The Tpl2 Kinase Regulates the COX-2/Prostaglandin E2 Axis in Adipocytes in Inflammatory Conditions

Flavien Berthou; Franck Ceppo; Karine Dumas; Fabienne Massa; Bastien Vergoni; Susana Alemany; Mireille Cormont; Jean-François Tanti

doi:10.1210/me.2015-1027

. 2015 May 28;29(7):1025–1036. doi: 10.1210/me.2015-1027

The Tpl2 Kinase Regulates the COX-2/Prostaglandin E2 Axis in Adipocytes in Inflammatory Conditions

Flavien Berthou ^1,^*, Franck Ceppo ^1,^*, Karine Dumas ¹, Fabienne Massa ¹, Bastien Vergoni ¹, Susana Alemany ¹, Mireille Cormont ¹, Jean-François Tanti ^1,^✉

PMCID: PMC5414708 PMID: 26020725

Abstract

Bioactive lipid mediators such as prostaglandin E2 (PGE2) have emerged as potent regulator of obese adipocyte inflammation and functions. PGE2 is produced by cyclooxygenases (COXs) from arachidonic acid, but inflammatory signaling pathways controlling COX-2 expression and PGE2 production in adipocytes remain ill-defined. Here, we demonstrated that the MAP kinase kinase kinase tumor progression locus 2 (Tpl2) controls COX-2 expression and PGE2 secretion in adipocytes in response to different inflammatory mediators. We found that pharmacological- or small interfering RNA-mediated Tpl2 inhibition in 3T3-L1 adipocytes decreased by 50% COX-2 induction in response to IL-1β, TNF-α, or a mix of the 2 cytokines. PGE2 secretion induced by the cytokine mix was also markedly blunted. At the molecular level, nuclear factor κB was required for Tpl2-induced COX-2 expression in response to IL-1β but was inhibitory for the TNF-α or cytokine mix response. In a coculture between adipocytes and macrophages, COX-2 was mainly increased in adipocytes and pharmacological inhibition of Tpl2 or its silencing in adipocytes markedly reduced COX-2 expression and PGE2 secretion. Further, Tpl2 inhibition in adipocytes reduces by 60% COX-2 expression induced by a conditioned medium from lipopolysaccharide (LPS)-treated macrophages. Importantly, LPS was less efficient to induce COX-2 mRNA in adipose tissue explants of Tpl2 null mice compared with wild-type and Tpl2 null mice displayed low COX-2 mRNA induction in adipose tissue in response to LPS injection. Collectively, these data established that activation of Tpl2 by inflammatory stimuli in adipocytes and adipose tissue contributes to increase COX-2 expression and production of PGE2 that could participate in the modulation of adipose tissue inflammation during obesity.

Obesity is associated with a state of low-grade inflammation of adipose tissue that contributes to its dysfunction. These alterations of adipose tissue predispose to insulin resistance development and to the onset of type 2 diabetes (1, 2). Macrophages and other immune cells infiltrate obese adipose tissue and a cross talk between macrophages and adipocytes perpetuates a vicious cycle that sustains adipose tissue inflammation (3, 4). Inflammatory cytokines, such as IL-1β and TNF-α, mainly produced by macrophages and free fatty acids or abnormal adipokines secretions by adipocytes are involved in this paracrine loop (3, 5, 6). Obesity is also characterized by an increase in lipopolysaccharide (LPS) circulating level due to modifications in gut microbiota. By activating macrophages in obese adipose tissue, LPS contributes to the chronic “low-grade” inflammation and to adipocyte dysfunction (7, 8). Thus, inflammatory mediators produced by macrophages or obese adipocytes contribute to the development of insulin resistance and other obesity-related metabolic complications.

Bioactive lipid mediators have emerged as potent regulators of adipose tissue inflammation and functions (9 –11). Among them, prostaglandins are synthetized from arachidonic acid by the cyclooxygenase (COX) pathway. Two isoforms of COX namely COX-1 and COX-2 exist but COX-1 is constitutively expressed in most tissues, whereas COX-2 is induced by different inflammatory cytokines, including IL-1β and TNF-α, or inflammatory signals such as LPS (9, 12). The most abundant COX-2 product in adipose tissue is prostaglandin E2 (PGE2) that is produced in adipose tissue by both adipocytes and stromal cells (9, 13). PGE2 and COX-2 are important actors in inflammation in various cell types (14) and pharmacological inhibition of COX-2 decreases adipose tissue inflammation and prevents the development of insulin resistance (15 –17). COX-2-dependent production of PGE2 was also shown to alter the resolution of inflammation in obesity (18). In addition to inflammation, COX-2 and PGE2 could also alter adipose tissue development by suppressing adipogenesis (19 –21). Thus, COX-2 expression and PGE2 production by adipocytes in response to inflammatory mediators may contribute to obese adipose tissue dysfunction. However, the signaling pathways activated by inflammatory mediators and involved in the increased expression of COX-2 in adipocytes are not well understood.

Deregulation in MAPK signaling is a common alteration in obese tissues, including adipose tissue (22). The kinase tumor progression locus 2 (Tpl2), also known as cancer Osaka thyroid, is a MAP kinase kinase kinase involved in the activation of ERK1/2 by phosphorylating mitogen/extracellular signal-regulated kinase, the ERK kinase (23). Tpl2 has been described as the only one serine/threonine kinase involved in the activation of ERK1/2 in response to inflammatory stimuli such as IL-1β, TNF-α, and LPS in immune cells. Furthermore, Tpl2 activation is required for the production of IL-1β and TNF-α by macrophages (24 –26). Deregulation of Tpl2 expression was found in several inflammatory diseases as well as in some cancers (27, 28). Tpl2 expression is up-regulated in obese adipose tissue and its activation by inflammatory cytokines increases lipolysis and alters insulin signaling in adipocytes (29). Moreover, involvement of Tpl2 in the cross talk between adipocytes and macrophages leading to the establishment of an inflammatory paracrine loop and to adipocyte insulin resistance has been recently described (30). Studies of Tpl2-deficient mice have revealed a role of Tpl2 in obese adipose tissue inflammation (31, 32), even if the implication of Tpl2 in insulin resistance is still a matter of debate (31 –33).

Tpl2 not only activates ERK1/2 signaling, but it is also implicated in cross talks with other signaling pathways, including nuclear factor κB (NF-κB) and nuclear factor of activated T-cells (NFAT) (23, 34 –36). These transcription factors are involved in the control of COX-2 expression in several cell types. However, the role of Tpl2 in the control of COX-2 expression is complex, because Tpl2 promotes or represses COX-2 expression and PGE2 production depending on the cell type (35, 37 –39). Thus, a role of Tpl2 in COX-2 regulation and PGE2 production in adipocytes in response to inflammatory mediators remains to be established.

Here, we demonstrate that Tpl2 positively control COX-2 expression and PGE2 secretion in adipocytes in response to IL-1β and TNF-α and in response to inflammatory mediators produced by LPS-activated macrophages. Further, we provide evidences that COX-2 induction by inflammatory mediators is blunted in adipose tissue of Tpl2-deficient mice.

Materials and Methods

Reagents and antibodies

DMEM, fetal bovine serum, and antibiotics were purchased from Invitrogen (Life Technologies SAS). INTERFERin transfection reagent was from Polyplus transfection. Protease inhibitors cocktails were obtained from Roche Diagnostics. The Tpl2 kinase inhibitor (4-(3-chloro-4-fluorophenylamino)-6-(pyridine-3-yl-methylamino)-3-cyano-[1,7]-napthyridine) (40, 41) was obtained from Calbiochem. The cell-permeable, irreversible pan-caspase inhibitor Z-Val-Ala-DL-Asp-FMK (fluoromethylketone) [Z-VAD-FMK] was purchased from Enzo Life Science. Murine IL-1β and TNF-α were purchased from PeproTech. LPS from Escherichia coli 0111:B4 strain and all other chemical reagents were purchased from Sigma. siRNAs against Tpl2 or p65 NF-κB the p65 subunit of NF-kB were purchased from Dharmacon (Thermo Fisher Scientific, Inc). Polyvinylidene difluoride membranes were purchased from Millipore. Bicinchoninic acid reagent was obtained from Pierce Biotechnology. Enhanced chemiluminescence reagent was purchased from PerkinElmer Life Sciences.

Antibodies against Tpl2, COX-2, heat shock protein of 90 kD (Hsp90), phospho-Ser276 p65NF-κB, and total-p65NF-κB were purchased from Santa Cruz Biotechnology, Inc (CliniSciences). All other antibodies were obtained from Cell Signaling Technology. Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson ImmunoResearch.

Culture of 3T3-L1 adipocytes and RAW264.7 macrophages

3T3-L1 preadipocytes were differentiated into adipocytes as previously described and used 10 days after the initiation of differentiation (42). RAW264.7 macrophages (thereafter referred as RAW macrophages) were maintained in DMEM containing 5% (vol/vol) heat-inactivated fetal bovine serum and antibiotics at 37°C and 5% CO₂/95% air atmosphere.

Measurement of 3T3-L1 apoptosis

3T3-L1 apoptosis was assessed by caspase-3/7 activity measurement using the Caspase-Glo 3/7 Assay (Promega Corp) after treatment of the cells with IL-1β (10 ng/mL), TNF-α (10 ng/mL), or a mix of the 2 cytokines (each at 10 ng/mL).

Coculture between adipocytes and macrophages

A direct coculture between 3T3-L1 adipocytes and RAW264.7 macrophages was performed as previously described (6, 30). Briefly, RAW macrophages (3 × 10⁵ cells) were seeded onto 12-well plates containing 3T3-L1 adipocytes (6 × 10⁵ cells). After 4 hours, the medium was changed for fresh medium with vehicle (dimethyl sulfoxide [DMSO]) or with the Tpl2 inhibitor (5μM). Twenty-four hours later, cells were lysed for mRNA or protein preparation. The medium was collected and centrifuged to eliminate potentially contaminating macrophages before the measurement of PGE2 secretion. As control, cells were cultured separately, treated exactly with the same conditions and mixed after harvest. In the transwell system, cells were cocultured as previously described (6). Briefly, transwell inserts with a 0.4-μm porous membrane (Corning) were used to separate adipocytes from macrophages. After incubation for 24 hours, adipocytes in the lower well were harvested.

Tpl2 silencing was performed in 3T3-L1 adipocytes by using Tpl2 siRNA (50nM) and INTERFERin following the reverse transfection protocol (43). RAW macrophages were seeded onto siTpl2-treated adipocytes 24 hours after transfection. The cells and the medium were harvested 24 hours later as described above.

Treatment of 3T3-L1 adipocytes with the CM from LPS-activated RAW264.7 macrophages

Adipocytes were treated with conditional medium (CM) from LPS-activated RAW macrophages (8 × 10⁵ cells/well for a 6-well plate) as previously described (30). Briefly, RAW macrophages were incubated with LPS (0.5 ng/mL) for 24 hours. Then, the CM was collected, centrifuged to eliminate potentially contaminating macrophages, and transferred for 24 hours onto 3T3-L1 adipocytes incubated without or with Tpl2 inhibitor (5μM) (1 mL of CM/well of a 12-well plate). As control, culture medium containing the same concentration of LPS was added onto adipocytes.

Injection of wild-type (WT) and Tpl2-deficient mice with LPS and treatment of adipose tissue explants with LPS

C57BL/6J WT and C57BL/6J Tpl2^−/− littermates were produced as previously described (38). Male mice were injected with LPS (2 μg/g of body weight) dissolved in NaCl 0.9% or were injected with NaCl 0.9% as control. After 5 hours, mice were killed by cervical dislocation and epididymal adipose tissue was removed, frozen in liquid nitrogen, and stored at −80°C before mRNA extraction.

Adipose tissue explants were prepared from epididymal fat pads of WT and Tpl2-deficient mice and incubated without or with LPS (100 ng/mL) for 24 hours in DMEM containing 10% fetal bovine serum and antibiotic at 37°C and 5% CO₂/95% air atmosphere. After 3 washes in PBS, adipose tissue explants were frozen in liquid nitrogen and store at −80°C before mRNA extraction.

Principles of laboratory animal care (NIH publication no. 85–23, revised 1985) were followed, as well those of the European Union guidelines on animal laboratory care. All procedures were approved by the Animal Care Committee of the Faculty of Medicine of the Nice Sophia Antipolis University (Comité Institutionnel d'Éthique Pour l'Animal de Laboratoire).

Western blot analysis

Proteins from cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes as previously described (42). Membranes were then incubated with the appropriate primary and secondary antibodies. Chemiluminescence was detected using Fujifilm LAS-3000 (Fujifilm Life Science). Quantifications were realized using MultiGauge software (Fujifilm Life Science).

Real-time RT-PCR

RNAs were prepared using the RNeasy total RNA kit (QIAGEN), treated with deoxyribonuclease (Applied Biosystems) and used to synthesize cDNAs using Transcriptor First Strand cDNA Synthesis kit (Roche). Real-time quantitative PCR was performed with sequence detection systems (Applied Biosystems StepOne or StepOnePlus Real-Time PCR Systems) and SYBR green dye as described (42). COX-2 mRNA expression was normalized with mouse Rplp0 mRNA. The relative amount of mRNA between 2 groups was determined by using the second derivative maximum method. Primers used were from SABiosciences (QIAGEN).

Quantification of PGE2

PGE2 production in culture medium was measured by enzyme immunoassay kit (Cayman Chemical) following the manufacturer's protocol.

Statistical analysis

Data are expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 software. Differences between groups were tested for significance by ANOVA with post hoc analysis or by Student's t test. P < .05 was considered to be statistically significant.

Results

Tpl2 in 3T3-L1 adipocytes regulates COX-2 expression and PGE2 secretion induced by inflammatory cytokines

To test the involvement of Tpl2 in COX-2 gene expression in response to inflammatory cytokines in adipocytes, 3T3-L1 adipocytes were incubated with a pharmacological inhibitor of Tpl2 (Tpl2-I) and then treated with IL-1β, TNF-α, or a mix of these 2 cytokines (MIX). IL-1β and TNF-α induced COX-2 mRNA expression by 4.7- and 6.5-fold, respectively, whereas when added together (MIX), IL-1β and TNF-α increased strongly this expression (22-fold), due to a synergistic effect between these 2 cytokines (Figure 1A). Pharmacological inhibition of Tpl2 prevented COX-2 gene expression by approximately 60% in response to the MIX, and by 25% and 50% in response to IL-1β or TNF-α treatment, respectively (Figure 1A). IL-1β and the cytokine mix also induced COX-2 protein by 1.6- and 2.5-fold, respectively, whereas the Tpl2 inhibitor totally abolished these effects (Figure 1B). Similar results were obtained in response to TNF-α treatment (data not shown). Moreover, the cytokine mix significantly increased PGE2 secretion which was reduced by around 30% after Tpl2 inhibition (Figure 1C). We observed a trend towards an increase in PGE2 secretion after IL-1β stimulation that did not reach statistically significance.

To confirm the involvement of Tpl2 in COX-2 mRNA expression, 3T3-L1 adipocytes were transfected with a siRNA against Tpl2. Tpl2 silencing (Figure 1D, inset) prevented by 50%–60% COX-2 gene expression induced by IL-1β, TNF-α, or the cytokine mix and markedly reduced the induction of COX-2 protein (Figure 1, D and E). PGE2 secretion in response to the cytokine mix was also reduced by 30% after Tpl2 silencing (Figure 1F).

Leukotrienes have been proposed as mediators of COX-2 mRNA induction under inflammatory conditions (44). We thus assessed the effect of cytokines on 5-lipoxygenase (Alox5) gene expression and whether Tpl2 inhibition could modulate Alox5 mRNA expression. In our experimental settings, the different inflammatory cytokines did not induce Alox5 mRNA expression (Figure 2A). Thus, it seems unlikely that the regulation of COX-2 mRNA by inflammatory cytokines and its modulation after Tpl2 inhibition were mediated by Alox5 products. TNF-α and COX-2 are involved in the regulation of apoptosis. We thus tested whether in our conditions TNF-α and/or IL-1β induced apoptosis and whether apoptosis affected the induction of COX2. IL-1β did not increase caspase-3 activity after 4 or 16 hours of stimulation, whereas TNF-α or the cytokine mix induced a transient increase in caspase-3 activity that was observed after 4 hours of treatment but not after 16 hours of stimulation (Figure 2, B and C). By contrast, staurosporine, a well-known apoptotic inducer, evoked a great and sustained increase in caspase-3 activity (Figure 2, B and C). However, blocking the caspase-3 activity with the caspase inhibitor, Z-VAD, did not modify the effect of TNF-α or the cytokine mix on COX-2 mRNA induction (Figure 2D).

Figure 2. — A, Expression of Alox5 mRNA in 3T3-L1 adipocytes treated without (vehicle, DMSO) or with a pharmacological Tpl2-I (10μM) and then stimulated or not with IL-1β or TNF-α alone (10 ng/mL) or a mix of these 2 cytokines (MIX, 10 ng/mL each) for 4 hours (n = 3 experiments). B, Caspase-3/7 activity in 3T3-L1 adipocytes treated for 4 hours with the indicated cytokines at the same concentration as in A. C, Caspase-3/7 activity in 3T3-L1 adipocytes treated for 16 hours with the indicated cytokines at the same concentration as in A. D, COX-2 mRNA expression in 3T3-L1 adipocytes incubated without or with the pan-caspase inhibitor Z-VAD-FMK (100μM) for 1 hour and then stimulated or not with TNF-α (10 ng/mL) or a mix of TNF-α and IL-1β (MIX, 10 ng/mL each) for 4 hours. Bars are mean ± SEM with *, P < .05; **, P < .01; and ***, P < .001 vs Ctrl condition.

The Tpl2/NF-κB axis positively regulates COX-2 mRNA expression in response to IL-1β but not to TNF-α or the cytokine mix

We then searched for the transcription factor(s) potentially involved in the induction of COX-2 in response to Tpl2 activation. The promoter of COX-2 contains binding sites for different transcription factors, including cAMP response element-binding protein (CREB), NF-κB, and NFAT, which have been shown to be regulated by Tpl2 in different cell types (23, 34 –36, 39). Especially, CREB has been reported to be activated by mitogen- and stress-activated protein kinase-1 and p90 ribosomal S6 kinase in macrophages after the activation of the Tpl2/ERK pathway (39). Our data addressing the implication of Tpl2 in the cytokine-induced activation of CREB in adipocytes revealed that pharmacological inhibition of Tpl2 did not markedly modify CREB phosphorylation, although ERK1/2 and p90 ribosomal S6 kinase activation were markedly reduced (Figure 3A). Thus, these data suggest that CREB do not significantly contribute to the induction of COX-2 in 3T3-L1 adipocytes after activation of Tpl2 by IL-1β or the cytokine mix.

Figure 3. — 3T3-L1 adipocytes were incubated without or with a Tpl2-I (30μM) (A and B) or treated with control or Tpl2 siRNA (siC and siT, respectively) (C). Then, cells were stimulated or not with IL-1β (10 ng/mL) or IL-1β and TNF-α (MIX, 10 ng/mL each) for 20 minutes. Cell were lysed for Western blot analysis with the indicated antibodies. Hsp90 was used as loading control in B and C. Representative immunoblots are shown.

NF-κB controls many inflammatory genes and Tpl2 is involved in the phosphorylation of NF-κB on Ser276 that stimulates NF-κB transactivation (36). We found that the pharmacological inhibition of Tpl2 (Figure 3B) or its silencing (Figure 3C) in 3T3-L1 adipocytes reduced the phosphorylation of NF-κB on this site in response to IL-1β or to the cytokine mix. We thus thought to determine whether NF-κB activation was required for the induction of COX-2 induced by the inflammatory cytokines. To this aim, 3T3-L1 adipocytes were transfected with a siRNA against p65NF-κB subunit and then treated with IL-1β, or the cytokine mix with or without the pharmacological Tpl2-I (10μM). The silencing of p65 (Figure 3A, inset) reduced by 35% the IL-1β-induced COX-2 mRNA expression (Figure 4A). This decrease was similar to the reduction in COX-2 mRNA expression obtained in 3T3-L1 adipocytes treated with the Tpl2 inhibitor and the control siRNA (Figure 4A). Furthermore, the pharmacological inhibition of Tpl2 had a significant but modest effect on COX-2 mRNA expression after p65 silencing (Figure 4A). By contrast, silencing of p65 increased the expression of COX-2 mRNA in response to a cytokine mix that was inhibited by the pharmacological Tpl2-I (Figure 4B). Similar results were obtained when the effect of TNF-α alone was investigated (Figure 4C). The secretion of PGE2 stimulated by TNF-α or the cytokine mix was also increased after p65 silencing, whereas secretion tended to decrease under IL-1β stimulation (Figure 4D). Together, these data indicate that the Tpl2-NF-κB pathway is involved, at least in part, in the induction of COX-2 expression in response to IL-1β. By contrast, NF-κB signaling has an inhibitory effect on COX-2 induction in response to TNF-α or the cytokine mix.

Figure 4. — A–C, COX-2 mRNA expression in 3T3-L1 adipocytes treated for 48 hours with control siRNA (siCtrl) or p65 NF-κB siRNA (sip65) and stimulated or not for 4 hours with IL-1β (A), the cytokine mix (B), or TNF-α (C) in the absence or presence of a Tpl2-I (10μM). Data are expressed as a percentage of COX-2 mRNA in siCtrl-treated adipocytes stimulated with IL-1β, TNF-α, or the cytokine mix (n = 4 experiments). The inset in A shows a representative immunoblot of the down-regulation of p65 NF-kβ in sip65-treated adipocytes with Hsp90 as loading control. D, PGE2 secretion in the culture medium of 3T3-L1 adipocytes treated with siCtrl or sip65 for 48 hours before stimulation for 16 hours with the indicated cytokines. Data in bar graphs represent mean ± SEM with *, P < .05; **, P < .01; and ***, P < .001.

Tpl2 activity in adipocytes regulates COX-2 expression and PGE2 secretion induced by a coculture between macrophages and adipocytes

Coculture experiments between adipocytes and macrophages suggest that a paracrine loop between these cell types could contribute to the production of inflammatory mediators in obese adipose tissue (6). We recently demonstrated that Tpl2 was involved in the cross talk between adipocytes and macrophages (30). We thus aimed at determining the impact of a direct coculture between RAW264.7 macrophages and 3T3-L1 adipocytes on COX-2 expression and PGE2 production and the effect of Tpl2 inhibition.

Contact coculture increased COX-2 mRNA and protein expression as compared with cells cultured separately (Figure 5, A and B). Importantly, Tpl2 inhibition in the coculture totally prevented COX-2 induction at both gene and protein levels (Figure 5, A and B). To determine in which cell types COX-2 induction occurred, we performed coculture in a transwell system in order to separate adipocytes and macrophages (6) before analyzing COX-2 expression. We found COX-2 mRNA induced in adipocytes cultured with macrophages (Figure 5, C and D), whereas we did not observe significant induction in macrophages (Figure 5C). Importantly, in the transwell system, pharmacological inhibition of Tpl2 or its silencing in adipocytes markedly reduced the induction of COX-2 in the adipocytes cultured with macrophages (Figure 5, C and D). Also, in the contact coculture system, Tpl2 knockdown in adipocytes markedly reduced COX-2 mRNA and protein (Figure 5, E and F). Accordingly, PGE2 secretion in the contact coculture system was reduced when Tpl2 was down-regulated in adipocytes by using specific siRNA (Figure 5G). These data indicate that the Tpl2-COX2 pathway in adipocytes plays a critical role in the stimulation of PGE2 secretion under coculture conditions.

Tpl2 activity in adipocyte is required for COX-2 induction in response to CM from LPS-activated macrophages

Obesity is associated with an increase in circulating LPS derived from gut microbiota and LPS participates to the inflammation of obese adipose tissue through activation of adipose tissue macrophages. Cytokines produced by LPS-activated macrophages up-regulate the expression of inflammatory mediators in adipocytes (7, 8). Using CM, we thus investigated whether proinflammatory cytokines secreted by LPS-activated macrophages could induce COX-2 expression in adipocytes by activating Tpl2. The expression of COX-2 mRNA was markedly induced when 3T3-L1 adipocytes were exposed to CM from LPS-treated RAW macrophages and this induction was nearly totally prevented when 3T3-L1 adipocytes were incubated with the pharmacological Tpl2 inhibitor or treated with a Tpl2 siRNA (Figure 6, A and B). Accordingly, COX-2 protein expression was induced by CM and this increase was severely blunted after pharmacological- or siRNA-mediated Tpl2 inhibition (Figure 6, C and D).

Figure 6. — A, COX-2 mRNA expression (n = 3 experiments) in 3T3-L1 adipocytes incubated without (vehicle, DMSO) or with a Tpl2-I (5μM) and treated for 24 hours with a CM from RAW macrophages activated with LPS (0.5 ng/mL) or with cultured medium containing the same concentration of LPS (control medium, Ctrl). B, COX-2 mRNA expression (n = 4 experiments) in 3T3-L1 adipocytes treated with control siRNA (Adipo siCtrl) or Tpl2 siRNA (Adipo siTpl2) and incubated with CM or control medium (Ctrl) as described in A. C, COX-2 expression with Hsp90 as loading control in the same experimental conditions as in A. Immunoblots from 2 independent experiments are shown. D, Immunoblot analysis and quantification (n = 3 experiments) of COX-2 protein expression with Hsp90 as loading control in the same experimental conditions as in B. E, COX-2 mRNA expression in adipose tissue explants from WT (n = 4) and Tpl2 KO mice (n = 4) incubated with or without LPS (100 ng/mL) for 24 hours. F, COX-2 mRNA expression in epididymal adipose tissue of WT (n = 9) and Tpl2 KO mice (n = 11) ip injected for 5 hours with LPS (2 μg/g of body weight) or NaCl 0.9% as control. Data in bar graphs represent mean ± SEM with *, P < .05 and **, P < .01.

This data indicate that Tpl2 activation in adipocytes is required for the induction of COX-2 by proinflammatory mediators produced by LPS-activated macrophages.

Tpl2 deficiency reduces LPS-stimulated COX-2 expression in adipose tissue

We next investigated whether Tpl2 was necessary for the expression of COX-2 in adipose tissue under inflammatory conditions. To this aim, we first used explants of adipose tissue from WT and Tpl2 knockout (Tpl2 KO) mice. LPS increased COX-2 mRNA expression in adipose tissue explants from WT mice and this effect was nearly abolished in adipose tissue explants from Tpl2 KO mice (Figure 6E).We then investigated whether Tpl2 deficiency could alter COX-2 expression in adipose tissue of LPS-treated mice. LPS injection significantly increased COX-2 mRNA expression in adipose tissue of WT mice and this effect was markedly blunted in adipose tissue of Tpl2-deficient mice (Figure 6F). Altogether, these data indicate that Tpl2 is necessary for the induction of COX-2 expression by LPS in adipose tissue.

Discussion

Prostaglandins are synthesized after the activation of the COX-2 by inflammatory mediators. Activation of COX-2 and enhanced production of PGE2 has been linked to obesity-induced inflammation and to alteration in adipose tissue development (9). Deregulation of the signaling pathways controlling COX-2 expression in adipocytes and adipose tissue during obesity could thus participate in adipose tissue inflammation and dysfunctions. The MAP kinase kinase kinase Tpl2 plays an important role in immune and inflammatory responses (28) and its expression is increased in obese adipose tissue (45). We recently found that Tpl2 was involved in an inflammatory paracrine loop between adipocytes and macrophages that could sustain adipose tissue inflammation (30).

Here, we provide evidences that Tpl2 activation by inflammatory cytokines is required for the induction of COX-2 and the secretion of PGE2 by adipocytes. We showed that Tpl2 inhibition in adipocytes markedly reduced the induction of COX-2 in response to IL-1β or to IL-1β and TNF-α. The secretion of PGE2 by adipocytes was also reduced in these experimental conditions. Thus, our data indicate that Tpl2 in adipocytes is a positive regulator of COX-2 expression and PGE2 production as previously shown in macrophages, T cells and intestinal myofibroblasts (35, 39, 46). By contrast, in other cellular contexts Tpl2 negatively regulates COX-2 expression and PGE2 production (37). Of note, Tpl2 inhibition only partially blocks the COX-2 induction as well as PGE2 production in response to cytokines. The absence of complete prevention of COX-2 induction could be due to residual activity of Tpl2 because we previously showed that siRNA knockdown or pharmacological inhibition of Tpl2 was not sufficient to completely shut down Tpl2 activity under IL-1β or TNF-α stimulation (29). Alternatively, we cannot exclude that other signaling pathways might be involved.

In addition to PGE2, adipocytes can secrete leukotrienes, and these proinflammatory lipid mediators have been suggested to regulate COX-2 expression (47). Moreover, leukotrienes are involved in obesity-induced adipose tissue inflammation and insulin resistance (11). However, it seems unlikely that in our conditions leukotrienes participated in the induction of COX-2, because we did not observe an effect of the cytokines on Alox5 gene expression. However, it remains to determine whether Tpl2 in adipocytes could control the synthesis of other proinflammatory prostaglandins species and also whether Tpl2 activation may alter the synthesis of different antiinflammatory and proresolving lipid mediators such as lipoxins, the cyclopentone prostaglandin of the D series, resolvins and protectins (9). This might add new insight for the role of Tpl2 in the commitment of adipocytes and adipose tissue inflammation by regulating the balance between the synthesis of proinflammatory lipid mediators and proresolving lipid mediators.

In macrophages, the ability of Tpl2 to regulate COX-2 in response to LPS is dependent on CREB (39). In adipocytes it seems unlikely that CREB was involved in the induction of COX-2 mediated by IL-1β or IL-1β and TNF-α, because we did not observe significant modifications in CREB phosphorylation after Tpl2 inhibition. The Tpl2/ERK pathway has been shown to cross talk with NF-κB by inducing the phosphorylation of p65 NF-κB on Ser276 that stimulates transactivation by NF-κB (36). Inhibition of Tpl2 markedly decreased the phosphorylation of this site in adipocytes in response to IL-1β or the cytokine mix suggesting that NF-κB could be involved in COX-2 induction downstream of Tpl2. However, the contribution of NF-κB to the expression of COX-2 in adipocytes appears complex with completely opposite effects depending on the cytokine used. Indeed, the silencing of p65 NF-κB reduced COX-2 induction in response to IL-1β to the same level than Tpl2 inhibitor in Ctrl siRNA-treated adipocytes (Figure 4A). Moreover, in p65 siRNA-treated adipocytes, the Tpl2 inhibitor only slightly reduced COX-2 expression. These findings suggest that Tpl2 controls COX-2 expression, at least in part, through NF-κB in response to IL-1β stimulation. We did not observe a complete prevention of COX-2 induction after p65 silencing suggesting another transcription factor in addition to NF-κB potentially involved in the control of COX-2 expression. Alternatively, the level of silencing of p65 NF-κB could not be enough to completely suppress COX-2 expression. Unexpectedly, it appears that NF-κB signaling strongly blocks COX-2 induction under TNF-α or the cytokine mix stimulation, because silencing of p65 NF-κB in this condition increased the expression of COX-2 and PGE2 secretion. Furthermore, pharmacological inhibition of Tpl2 in p65 siRNA-treated adipocytes markedly decreased COX-2 mRNA level. The net effect on COX-2 expression under TNF-α stimulation could be the result of a positive effect of a transcription factor under the control of Tpl2 and a negative effect exerted by NF-κB that could be mediated for instance through association with corepressors. This could explain why silencing of p65 NF-κB markedly increased COX-2 expression under TNF-α stimulation and why Tpl2 inhibition reduced COX-2 expression in this condition. The transcription factor NFAT could be a potential candidate to be controlled by Tpl2, because it has been reported that Tpl2 induced COX-2 expression in T cells through NFAT activation (35). Under the cytokine mix stimulation, the negative effect of NF-κB seems to be dominant over its positive effect, because we found that p65 silencing markedly potentiated COX-2 expression and PGE2 secretion. Further analyses are required to precisely decipher the complex role of NF-κB and to identify other transcription factors under the control of Tpl2 involved in the regulation of COX-2 mRNA expression that could explain the synergistic effect of the 2 cytokines.

PGE2 is well known to have proinflammatory effects on macrophages (12) and cross talk between adipocytes and macrophages is a potential mechanism sustaining the inflammation of obese adipose tissue (6). Coculture between adipocytes and macrophages provides experimental model to investigate actors involved in this cross talk, because adipocytes induce inflammatory action on macrophages resulting in an increased production of inflammatory mediators such as IL-1β, TNF-α, IL-6, and monocyte chemotactic protein-1 (6, 30). We found that a coculture between adipocytes and macrophages increased COX-2 expression and PGE2 secretion and we provided evidence that the Tpl2-COX-2 pathway in adipocytes is involved in the production of PGE2. By contrast, we previously demonstrated that the Tpl2 pathway in macrophages is involved in the production of inflammatory cytokines in this coculture system (30). Thus, our data suggest that secreted inflammatory mediators produced mainly by macrophages may activate the Tpl2-COX-2 pathway in adipocytes leading to an increase production of PGE2 by the adipocytes. It has been reported that saturated free fatty acids derived from adipocytes are important paracrine actors involved in the inflammation of macrophages (6, 48). In addition to free fatty acids, the increased production of PGE2 mediated by the Tpl2-COX-2 pathway in adipocytes could participate in the establishment of an inflammatory paracrine loop between adipocytes and macrophages, because PGE2 may exert inflammatory stimulus on macrophages (12).

LPS derived from gut microbiota is another important contributor of adipose tissue macrophage activation and we found that CM from LPS-activated macrophages increased COX-2 expression in adipocytes. Similar to the coculture findings, we found that Tpl2 inhibition in adipocytes markedly prevented the up-regulation of COX-2 expression induced by the CM from LPS-treated macrophages. It is therefore likely that inflammatory cytokines produced mainly by macrophages in the coculture or from LPS-activated macrophages increased COX-2 expression and PGE2 synthesis in adipocytes by activating Tpl2. Thus, we postulated that Tpl2 was necessary for COX-2 induction in adipose tissue under inflammatory conditions. Accordingly, we found that the COX-2 induction by LPS was lower in adipose tissue from Tpl2 KO mice compared with WT highlighting an important role of Tpl2 in adipose tissue for the regulation of COX-2 expression.

In conclusion, this study demonstrates that Tpl2 in adipocytes regulates the expression of COX-2 and the production of PGE2 in response to IL-1β and TNF-α and also in response to inflammatory mediators produced by LPS-activated macrophages. Further, our study indicates that Tpl2 is necessary for COX-2 induction by inflammatory mediators in adipose tissue. The Tpl2-NF-κB axis positively regulates COX-2 expression in response to IL-1β, but NF-κB strongly blocks COX-2 expression in response to TNF-α or to the cytokine mix, suggesting that other transcription factors under the control of Tpl2 are also involved. Because it was recently shown that Tpl2 affects the expression of a specific set of inflammatory mediators in adipose tissue (32), we propose that PGE2 produced by the activation of COX-2 could be added to the subset of these inflammatory mediators controlled by Tpl2 in adipocytes and adipose tissue.

Acknowledgments

We thank Frédéric Bost, Jean-François Louet, Jérome Gilleron, and Sophie Giorgetti-Péraldi (Inserm, Unit 1065, Centre Méditerranéen de Médecine Moléculaire, Nice) for critical reading of the manuscript and helpful discussions and Véronique Corcelle and the animal facility staff (Inserm, U1065, C3M, Nice) for animal care and breeding.

This work was supported by Inserm, the Université de Nice Sophia Antipolis, and Agence Nationale de la Recherche Grant 2010-BLAN-1117–01 (to J.-F.T.); LABEX SIGNALIFE Grant ANR-11-LABX-0028–01 and an Alfediam-Abbott grant (J.-F.T.). F.C. is supported by an Inserm/Région Provence Alpes-Cote d'Azur/FEDER doctoral fellowship and by a grant from the Société Francophone du Diabète. F.M. is supported by a postdoctoral fellowship from the Institut Thématique Multi-Organismes Cancer.

Disclosure Summary: The authors have nothing to disclose.

Funding Statement

Footnotes

Abbreviations:

Alox5: 5-lipoxygenase
CM: conditioned medium
COX: cyclooxygenase
CREB: cAMP response element-binding protein
DMSO: Dimethyl sulfoxide
Hsp90: Heat shock protein of 90 kD
LPS: lipopolysaccharide
NFAT: nuclear factor of activated T-cells
NF-κB: nuclear factor κB
PGE2: prostaglandin E2
siRNA: small interfering RNA
Tpl2: tumor progression locus 2
Tpl2 KO: Tpl2 knockout
WT: wild-type
Z-VAD-FMK: Z-Val-Ala-DL-Asp-fluoromethylketone.

References

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[B1] 1. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev Immunol. 2011;29:415–445. [DOI] [PubMed] [Google Scholar]

[B2] 2. Sun S, Ji Y, Kersten S, Qi L. Mechanisms of inflammatory responses in obese adipose tissue. Annu Rev Nutr. 2012;32:261–286. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Exley MA, Hand L, O'Shea D, Lynch L. Interplay between the immune system and adipose tissue in obesity. J Endocrinol. 2014;223:R41–R48. [DOI] [PubMed] [Google Scholar]

[B4] 4. McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity. 2014;41:36–48. [DOI] [PubMed] [Google Scholar]

[B5] 5. Suganami T, Mieda T, Itoh M, Shimoda Y, Kamei Y, Ogawa Y. Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a Toll-like receptor 4 mutation. Biochem Biophys Res Commun. 2007;354:45–49. [DOI] [PubMed] [Google Scholar]

[B6] 6. Suganami T, Nishida J, Ogawa Y. A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor α. Arterioscler Thromb Vasc Biol. 2005;25:2062–2068. [DOI] [PubMed] [Google Scholar]

[B7] 7. Cani PD, Amar J, Iglesias MA, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007;56:1761–1772. [DOI] [PubMed] [Google Scholar]

[B8] 8. Nicholson JK, Holmes E, Kinross J, et al. Host-gut microbiota metabolic interactions. Science. 2012;336:1262–1267. [DOI] [PubMed] [Google Scholar]

[B9] 9. Gonzàlez-Périz A, Claria J. Resolution of adipose tissue inflammation. Scientific WorldJournal. 2010;10:832–856. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Horrillo R, González-Périz A, Martínez-Clemente M, et al. 5-lipoxygenase activating protein signals adipose tissue inflammation and lipid dysfunction in experimental obesity. J Immunol. 2010;184:3978–3987. [DOI] [PubMed] [Google Scholar]

[B11] 11. Mothe-Satney I, Filloux C, Amghar H, et al. Adipocytes secrete leukotrienes: contribution to obesity-associated inflammation and insulin resistance in mice. Diabetes. 2012;61:2311–2319. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31:986–1000. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Fain JN, Kanu A, Bahouth SW, Cowan GS Jr, Hiler ML, Leffler CW. Comparison of PGE2, prostacyclin and leptin release by human adipocytes versus explants of adipose tissue in primary culture. Prostaglandins Leukot Essent Fatty Acids. 2002;67:467–473. [DOI] [PubMed] [Google Scholar]

[B14] 14. Kawahara K, Hohjoh H, Inazumi T, Tsuchiya S, Sugimoto Y. Prostaglandin E2-induced inflammation: relevance of prostaglandin E receptors. Biochim Biophys Acta. 2015;1851:414–421. [DOI] [PubMed] [Google Scholar]

[B15] 15. Ghoshal S, Trivedi DB, Graf GA, Loftin CD. Cyclooxygenase-2 deficiency attenuates adipose tissue differentiation and inflammation in mice. J Biol Chem. 2011;286:889–898. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Hsieh PS, Lu KC, Chiang CF, Chen CH. Suppressive effect of COX2 inhibitor on the progression of adipose inflammation in high-fat-induced obese rats. Eur J Clin Invest. 2010;40:164–171. [DOI] [PubMed] [Google Scholar]

[B17] 17. Hsieh PS, Jin JS, Chiang CF, Chan PC, Chen CH, Shih KC. COX-2-mediated inflammation in fat is crucial for obesity-linked insulin resistance and fatty liver. Obesity (Silver Spring). 2009;17:1150–1157. [DOI] [PubMed] [Google Scholar]

[B18] 18. Hellmann J, Zhang MJ, Tang Y, Rane M, Bhatnagar A, Spite M. Increased saturated fatty acids in obesity alter resolution of inflammation in part by stimulating prostaglandin production. J Immunol. 2013;191:1383–1392. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Fujimori K, Yano M, Miyake H, Kimura H. Termination mechanism of CREB-dependent activation of COX-2 expression in early phase of adipogenesis. Mol Cell Endocrinol. 2014;384:12–22. [DOI] [PubMed] [Google Scholar]

[B20] 20. Chu X, Nishimura K, Jisaka M, Nagaya T, Shono F, Yokota K. Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction. Prostaglandins Other Lipid Mediat. 2010;91:1–9. [DOI] [PubMed] [Google Scholar]

[B21] 21. Fujimori K, Yano M, Ueno T. Synergistic suppression of early phase of adipogenesis by microsomal PGE synthase-1 (PTGES1)-produced PGE2 and aldo-keto reductase 1B3-produced PGF2α. PLoS One. 2012;7:e44698. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Tanti JF, Ceppo F, Jager J, Berthou F. Implication of inflammatory signaling pathways in obesity-induced insulin resistance. Front Endocrinol (Lausanne). 2013;3:181. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Gantke T, Sriskantharajah S, Sadowski M, Ley SC. IκB kinase regulation of the TPL-2/ERK MAPK pathway. Immunol Rev. 2012;246:168–182. [DOI] [PubMed] [Google Scholar]

[B24] 24. Dumitru CD, Ceci JD, Tsatsanis C, et al. TNF-α induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway. Cell. 2000;103:1071–1083. [DOI] [PubMed] [Google Scholar]

[B25] 25. Rousseau S, Papoutsopoulou M, Symons A, et al. TPL2-mediated activation of ERK1 and ERK2 regulates the processing of pre-TNF α in LPS-stimulated macrophages. J Cell Sci. 2008;121:149–154. [DOI] [PubMed] [Google Scholar]

[B26] 26. Mielke LA, Elkins KL, Wei L, et al. Tumor progression locus 2 (Map3k8) is critical for host defense against listeria monocytogenes and IL-1β production. J Immunol. 2009;183:7984–7993. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Vougioukalaki M, Kanellis DC, Gkouskou K, Eliopoulos AG. Tpl2 kinase signal transduction in inflammation and cancer. Cancer Lett. 2011;304:80–89. [DOI] [PubMed] [Google Scholar]

[B28] 28. Gantke T, Sriskantharajah S, Ley SC. Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase. Cell Res. 2011;21:131–145. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Jager J, Grémeaux T, Gonzalez T, et al. The Tpl2 kinase is up-regulated in adipose tissue in obesity and may mediate IL-1β and TNF-α effects on ERK activation and lipolysis. Diabetes. 2010;59:61–70. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Ceppo F, Berthou F, Jager J, Dumas K, Cormont M, Tanti JF. Implication of the Tpl2 kinase in inflammatory changes and insulin resistance induced by the interaction between adipocytes and macrophages. Endocrinology. 2014;155:951–964. [DOI] [PubMed] [Google Scholar]

[B31] 31. Perfield JW 2nd, Lee Y, Shulman GI, et al. Tumor progression locus 2 (TPL2) regulates obesity-associated inflammation and insulin resistance. Diabetes. 2011;60:1168–1176. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Ballak DB, van Essen P, van Diepen JA, et al. MAP3K8 (TPL2/COT) affects obesity-induced adipose tissue inflammation without systemic effects in humans and in mice. PLoS One. 2014;9:e89615. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Lancaster GI, Kowalski GM, Estevez E, et al. Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease. PLoS One. 2012;7:e39100. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34. Gómez-Casero E, San-Antonio B, Iñiguez MA, Fresno M. Cot/Tpl2 and PKCζ cooperate in the regulation of the transcriptional activity of NFATc2 through the phosphorylation of its amino-terminal domain. Cell Signal. 2007;19:1652–1661. [DOI] [PubMed] [Google Scholar]

[B35] 35. de Gregorio R, Iñiguez MA, Fresno M, Alemany S. Cot kinase induces cyclooxygenase-2 expression in T cells through activation of the nuclear factor of activated T cells. J Biol Chem. 2001;276:27003–27009. [DOI] [PubMed] [Google Scholar]

[B36] 36. Das S, Cho J, Lambertz I, et al. Tpl2/cot signals activate ERK, JNK, and NF-κB in a cell-type and stimulus-specific manner. J Biol Chem. 2005;280:23748–23757. [DOI] [PubMed] [Google Scholar]

[B37] 37. DeCicco-Skinner KL, Nolan SJ, et al. Altered prostanoid signaling contributes to increased skin tumorigenesis in Tpl2 knockout mice. PLoS One. 2013;8:e56212. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38. Soria-Castro I, Krzyzanowska A, Pelaéz ML, et al. Cot/tpl2 (MAP3K8) mediates myeloperoxidase activity and hypernociception following peripheral inflammation. J Biol Chem. 2010;285:33805–33815. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Eliopoulos AG, Dumitru CD, Wang CC, Cho J, Tsichlis PN. Induction of COX-2 by LPS in macrophages is regulated by Tpl2-dependent CREB activation signals. EMBO J. 2002;21:4831–4840. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Hatziapostolou M, Polytarchou C, Panutsopulos D, Covic L, Tsichlis PN. Proteinase-activated receptor-1-triggered activation of tumor progression locus-2 promotes actin cytoskeleton reorganization and cell migration. Cancer Res. 2008;68:1851–1861. [DOI] [PubMed] [Google Scholar]

[B41] 41. Gavrin LK, Green N, Hu Y, et al. Inhibition of Tpl2 kinase and TNF-α production with 1,7-naphthyridine-3-carbonitriles: synthesis and structure-activity relationships. Bioorg Med Chem Lett. 2005;15:5288–5292. [DOI] [PubMed] [Google Scholar]

[B42] 42. Jager J, Grémeaux T, Cormont M, Le Marchand-Brustel Y, Tanti JF. Interleukin-1β-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression. Endocrinology. 2007;148:241–251. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43. Kilroy G, Burk DH, Floyd ZE. High efficiency lipid-based siRNA transfection of adipocytes in suspension. PLoS One. 2009;4:e6940. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44. González-Périz A, Clària J. New approaches to the modulation of the cyclooxygenase-2 and 5-lipoxygenase pathways. Curr Top Med Chem. 2007;7:297–309. [DOI] [PubMed] [Google Scholar]

[B45] 45. Jager J, Corcelle V, Grémeaux T, et al. Deficiency in the extracellular signal-regulated kinase 1 (ERK1) protects leptin-deficient mice from insulin resistance without affecting obesity. Diabetologia. 2011;54:180–189. [DOI] [PubMed] [Google Scholar]

[B46] 46. Roulis M, Nikolaou C, Kotsaki E, et al. Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis. Proc Natl Acad Sci USA. 2014;111:E4658–E4667. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

The Tpl2 Kinase Regulates the COX-2/Prostaglandin E2 Axis in Adipocytes in Inflammatory Conditions

Flavien Berthou

Franck Ceppo

Karine Dumas

Fabienne Massa

Bastien Vergoni

Susana Alemany

Mireille Cormont

Jean-François Tanti

Abstract

Materials and Methods

Reagents and antibodies

Culture of 3T3-L1 adipocytes and RAW264.7 macrophages

Measurement of 3T3-L1 apoptosis

Coculture between adipocytes and macrophages

Treatment of 3T3-L1 adipocytes with the CM from LPS-activated RAW264.7 macrophages

Injection of wild-type (WT) and Tpl2-deficient mice with LPS and treatment of adipose tissue explants with LPS

Western blot analysis

Real-time RT-PCR

Quantification of PGE2

Statistical analysis

Results

Tpl2 in 3T3-L1 adipocytes regulates COX-2 expression and PGE2 secretion induced by inflammatory cytokines

Figure 1. Tpl2 inhibition in 3T3-L1 adipocytes decreases the induction of COX-2 and the secretion of PGE2 in response to IL-1β, TNF-α, or to a cytokine mix.

Figure 2. Effect of IL-1β, TNF-α or a cytokine mix on 5-lipooxygenase mRNA expression and on caspase-3 activity in 3T3-L1 adipocytes.

The Tpl2/NF-κB axis positively regulates COX-2 mRNA expression in response to IL-1β but not to TNF-α or the cytokine mix

Figure 3. Tpl2 is involved in NF-κB but not CREB phosphorylation in response to IL-1β or the cytokine mix.

Figure 4. NF-κB is involved in Tpl2-induced COX-2 mRNA expression in response to IL-1β but not to TNF-α or the cytokine mix.

Tpl2 activity in adipocytes regulates COX-2 expression and PGE2 secretion induced by a coculture between macrophages and adipocytes

Figure 5. The pharmacological inhibition of Tpl2 or its silencing in adipocytes prevents the induction of COX-2 and the secretion of PGE2 induced by a coculture between adipocytes and macrophages.

Tpl2 activity in adipocyte is required for COX-2 induction in response to CM from LPS-activated macrophages

Figure 6. Tpl2 activity in adipocytes or in adipose tissue is required for LPS-induced COX-2 expression.

Tpl2 deficiency reduces LPS-stimulated COX-2 expression in adipose tissue

Discussion

Acknowledgments

Funding Statement

Footnotes

References

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