 to )140 did not result in any significant change from that observed when the )500 ⁄ +124 construct was used for the transfection (Fig 5A), indicating that the region from )500 to )140 of COX-2 promoter
0.01
0.0075
0.0025
0.005
COX-2 mRNA level (/TBP mRNA level)
0.03
* *
0
+Fluprostenol AL
0.03
*
NS I
*
*
0.01
0.02
0.01 0.02
COX-2 mRNA level (/TBP mRNA level)
COX-2
COX-2
0
Fluprostneol (n M )
Fluprostenol (n M ) PD98059 (μ M )
2
3
* * *
1
0
Fluprostneol (n M )
Fluprostenol (n M ) PD98059 (μ M ) 0
1 Relative band intensity Relative band intensity
0 0.5
A
B
C
Fig 2 Involvement of MEK signaling in
the enhancement of COX-2 expression (A)
Inhibition of Fluprostenol-activated COX-2
expression by kinase and COX inhibitors.
3T3-L1 cells were cultured in the presence
of Fluprostenol (0.5 n M ) with or without
AL8810 (50 l M ; Cayman Chemical), which
is an FP receptor antagonist, each of various
protein kinases: the MEK inhibitor, PD98059
(PD) (10 l M , Calbiochem), the PLC inhibitor
U73122 (U) (1 l M , Calbiochem), the PKC
inhibitor bisindolylmaleimide I (Bis) (0.1 l M ,
Calbiochem), the general COX inhibitor
indomethacin (I) (2 l M , Cayman Chemical)
and the COX-2 specific inhibitor NS-398
(NS) (1 l M , Cayman Chemical) for 5 min.
Cells were further incubated for 1 h with
Fluprostenol (0.5 n M ) in the presence of
each inhibitor COX-2 mRNA levels were
measured by quantitative PCR Data are
the mean ± SD of three independent
experiments *P < 0.01, as indicated by the
brackets (B) Suppression of
Fluprostenol-activated COX-2 expression by AL8810 or
PD98059 Cells were incubated with
AL8810 (0–50 l M ) or PD98059 (0–10 l M )
together with Fluprostenol (5 n M ) The
expression level of COX-2 gene was
quanti-fied by PCR Data are the mean ± SD of
three independent experiments *P < 0.01,
as indicated by the brackets (C) Western
blot analysis Cells were treated as
described in (B) Crude cell extracts (15 lg)
were loaded in each lane Data are the
mean ± SD of three independent
experiments *P < 0.01, as indicated by the
brackets.
Trang 6containing the nuclear factor jB element was not
involved in the activation of COX-2 promoter activity
In contrast, when the region from )140 to )110 was
deleted, the luciferase reporter activity was 24%
decreased compared with that of the )140 ⁄ +124
con-struct (Fig 5A) However, the responsiveness to
Flupro-stenol and AL8810 was still observed, suggesting the
existence of a cis-regulatory element for the basal
expression of the COX-2 gene in the region from)140
to )110 of the COX-2 promoter (Fig 5A) This result was consistent with previous results showing that the binding element for CCAAT⁄ enhancer binding protein
at )135 of the COX-2 promoter acts as a positive cis-regulatory element in basal COX-2 gene expression
in 3T3-L1 cells [29] Furthermore, when the region from )110 to )50 was deleted, the luciferase reporter activity was significantly decreased, and the responses
to Fluprostenol and AL8810 disappeared (Fig 5A),
P-ERK1/2
ERK1/2
A
P-ERK1/2 ERK1/2
B
Time after addition of Fluprostenol (min)
Fluprostenol AL8810 PD98059
– – ––
+ –
+ +– + 5
4
3
2
*
*
*
Time after addition of Fluprostenol (min)
Fluprostenol AL8810 –– +– ++ +– 0
2 3
1 Relative band
0 1
*
Fig 3 Time course of PGF 2a -stimulated ERK1 ⁄ 2 phosphorylation (A) Fluprostenol-induced phosphorylation of ERK1 ⁄ 2 in 3T3-L1 cells Cells were cultured with Fluprostenol (0.5 n M ) for 0–60 min Cell lysates (20 lgÆlane)1) were subjected to SDS ⁄ PAGE and western blot analysis for phosphorylated ERK1 ⁄ 2 and total ERK1 ⁄ 2 proteins Band intensity was measured by the use of MULTI GAUGE software The data shown are representative of three independent experiments (B) The MEK inhibitor, PD98059, prevented Fluprostenol-mediated phosphorylation of ERK1 ⁄ 2 in 3T3-L1 cells Cells were cultured with Fluprostenol (0.5 n M ) in the presence or absence of AL8810 (50 l M ) or PD98059 (10 l M ) for 10 min Cell lysates (20 lgÆlane)1) were used for SDS ⁄ PAGE and western blot analysis to determine the levels of phospho-ERK1 ⁄ 2 and total ERK1 ⁄ 2 proteins Band intensity was measured by using MULTI GAUGE software The data shown are representative of three indepen-dent experiments *P < 0.01, as indicated by the brackets.
1.2 1.5 1.8
20 25 30
*
*
–1 )
–1 )
0 0.3 0.6 0.9
V V AL PD I NS U Bis
5 10 15
0
V V AL PD I NS U Bis
Fig 4 Effect of kinase inhibitors or COX inhibitors on Fluprostenol-stimulated PGF2aand PGE2production 3T3-L1 cells were incubated in DMEM containing AL8810 (AL) (10 l M ), PD98059 (PD) (10 l M ), U73122 (U) (1 l M ), bisindolylmaleimide I (Bis) (0.1 l M ), COX inhibitors indo-methacin (I) (2 l M ) or NS-398 (NS) (1 l M ) together with Fluprostenol (5 n M ) for 1 h The medium was then removed and replaced with fresh medium containing inhibitor and A23187 (5 l M ), and the cells were further incubated for 10 min The medium was collected for the mea-surement of PGF 2a and PGE 2 levels by performing the respective EIAs Data are expressed as the mean ± SD of three independent experi-ments *P < 0.01, as indicated by the brackets.
Trang 7revealing that the region from)110 to )50 contained
a critical cis-regulatory element for the basal and
PGF2a-derived activation of COX-2 gene expression in
3T3-L1 cells
We searched for putative cis-regulatory elements in
the promoter region from )110 to )50 of the COX-2
promoter by using matinspector software [30] and
found one putative CRE at )59 of the COX-2
pro-moter (Fig 5A) To confirm the importance of the
CRE at )59 in the PGF2a-derived elevation of COX-2 gene expression in 3T3-L1 cells, we introduced a muta-tion at this CRE at)59 of the COX-2 promoter of the )500 ⁄ +124 construct: )500 ⁄ +124(mu) When the cells were transfected with the )500 ⁄ +124(mu) con-struct, the luciferase reporter activity was significantly decreased compared with that of the wild-type )500 ⁄ +124 construct (Fig 5A) In addition, the responsiveness to Fluprostenol and AL8810 was lost
+124 –500/+124
A
+1 –500
CRE (–59) CCAAT (–135) NF-κB (–224)
*
–110/+124 –50/+124
–300/+124 –140/+124
*
*
*
*
*
*
*
*
*
*
–500/+124 (mu) pGL4.10
Relative luminescence
0 WT
CagtaggA
Vehicle Fluprostenol Fluprostenol + AL8810
B
Input
CRE –59 –52
168-bp
3′
5′
Fluprostenol – +
ChIP: anti-CREB
PD98059 – – – +
3 4 6
2
*
0
1
Fig 5 Enhancement of PGF 2a -mediated COX-2 gene expression through binding of CREB to COX-2 promoter (A) Deletion and mutation analyses of mouse COX-2 promoter region in 3T3-L1 cells Transfected cells were treated with Fluprostenol (0.5 n M , black column), Flupros-tenol and AL8810 (10 l M , gray columns) or incubated without any treatment (white columns) for 6 h; then luciferase reporter activities were measured (right panel) The data represent the mean ± SD of three independent assays The putative cis-regulatory elements are indicated
at the top of the diagram, and mutated nucleotides by small characters (left panel) *P < 0.01, as indicated by the brackets (B) ChIP assay
of the CRE of mouse COX-2 promoter in 3T3-L1 cells The scheme for the ChIP assay for the mouse COX-2 promoter is shown at the left Cells were treated with Fluprostenol (0.5 n M ) with or without AL8810 (10 l M ) or PD98059 (10 l M ) for 1 h, and the ChIP assay was then car-ried out The profile of the amplicon is shown at the right and the input control (input) means that a small aliquot before immunoprecipitation was used for PCR amplification The data are representative of three independent experiments *P < 0.01, as indicated by the brackets.
Trang 8(Fig 5A) These results indicate that PGF2a activated
COX-2 gene expression through the CRE at )59 of
the mouse COX-2 promoter in 3T3-L1 cells
Next, we examined the binding of CREB to the
CRE at )59 of the COX-2 promoter by performing a
chromatin immunoprecipitation (ChIP) assay with
anti-CREB antibody The expected size (168 bp;
Fig 5B, left panel) of an amplicon containing the
CRE at )59 was detected in the
formaldehyde-medi-ated DNA–protein complexes immunoprecipitated
with anti-CREB antibody (Fig 5B, right panel)
More-over, the binding efficiency was enhanced when the
cells were treated with Fluprostenol (Fig 5B, right
panel), and the Fluprostenol-derived increase in the
efficiency of binding of CREB to the CRE was clearly
suppressed by the co-treatment with either AL8810 or
PD98059 (Fig 5B, right panel) In contrast, there was
no detectable signal when rabbit normal IgG was
added, although the signals were detected in both
input controls (data not shown) These results, taken
together, indicate that Fluprostenol-mediated COX-2
expression via the FP receptor and MEK⁄ ERK
cas-cade was enhanced through the binding of CREB to
the CRE at )59 of the COX-2 promoter in 3T3-L1
cells
Discussion
PGs are lipid mediators involved in the regulation of
cell growth, differentiation and homeostasis [5] as well
as that of adipogenesis PGD2 accelerates adipogenesis
[7] and PGD2-overexpressing mice become obese when
given a high fat diet [31] In contrast, PGF2a and
PGE2suppress adipogenesis through specific receptors,
i.e FP [9–12] and EP4 [8], respectively, and inhibit the
functions of peroxisome proliferator-activated receptor
(PPAR)c, which is a key transcription factor that
regu-lates adipogenesis and is expressed in the mid-late
phase of adipogenesis [32,33] A recent study
demon-strated that PGF2a was detected in pre-adipocytes and
that its level was enhanced with a peak at 3 h after the
initiation of adipogenesis and then decreased [15],
indi-cating that PGF2a-mediated suppression of
adipogene-sis is an early event during adipogeneadipogene-sis In this study,
we found an increase in the PGF2a level along with
enhanced expression of COX-2 when the FP receptor
in adipocytes was activated by a stable PGF2a analog,
Fluprostenol (Fig 1A) Thus, we suspect the existence
of a regulatory loop for the enhancement of PGF2a
production by activation of FP receptor signaling Our
present study demonstrated that PGF2aenhanced
anti-adipogenic PGF2a and PGE2 production through
the FP receptor-activated COX-2 expression via the
MEK⁄ ERK-CREB cascade to form a positive feed-back loop, one that probably plays an important role
in the regulation of the early phase of adipogenesis (Fig 6)
PGs have numerous functions, and their activities are achieved by their binding to their specific receptors PGF2a binds to the FP receptor, which is a G-protein-coupled receptor; and this binding activates the downstream signaling pathways including the Gq heterotrimeric G protein, and then further increases the intracellular calcium level and activates various kinases including MEK [16,18–20] MEK, also known
as MAPK kinase [34], is an activator of ERK, a MAPK The MEK⁄ ERK pathway is a critical signal-ing pathway that regulates a number of cellular func-tions such as cell growth and differentiation [34] The upstream component of the MEK⁄ ERK pathway is the Ras GTPase, which activates the serine⁄ threonine kinase Raf, which in turn phosphorylates ERK1⁄ 2 through the Ras⁄ Raf ⁄ MEK ⁄ ERK signal transduction pathway [34] PGF2a stimulates Raf⁄ MEK ⁄ ERK sig-naling in luteal [35], endometrial [36], pulp cells [37] and osteoblasts [38,39] Previously, PGF2a was shown
to regulate COX-2 expression in an autocrine⁄ para-crine manner to establish a positive feedback system in Ishikawa endometrial adenocarcinoma cells [36,40] Here, we demonstrated the PGF2a–FP receptor-derived activation of COX-2 expression via the activation of the MEK⁄ ERK cascade Treatment of 3T3-L1 cells with the FP receptor antagonist or MEK inhibitor significantly reduced the Fluprostenol-activated COX-2 expression To determine whether the enhanced COX-2
FP
MEK
ERK
P
PPARγ
CREB
Fig 6 Schematic representation of a novel suppression mecha-nism operating in the early phase of adipogenesis through a posi-tive feedback loop by enhancement of PGF2a-mediated COX-2 expression via FP receptor-activated MEK ⁄ ERK-CREB cascade.
Trang 9expression induced after the PGF2a–FP receptor
inter-action could lead to de novo biosynthesis of
prosta-noids, we treated 3T3-L1 cells with Fluprostenol for
1 h to induce COX-2 expression in the absence or
presence of the selective inhibitors of COX-2 and
MEK or FP receptor antagonist We found that
PGF2a and PGE2were de novo synthesized by the
acti-vation of FP receptor and that this effect was
abol-ished by the co-treatment with the FP receptor
antagonist or MEK inhibitor (Fig 4) The inhibition
of PG biosynthesis by the specific COX-2 inhibitor
NS-398 and the MEK inhibitor PD98059 confirmed
that the increased production of PGF2aand PGE2 was
a consequence of the elevated expression of the COX-2
gene, which was dependent on the phosphorylation of
ERK1⁄ 2 (Fig 3B) Thus, PGF2a and PGE2
produc-tion through PGF2a–FP receptor-MEK⁄ ERK-activated
COX-2 expression was involved in the regulation of
the early phase of adipogenesis Hence, adipogenesis
could be regulated through a self-amplifying loop,
trig-gered by PGF2a–FP receptor coupling and activation
of the COX-2 gene expression via the MEK⁄ ERK
cas-cade However, this PGF2a-mediated acceleration of
suppression of the early phase of adipogenesis was
transient, returning to basal levels within 3 h after the
treatment with PGF2a, indicating that this signaling
cascade was rapidly desensitized in the presence of
PGF2a The desensitization mechanism to PGF2a
should be elucidated to fully understand the PGF2a
-derived suppression of adipogenesis in its early phase
The regulatory mechanism governing the
transcrip-tion of the COX-2 gene has been extensively studied
[41], and various transcription factors are now known
to be involved in the regulation of COX-2 gene
expres-sion [41] CRE was first identified as an element in the
promoter of genes transcribed in response to the
eleva-tion of the cAMP level [42] CREB is also involved in
the regulation of COX-2 gene expression in various
cells [43] through activation of protein kinase C and
Ca2+ signaling cascades in ileal epithelial IEC-18 cells
[44] or the protein kinase A pathway in human amnion
fibroblasts [45] In our present study, we demonstrated
CREB to be a critical transcription factor in the
PGF2a-derived enhancement of COX-2 gene expression
through the FP receptor-activated ERK⁄ MEK cascade
in 3T3-L1 cells (Fig 5A,B)
In summary, our data provide a novel regulatory
loop of the PGF2a-activated FP receptor-MEK⁄
ERK-CREB-COX-2 cascade that coordinately suppresses
the early phase of adipogenesis via de novo synthesis of
anti-adipogenic PGF2a and PGE2 Therefore, PGF2a
plays a critical role in suppressing the progression
of the early phase of adipogenesis, and this positive
feedback loop may provide a novel therapeutic strat-egy for the treatment of obesity
Experimental procedures
Cell culture Mouse 3T3-L1 cells were obtained from the Human Science Research Resources Bank (Osaka, Japan), cultured in DMEM (Sigma, St Louis, MO, USA) containing 10%
RNA preparation and quantification of RNA level Total RNA was extracted with Sepasol-RNAI (Nacalai Tesque, Kyoto, Japan), and was then further purified with
an RNeasy Purification System (Qiagen, Hilden, Germany) The first-strand cDNAs were synthesized from 1 lg of total RNA with random hexamer and ReverTra Ace Reverse
cDNAs were diluted and further utilized as the templates for quantitative PCR analysis
Expression levels were quantified by using a LightCycler system (Roche Diagnostics, Mannheim, Germany) with Thunderbird SYBR qPCR Mix (Toyobo) and the primer sets shown in Table S1 The expression level of the target genes was estimated by the use of concentration-known standard DNA, and normalized to that of SASA-binding protein
Western blot analysis
mix-ture (Nacalai Tesque) and phosphatase inhibitors, 50 lm
soni-cation, cell extracts were prepared by centrifugation for
tein concentrations were measured with a Pierce BCA Pro-tein Assay Reagent (Thermo Scientific, Rockford, IL,
transferred onto an Immobilon PVDF membrane (Milli-pore, Bedford, MA, USA) Blots were first incubated with primary antibodies, i.e anti-COX-2 polyclonal antibody (pAb) (1 : 200, C-20; Santa Cruz Biotech., Santa Cruz, CA,
Cell Signaling, Danvers, MA, USA) or anti-actin monoclo-nal antibody (1 : 5000, AC-15; Sigma), followed by incuba-tion with anti-rabbit, anti-goat or anti-mouse IgG antibody
Trang 10conjugated with horseradish peroxidase (Santa Cruz
Bio-tech.) Immunoreactive signals were detected by the use of
an Immobilon Western Detection Reagent (Millipore) and
LAS-3000 Luminoimage Analyzer (Fujifilm, Tokyo, Japan),
and analyzed with multi gauge software (Fujifilm)
Measurement of PGF2aand PGE2by enzyme
immunoassay (EIA)
Cells were incubated in DMEM containing A23187 (5 lm;
Calbiochem, San Diego, CA, USA), a calcium ionophore,
Chem-ical, Ann Arbor, MI, USA) according to the
manufac-turer’s instructions
Construction of luciferase reporter vectors and
luciferase assay
The luciferase reporter vectors carrying the mouse COX-2
sequentially deleted region of the COX-2 promoter was
cloned into the pGL4.10[luc2] vector (Promega, Madison,
WI, USA) Site-directed mutation was introduced by using
a QuikChange Site-directed Mutagenesis Kit (Stratagene,
La Jolla, CA, USA) according to the manufacturer’s
instruction Nucleotide sequences of the constructs were
determined to verify the correct sequences
3T3-L1 cells were co-transfected with each construct
(0.9 lg) and pRL-SV40 (0.1 lg, Promega) in six-well plates,
the latter plasmid carrying the Renilla luciferase gene under
the control of the SV40 promoter as the transfection
con-trol, along with FuGENE6 Transfection Reagent (Roche
Diagnostics) according to the manufacturer’s instructions
The cells were cultured for a further 48 h, and the luciferase
activities were measured by using a Dual-Glo Luciferase
Assay System (Promega) The reporter activity was
calcu-lated relative to that of pGL4.10[luc2] vector, which was
defined as 1 Data were obtained from three independent
experiments, and each experiment was performed in
tripli-cate The relative promoter activities are presented as the
mean ± SD
Chromatin immunoprecipitation assay
The ChIP assay was performed as described previously [7]
by the use of CREB pAb (SC-25785X; Santa Cruz
Bio-tech.) Immunoprecipitated DNA–protein complexes were
reverse crosslinked, and the DNAs were purified by using a
MinElute PCR Purification Kit (Qiagen) and used for
sub-sequent PCR amplification with KOD FX DNA
Polymer-ase (Toyobo) and the following specific primer set for CRE
AAGTTGG-3¢ and 5¢-GAGCAGAGTCCTGACTGACTC-3¢ PCR was conducted under the following conditions: initial
amplified PCR products (expected size 168 bp) were ana-lyzed by performing agarose gel electrophoresis
Statistical analysis Differences between two groups were analyzed by the unpaired t-test or Welch t-test P-values < 0.05 were con-sidered significant
Acknowledgements
We acknowledge Dr Fumio Amano (Osaka University
of Pharmaceutical Sciences) for valuable discussions This work was supported in part by a Grant-in-Aid for Scientific Research and Scientific Research on Innovative Areas from the Ministry of Education, Cul-ture, Sports, Science and Technology of Japan, and by grants from Suzuken Memorial Foundation, the Sumi-tomo Foundation, Gushinkai Foundation, Japan Foundation for Applied Enzymology, Takeda Science Foundation and the Research Foundation for Pharma-ceutical Sciences (to K.F.)
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