Novel suppression mechanism operating in early phase of adipogenesis by positive feedback loop for enhancement of cyclooxygenase-2 expression through prostaglandin F2a receptor mediated activation of MEK ⁄ ERK-CREB cascade 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 April 2011, revised 31 May 2011, accepted June 2011) doi:10.1111/j.1742-4658.2011.08213.x Prostaglandin (PG) F2a suppresses 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 PGE2 by enhancing cyclooxygenase (COX) expression through the PGF2aactivated FP receptor ⁄ extracellular-signal-regulated kinase (ERK) ⁄ cyclic AMP response element binding protein (CREB) cascade COX-2 expression was enhanced with a peak at h for the mRNA level and at h for the protein 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-activated protein kinase (MEK; ERK kinase) inhibitor, PD98059 ERK was phosphorylated within 10 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 Furthermore, promoter-luciferase and chromatin immunoprecipitation assays demonstrated 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 promoter via FP receptor activated MEK ⁄ ERK cascade Thus, PGF2a forms a positive feedback loop that coordinately suppresses the early phase of adipogenesis through the increased production of anti-adipogenic PGF2a and 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 associated 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; PLA2, phospholipase A2; PPAR, peroxisome proliferator-activated receptor FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS 2901 Suppression of adipogenesis via FP-ERK-CREB-COX-2 T Ueno and K Fujimori Adipocyte 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 PGH2 by either cyclooxygenase (COX) or COX-2 Third, PGH2, which is a common precursor of all prostanoids, is metabolized to various PGs by the action of specific PG synthases [5,6] PGs are involved in the regulation of adipocyte differentiation Lipocalin-type PGD-synthase-produced PGD2 enhances adipocyte differentiation [7], whereas PGE2 and PGF2a suppress the differentiation of adipocytes through EP4 [8] and FP [9–12] receptors, respectively, indicating that PGE2 and PGF2a are anti-adipogenic factors PGF2a plays a variety of physiological roles in the body and is synthesized 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 synthase in adipocytes and that AKR1B3-derived PGF2a suppresses the early phase of adipogenesis [14,15] through a specific cell surface G-protein-coupled receptor, 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 conversion of arachidonic acid into endoperoxide intermediates 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 stimuli 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 production of anti-adipogenic prostanoids [23] In contrast, selective COX-2 inhibitors impair adipocyte differentiation through inhibition of the clonal expansion phase [24] Moreover, COX-2-deficient mice exhibit suppressed adipocyte differentiation [25] Thus, arachidonic 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 COX2-derived PGs play suppressive roles in the early phase of adipogenesis [14,15], while COX-2 and lipocalintype PGD-synthase-derived PGD2 activates the late 2902 phase of adipogenesis [7] The regulation of adipogenesis 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 production of anti-adipogenic PGF2a and PGE2 by enhancing the expression of COX-2 through FP-receptor-activated mitogen-activated protein kinase ⁄ extracellular signalregulated kinase kinase (MEK) ⁄ extracellular signalregulated 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-CREBCOX-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 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 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 production in adipocytes, at first we investigated the expression of genes involved in the biosynthesis of PGF2a and FP receptor in Fluprostenol-treated 3T3-L1 cells by performing quantitative PCR analysis The transcription level of the COX-2 gene was increased  3.7fold, 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 cytosolic PLA2 (cPLA2), COX-1 and AKR1B3 genes were not altered by the treatment of the cells with Fluprostenol (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 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 h after the addition of Fluprostenol and then decreased FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS T Ueno and K Fujimori Suppression of adipogenesis via FP-ERK-CREB-COX-2 A B 1.6 mRNA level (/TBP mRNA level) PGF2α(ng·mL–1) 0.8 0.4 0.08 ** 0.5 0.06 0.04 0.25 0.02 + – FP 0.75 * * 1.2 COX-2 0.1 + – – Fluprostenol Fluprostenol C 0.06 cPLA2 10 + Fluprostenol COX-1 10 10 AKR1B3 mRNA level (/TBP mRNA level) 0.04 0.02 7.5 7.5 7.5 5 2.5 2.5 2.5 + – PGF2α COX-2mRNA level (/TBP mRNA level) D – Fluprostenol E 0.2 * 0.1 0.05 * + – Fluprostenol – + Fluprostenol COX-2 Actin 0.15 0 + * * 12 Time after addition of Fluprostenol (h) Relative band intensity COX-2mRNA level (/TBP mRNA level) * 1.5 * * 0.5 0 12 Time after addition of Fluprostenol (h) Fig Enhancement of COX-2 expression by Fluprostenol, an FP receptor agonist (A) Induction of PGF2a production by treatment with Fluprostenol Pre-adipocytic 3T3-L1 cells were incubated with Fluprostenol (0.5 nM; Cayman Chemical) or vehicle for h, followed by treatment with A23187 (5 lM) for 10 at 37 °C, after which the medium was collected to measure the PGF2a level by EIA *P < 0.01, compared with vehicle-treated cells (B) Expression levels of COX-2, FP receptor, cPLA2, COX-1 and AKR1B3 genes in the Fluprostenol-treated 3T3-L1 cells Cells were incubated with Fluprostenol (0.5 nM) for 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 nM) for 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 nM) 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 FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS 2903 Suppression of adipogenesis via FP-ERK-CREB-COX-2 T Ueno and K Fujimori to almost the same level as detected in the vehicle-treated cells, although the actin levels were not altered in each sample (Fig 1E) These results, all taken together, reveal that Fluprostenol-activated FP receptors increased PGF2a production 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 inhibitor, PD98059; a PLC inhibitor, U73122; a PKC inhibitor, bisindolylmaleimide I; and a general COX inhibitor, indomethacin, or a selective COX-2 inhibitor, NS-398, in the presence of Fluprostenol for h COX-2 expression was enhanced by the treatment with Fluprostenol compared with that obtained with the vehicle control (Fig 2A) Fluprostenol FP receptoractivated 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 elevation 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 expression occurred through FP receptor and MEK signaling in 3T3-L1 cells Phosphorylation of ERK1 ⁄ 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 ⁄ was activated by MEK in the Fluprostenol-treated 3T3-L1 cells ERK1 ⁄ was expressed constitutively even in the absence of Fluprostenol, and slightly phosphorylated in the vehicle-treated cells (Fig 3A) When the cells were cultured in DMEM containing Fluprostenol, phosphorylation of ERK1 ⁄ was enhanced with a peak 10 after the addition of Fluprostenol and then decreased (Fig 3A) To confirm the activation of ERK1 ⁄ via Fluprostenol-activated FP receptors, we cultured the cells in the 2904 presence of AL8810 or PD98059 and Fluprostenol, and assessed the level of phosphorylated ERK1 ⁄ protein by western blot analysis Fluprostenol-mediated phosphorylation of ERK1 ⁄ 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 through the FP receptor Enhanced de novo synthesis of PGF2a and PGE2 by activation of FP receptor MEK ⁄ ERK cascade Next we examined whether FP receptor-mediated activation of the MEK ⁄ ERK cascade would enhance PGF2a production in 3T3-L1 cells PGF2a production was increased by the treatment with Fluprostenol (Figs and 4, left panel) Fluprostenol-derived elevation of PGF2a production was decreased by the treatment with AL8810, PD98059, indomethacin or NS-398 (Fig 4, left panel), whereas U73122 and bisindolylmaleimide 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 Fluprostenolinduced PGE2 production (Fig 4, right panel) In contrast, 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 Fluprostenol (data not shown) These results reveal that Fluprostenol-derived activation of the FP receptor MEK ⁄ ERK cascade enhanced de novo synthesis of anti-adipogenic PGF2a and PGE2 in 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 reporter assays using various deleted or mutated promoterreporter 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 FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS T Ueno and K Fujimori Suppression of adipogenesis via FP-ERK-CREB-COX-2 A 0.01 COX-2 mRNA level (/TBP mRNA level) # 0.0075 0.005 0.0025 V V AL PD U Bis I NS +Fluprostenol B 0.03 COX-2 mRNA level (/TBP mRNA level) 0.03 * * * * 0.02 0.01 * 0.02 0.01 Fluprostneol (nM) 5 Fluprostenol (nM) 5 AL 8810 (μM) 0 10 50 PD98059 (μM) 0 10 C COX-2 COX-2 Actin Actin 1.5 ** * Relative band intensity Relative band intensity Fig 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 nM) with or without AL8810 (50 lM; Cayman Chemical), which is an FP receptor antagonist, each of various protein kinases: the MEK inhibitor, PD98059 (PD) (10 lM, Calbiochem), the PLC inhibitor U73122 (U) (1 lM, Calbiochem), the PKC inhibitor bisindolylmaleimide I (Bis) (0.1 lM, Calbiochem), the general COX inhibitor indomethacin (I) (2 lM, Cayman Chemical) and the COX-2 specific inhibitor NS-398 (NS) (1 lM, Cayman Chemical) for Cells were further incubated for h with Fluprostenol (0.5 nM) 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 Fluprostenolactivated COX-2 expression by AL8810 or PD98059 Cells were incubated with AL8810 (0–50 lM) or PD98059 (0–10 lM) together with Fluprostenol (5 nM) The expression level of COX-2 gene was quantified 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 * * * * * 0.5 Fluprostneol (nM) 5 Fluprostenol (nM) 5 AL 8810 (μM) 0 10 50 PD98059 (μM) 0 10 transfected cells were treated with Fluprostenol, luciferase reporter activity was  138% (black column) of that of the vehicle (white column); and this Fluprostenol-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 FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS 2905 Suppression of adipogenesis via FP-ERK-CREB-COX-2 T Ueno and K Fujimori B A P-ERK1/2 P-ERK1/2 ERK1/2 ERK1/2 10 30 60 Time after addition of Fluprostenol (min) * * – – – * 10 30 60 Time after addition of Fluprostenol (min) Relative band intensity Relative band intensity Fluprostenol AL8810 PD98059 + – – + + – + – + * * * Fluprostenol AL8810 PD98059 – – – + – – + + – + – + Fig Time course of PGF2a-stimulated ERK1 ⁄ phosphorylation (A) Fluprostenol-induced phosphorylation of ERK1 ⁄ in 3T3-L1 cells Cells were cultured with Fluprostenol (0.5 nM) for 0–60 Cell lysates (20 lgỈlane)1) were subjected to SDS ⁄ PAGE and western blot analysis for phosphorylated ERK1 ⁄ and total ERK1 ⁄ 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 ⁄ in 3T3-L1 cells Cells were cultured with Fluprostenol (0.5 nM) in the presence or absence of AL8810 (50 lM) or PD98059 (10 lM) for 10 Cell lysates (20 lgỈlane)1) were used for SDS ⁄ PAGE and western blot analysis to determine the levels of phospho-ERK1 ⁄ and total ERK1 ⁄ proteins Band intensity was measured by using MULTI GAUGE software The data shown are representative of three independent experiments *P < 0.01, as indicated by the brackets 1.8 * * * 30 * 25 PGE2 (ng·mL–1) PGF2α (ng·mL–1) 1.5 * 1.2 0.9 0.6 0.3 * * * * * 20 15 10 V V AL PD I NS U Bis + Fluprostenol V V AL PD I NS U Bis + Fluprostenol Fig Effect of kinase inhibitors or COX inhibitors on Fluprostenol-stimulated PGF2a and PGE2 production 3T3-L1 cells were incubated in DMEM containing AL8810 (AL) (10 lM), PD98059 (PD) (10 lM), U73122 (U) (1 lM), bisindolylmaleimide I (Bis) (0.1 lM), COX inhibitors indomethacin (I) (2 lM) or NS-398 (NS) (1 lM) together with Fluprostenol (5 nM) for h The medium was then removed and replaced with fresh medium containing inhibitor and A23187 (5 lM), and the cells were further incubated for 10 The medium was collected for the measurement of PGF2a and PGE2 levels by performing the respective EIAs Data are expressed as the mean ± SD of three independent experiments *P < 0.01, as indicated by the brackets containing 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 construct (Fig 5A) However, the responsiveness to Fluprostenol 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 2906 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), FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS T Ueno and K Fujimori Suppression of adipogenesis via FP-ERK-CREB-COX-2 NF-κB CCAAT CRE (–224) (–135) (–59) A –500 +1 +124 * * –500/+124 * * –300/+124 * * * –140/+124 * * * –110/+124 * * * * * –50/+124 –500/+124 (mu) Vehicle Fluprostenol Fluprostenol + AL8810 pGL4.10 WT mu Relative luminescence CTACGTCA CagtaggA B ChIP: anti-CREB –59 5′ –52 CRE 3′ Relative band intensity 168-bp Input Fluprostenol AL8810 PD98059 + – – – – – + + – * * + – + * Fig Enhancement of PGF2a-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 nM, black column), Fluprostenol and AL8810 (10 lM, gray columns) or incubated without any treatment (white columns) for 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 nM) with or without AL8810 (10 lM) or PD98059 (10 lM) for h, and the ChIP assay was then carried 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 revealing 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 promoter (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 mutation 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) construct, 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 FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS 2907 Suppression of adipogenesis via FP-ERK-CREB-COX-2 T Ueno and K Fujimori (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-mediated DNA–protein complexes immunoprecipitated with anti-CREB antibody (Fig 5B, right panel) Moreover, 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 cascade 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 PGE2 suppress 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 regulates adipogenesis and is expressed in the mid-late phase of adipogenesis [32,33] A recent study demonstrated that PGF2a was detected in pre-adipocytes and that its level was enhanced with a peak at h after the initiation of adipogenesis and then decreased [15], indicating that PGF2a-mediated suppression of adipogenesis is an early event during adipogenesis 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 PGF2a enhanced antiadipogenic PGF2a and PGE2 production through the FP receptor-activated COX-2 expression via the 2908 MEK ⁄ ERK-CREB cascade to form a positive feedback 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-proteincoupled 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 signaling pathway that regulates a number of cellular functions 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 ⁄ through the Ras ⁄ Raf ⁄ MEK ⁄ ERK signal transduction pathway [34] PGF2a stimulates Raf ⁄ MEK ⁄ ERK signaling in luteal [35], endometrial [36], pulp cells [37] and osteoblasts [38,39] Previously, PGF2a was shown to regulate COX-2 expression in an autocrine ⁄ paracrine 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 PGF2α α FP MEK P ERK PPARγ CREB Adipogenesis COX-2 PGF2α AKR1B3 PGE2 Fig Schematic representation of a novel suppression mechanism operating in the early phase of adipogenesis through a positive feedback loop by enhancement of PGF2a-mediated COX-2 expression via FP receptor-activated MEK ⁄ ERK-CREB cascade FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS T Ueno and K Fujimori expression induced after the PGF2a–FP receptor interaction could lead to de novo biosynthesis of prostanoids, we treated 3T3-L1 cells with Fluprostenol for 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 PGE2 were de novo synthesized by the activation of FP receptor and that this effect was abolished 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 PGF2a and PGE2 was a consequence of the elevated expression of the COX-2 gene, which was dependent on the phosphorylation of ERK1 ⁄ (Fig 3B) Thus, PGF2a and PGE2 production 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, triggered by PGF2a–FP receptor coupling and activation of the COX-2 gene expression via the MEK ⁄ ERK cascade However, this PGF2a-mediated acceleration of suppression of the early phase of adipogenesis was transient, returning to basal levels within 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 PGF2aderived suppression of adipogenesis in its early phase The regulatory mechanism governing the transcription 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 expression [41] CRE was first identified as an element in the promoter of genes transcribed in response to the elevation 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 ⁄ ERKCREB-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 Suppression of adipogenesis via FP-ERK-CREB-COX-2 feedback loop may provide a novel therapeutic strategy 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% (v ⁄ v) fetal bovine serum and antibiotics, and maintained in a humidified atmosphere of 5% CO2 at 37 °C [15] 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 lg of total RNA with random hexamer and ReverTra Ace Reverse Transcriptase (Toyobo, Osaka, Japan) at 42 °C for 60 after the initial denaturation at 72 °C for min, followed by heat inactivation of enzyme at 99 °C for The 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 Cells were lysed in RIPA buffer containing 50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 0.1% (w ⁄ v) SDS, 0.5% (w ⁄ v) sodium deoxycholate, 1% (v ⁄ v) Nonidet P-40 and 1% (v ⁄ v) Triton X-100 along with a protease inhibitor mixture (Nacalai Tesque) and phosphatase inhibitors, 50 lm Na2MoO4, mm NaF and mm Na3VO4 After mild sonication, cell extracts were prepared by centrifugation for 20 at 12 000 g at °C to remove the cell debris Protein concentrations were measured with a Pierce BCA Protein Assay Reagent (Thermo Scientific, Rockford, IL, USA) Proteins were separated on SDS ⁄ PAGE and then transferred onto an Immobilon PVDF membrane (Millipore, 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, USA), anti-ERK1 ⁄ 2, anti-phospho ERK1 ⁄ pAbs (1 : 500; Cell Signaling, Danvers, MA, USA) or anti-actin monoclonal antibody (1 : 5000, AC-15; Sigma), followed by incubation with anti-rabbit, anti-goat or anti-mouse IgG antibody FEBS Journal 278 (2011) 2901–2912 ª 2011 The Authors Journal compilation ª 2011 FEBS 2909 Suppression of adipogenesis via FP-ERK-CREB-COX-2 T Ueno and K Fujimori conjugated with horseradish peroxidase (Santa Cruz Biotech.) 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 PGF2a and PGE2 by enzyme immunoassay (EIA) Statistical analysis Cells were incubated in DMEM containing A23187 (5 lm; Calbiochem, San Diego, CA, USA), a calcium ionophore, along with any inhibitors for 10 at 37 °C; then the medium was collected and measured for PGF2a and PGE2 levels by using PGF2a and PGE2 EIA Kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions Construction of luciferase reporter vectors and luciferase assay The luciferase reporter vectors carrying the mouse COX-2 promoter were generated as follows An  600 bp and 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 control, 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 calculated relative to that of pGL4.10[luc2] vector, which was defined as Data were obtained from three independent experiments, and each experiment was performed in triplicate 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 Biotech.) Immunoprecipitated DNA–protein complexes were reverse crosslinked, and the DNAs were purified by using a MinElute PCR Purification Kit (Qiagen) and used for subsequent PCR amplification with KOD FX DNA Polymerase (Toyobo) and the following specific primer set for CRE at )59 in the COX-2 promoter: 5¢-CAGAGAGGGGGAA 2910 AAGTTGG-3¢ and 5¢-GAGCAGAGTCCTGACTGACTC-3¢ PCR was conducted under the following conditions: initial denaturation at 94 °C for min, followed by 32 cycles of 98 °C for 10 s, 55 °C for 20 s and 60 °C for 20 s The amplified PCR products (expected size 168 bp) were analyzed by performing agarose gel electrophoresis Differences between two groups were analyzed by the unpaired t-test or Welch t-test P-values < 0.05 were considered 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, Culture, Sports, Science and Technology of Japan, and by grants from Suzuken Memorial Foundation, the Sumitomo Foundation, Gushinkai Foundation, Japan Foundation for Applied Enzymology, Takeda Science Foundation and the Research Foundation for Pharmaceutical Sciences (to K.F.) 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CREB Adipogenesis COX-2 PGF2α AKR1B3 PGE2 Fig Schematic representation of a novel suppression mechanism operating in the early phase of adipogenesis through a positive feedback loop by enhancement. .. molecular mechanism of this Fluprostenol -mediated increase in PGF2a production in adipocytes, at first we investigated the expression of genes involved in the biosynthesis of PGF2a and FP receptor in. .. self-amplifying loop, triggered by PGF2a–FP receptor coupling and activation of the COX-2 gene expression via the MEK ⁄ ERK cascade However, this PGF2a -mediated acceleration of suppression of the early phase