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Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
TSH signaling pathways that regulate MCP-1 in human differentiated adipocytes AnneMarie Gagnon, Melanie L. Langille, Seham Chaker, Tayze T. Antunes, Jason Durand, Alexander Sorisky⁎ Chronic Disease Program, Ottawa Hospital Research Institute, Departments of Medicine and of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa, Ontario, Canada Chronic Disease Program, Ottawa Hospital Research Institute, Department of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa, Ontario, Canada
A R T I C LE I N FO Article history:
AB S T R A C T Objective. Adipose tissue is an extra-thyroidal thyroid-stimulating hormone (TSH) target.
Received 11 November 2013
Increases in lipolysis and in expression and release of interleukin-6 (IL-6) occur in TSH-
Accepted 25 February 2014
stimulated adipocytes, and levels of circulating free fatty acids and IL-6 rise following TSH administration to patients with previous thyroidectomy and radioablation for thyroid
Keywords:
cancer. Our first objective was to compare how TSH stimulates protein kinase A (PKA) and
Thyroid-stimulating hormone
inhibitor of κB (IκB) kinase (IKK)-β. Our second objective was to investigate whether TSH
Monocyte chemoattractant protein-1
induces other cytokines besides IL-6.
Inhibitor of κB kinase-β Protein kinase C-δ NADPH oxidase
Methods. TSH stimulation of either CHO cells expressing human TSH receptor or human abdominal subcutaneous differentiated adipocytes. Results. Signaling studies showed TSH increased NADPH oxidase activity, and either diphenyleneiodonium (oxidase inhibitor) or N-acetyl cysteine (scavenger of reactive oxygen species) reduced IKKβ phosphorylation. Phosphorylation of protein kinase C-δ, an upstream regulator of NADPH oxidase, was increased by TSH, and rottlerin (PKCδ inhibitor) reduced TSH-stimulated IKKβ phosphorylation. TSH upregulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression and the release of MCP-1 protein in human abdominal differentiated adipocytes. H89 (PKA inhibitor) and sc-514 (IKKβ inhibitor) each blocked TSH-stimulated MCP-1 mRNA expression and protein release, suggesting PKA and IKKβ participate in this pathway. Conclusions. These data provide new information about TSH signaling in human differentiated adipocytes, and add to the evidence that TSH is a pro-inflammatory stimulus of adipocytes. © 2014 Elsevier Inc. All rights reserved.
Abbreviations: BSA, bovine serum albumin; CHO, Chinese hamster ovarian; CREB, cAMP response element-binding protein; DMEM, Modified Eagle’s Medium; DPI, diphenyleneiodonium; FBS, fetal bovine serum; FFA, free fatty acids; GPCR, G protein-coupled receptor; IκB, inhibitor of κB; IKK, inhibitor of IκB kinase; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; PKA, protein kinase A; PKC, protein kinase C; RBP4, retinol binding protein 4; rh, recombinant human; RT, reverse transcriptase; TSH, thyroidstimulating hormone; TSHR, TSH receptor. ⁎ Corresponding author at: Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada. E-mail address: [email protected] (A. Sorisky). http://dx.doi.org/10.1016/j.metabol.2014.02.015 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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1.
Introduction
In thyrocytes, thyroid-stimulating hormone (TSH) binds to its receptor (TSHR), a G protein-coupled receptor (GPCR) that can communicate with several G proteins, including Gs and G q . A variety of downstream signaling pathways are consequently activated, regulating thyrocyte growth and hormone production [1–4]. Extra-thyroidal expression of TSHR in adipocytes has been documented [5–8], but little is known about how TSH influences adipocyte signaling and cellular responses. In vivo, TSH stimulates the release of pro-inflammatory molecules and free fatty acids (FFA) when administered acutely to patients previously treated for thyroid cancer by total thyroidectomy and radioablation [9–11]. Furthermore, serum levels of interleukin-6 (IL-6), tumor necrosis factor-α, C-reactive protein, and FFA are elevated in patients with subclinical hypothyroidism, a condition whereby chronically elevated TSH serum levels compensate for mild thyroid failure to maintain normal thyroid hormone levels [12–15]. These inflammatory and metabolic disturbances might explain the epidemiological association of accelerated cardiovascular disease with subclinical hypothyroidism [16,17]. Studies on adipocyte cell cultures reveal similar TSHstimulated responses to the ones described above for in vivo studies. TSH stimulates release of FFA in human differentiated adipocytes and neonatal adipocytes in culture, and requires protein kinase A (PKA) as well as protein kinase C (PKC) [11,18,19]. TSH also dose-dependently stimulates IL-6 production and release in human abdominal differentiated adipocytes, and this depends on the inhibitor of κB kinase (IKK) β/nuclear factor (NF) κB pathway [9,20]. This pathway is also activated by TSH in orbital adipose cells and other cell types [21–24]. The downstream activation of IKKβ by other GPCR may occur via the activation of NADPH oxidase [25]. The generation of reactive oxygen species by NADPH oxidase results in the activation of IKKβ [26]. Our objectives were to investigate how TSHR activates the IKKβ pathway, and to evaluate whether adipokines other than IL-6 are regulated by TSH. We examined if there is a role for NADPH oxidase and PKC-δ upstream of TSH-stimulated IKKβ, and also whether monocyte chemoattractant protein-1 (MCP-1) is a TSH-induced adipokine.
2.
Material and methods
2.1.
Chinese hamster ovarian (CHO) cell culture
Parental CHO cells (CHO-JP02) and human TSHR overexpressing CHO cells (CHO-JP2626, or CHO-hTSHR) were kindly provided by J. E. Dumont, Erasme University Hospital, Free University of Brussels, Brussels, Belgium [27]. Cells were plated and grown in Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS), antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and 400 μg/ml G418 (all from Life Technologies, Burlington, ON, Canada). Medium was changed every 2 days until confluence.
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2.2. Isolation, culture, and differentiation of human abdominal subcutaneous stromal cells Human abdominal subcutaneous adipose tissue was obtained from 22 patients (19 female, 3 male), with mean age 49 ± 9 years (± S.D.) and mean body mass index 27 ± 6 (±S.D.), undergoing elective abdominal surgery (approved by the Ottawa Hospital Research Ethics Board, protocol 1995023-01H). They were weight-stable, did not have diabetes, and were not on steroid therapy. The stromal preadipocytes were isolated as previously described [28]. Briefly, blood vessels and fibrous tissue were carefully removed, followed by collagenase digestion (CLS type I; 600 U/g of tissue; Worthington Biochemical Corporation, Lakewood, NJ, USA), and sequential centrifugation, size filtration, and treatment with erythrocyte lysis buffer. Preadipocytes were cultured in growth medium which was Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies) supplemented with 10% FBS and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 U/ml nystatin; EMD Millipore, Billerica, MA, USA). Upon reaching 80%–90% confluence, cells were either re-plated for a maximum of three passages or cryopreserved until needed. The presence of macrophages (CD14) or endothelial cells (CD31) is < 2% in our culture conditions. For differentiation, preadipocytes were seeded at a density of 3 × 104 cells/cm2 and allowed to adhere overnight. Adipogenesis was induced with growth medium supplemented with 0.85 μmol/L insulin, 0.25 mmol/L isobutylmethylxanthine, 100 μmol/L indomethacin, and 0.5 μmol/L dexamethasone for 14 days. Cells were then placed in growth medium for 2 days prior to stimulation studies.
2.3.
Acute stimulation
CHO cells, CHO-hTSHR cells, or human differentiated adipocytes, in DMEM supplemented with 4% fatty acid-free bovine serum albumin (BSA) or 1% calf serum (Life Technologies), were stimulated with 50 mU/ml Calbiochem bovine TSH (C-bTSH; EMD Millipore), Sigma bovine TSH (S-bTSH; Sigma-Aldrich, Oakville, ON, Canada), an analog of recombinant human (rh) TSH (TR1401, a kind gift from MW Szkudlinski,Trophogen, Rockville, MD), or vehicle (H2O) for 0 to 4 h, as shown. Where indicated, cells were pre-treated for 30 min with 10 μmol/L diphenyleneiodonium (DPI; Sigma-Aldrich), 10 μmol/L rottlerin (EMD Millipore), or vehicle (0.1% DMSO); for 1 h with 100 μmol/L sc-514, 20 μmol/L H-89 (both from EMD Millipore), or vehicle (0.1% DMSO); or for 2 h with 10 mmol/L N-acetylcysteine (NAC; SigmaAldrich) or vehicle (H2O), prior to stimulation with TSH. Cells were then lysed and processed for immunoblot analysis, NADPH oxidase activity, or RNA isolation and qPCR. Conditioned medium was collected and assessed for cytokine content by ELISA.
2.4.
Immunoblot analysis
Cells were lysed in Laemmli buffer [29] containing 50 mmol/L sodium fluoride, 5 mmol/L sodium pyrophostate, 5 mmol/L EGTA, and 1 mmol/L sodium orthovanadate. Protein was quantified by the modified Lowry assay (BioRad; Bio-Rad, Hercules, CA, USA), with BSA as standard. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose. Non-specific antigenic sites were blocked and
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membranes were probed with the indicated primary antibodies: anti-phospho-cAMP response element-binding protein (CREB; Ser133; 1:1000), anti-CREB (1:1000), anti-phospho-IKK α Ser180/IKKβ Ser181 (1:1000), anti-inhibitor of κBα (1:1000), antiIKKβ (1:500), anti-phospho-PKC δ (Thr505; 1:500), anti-PKCδ (1:1000, all from Cell Signaling; Danvers, MA, USA), or anti-actin (serves as a loading control, Santa Cruz Biotech., Santa Cruz, CA, USA). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch, Laboratories, West Grove, PA, USA), and immunoreactivity was visualized by enhanced chemiluminescence (EMD Millipore). Densitometric analysis of immunoreactive bands was performed with AlphaEaseFC software (Alpha Innotec Co, San Leandro, CA, USA), and data were expressed as integrated optical density (IOD) units.
2.5.
NADPH oxidase activity
Following stimulation of cells with TSH as described, cells were lysed in 20 mmol/L KH2PO4, 1 mmol/L EGTA pH 7.4, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mmol/L PMSF. Reaction tubes included 175 μl of assay buffer (20 mmol/L KH2PO4, 1 mmol/L EGTA, 150 mmol/L sucrose pH 7.4), 25 μL of cellular lysates, and 5 μmol/L lucigenin. Lucigenin-enhanced chemiluminescence measurements were performed in the presence versus absence of 1 mmol/L NAPDH, over 3 min in a FluoStar Optima reader (BMG Labtech, Cary, NC, USA). After subtraction of a buffer blank, the results were expressed as foldstimulation of control conditions.
2.6.
qPCR
Cells were lysed in Qiazol (Qiagen, Toronto, ON, Canada), and RNA was isolated and treated with DNA-free (Life Technologies), as per manufacturer’s instructions. qPCR was performed as previously described [30]. Briefly, total RNA (1 μg per sample) was heat-denatured, then reverse-transcribed using the Retroscript kit with random primers (Life Tehcnologies). Control reactions, without transcriptase, were performed for all samples. Real-time qPCR assays were performed using the QuantiTect SYBR Green RT-PCR kit (for target genes) or the QuantiTect Probe RT-PCR kit (for 18S rRNA; both from Qiagen), according to manufacturer’s instructions. Reactions were run on the Roche Light Cycler RealTime PCR System (Roche, Laval, QC, Canada), and data were analyzed with Light Cycler Software, version 3.0 (Roche). Primer sequences were: adiponectin: forward: 5’-GCAGAGATGG CACCCCTG-3’, reverse: 5’-GGTTTCACCGATGTCTCCCTTA-3’; MCP-1: forward: 5’-CAGCCAGATGCAATCAATGC-3’, reverse: 5’GTGGTCCATGGAATCCTGAA-3’; and retinol-binding protein 4 (RBP4): forward: 5’-GCCTCTTTCTGCAGGACAAC-3’, reverse: 5’-GCACACGTCCCAGTTATTCA-3’.
2.7.
ELISA
Conditioned medium was collected and centrifuged at 500 ×g, 5 min at 4 °C, to remove any cellular debris. Supernatant was collected and MCP-1 protein was quantified using a Quantikine ELISA kit (MCP-1; R&D systems, Minneapolis, MN, USA). The cell monolayer was lysed in Laemmli buffer [29] and protein was quantified using the modified Lowry assay, with BSA as
standard. MCP-1 release into the medium was normalized to cellular protein content.
2.8.
Statistical analysis
Statistical analysis was performed with Student’s t-test for paired samples (two means compared), one-way ANOVA followed by Student–Newman–Keuls post hoc test (three or more means with one variable compared), or with two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was defined as P < 0.05.
3.
Results
To clarify the activation of the PKA and IKKβ signaling pathways by TSH, we compared bovine TSH (bTSH) from Calbiochem (C-bTSH) versus Sigma (S-bTSH). Since the CbTSH and S-bTSH preparations are not completely purified, other factors present in the reagents could possibly be responsible for the activation of these signaling molecules. The JP2626 CHO-hTSHR and JP02 CHO control cell lines stably transfected with either human TSHR [27] or control vector, respectively, were used. CREB is a direct substrate of PKA and its phosphorylation indicates PKA activation [20]. Each TSH preparation (50 mU/ml) significantly increased the levels of phospho-CREB and phospho-IKKβ in the CHO-hTSHR cells (Fig. 1). There was no consistent change in total CREB levels. In the CHO controls, the small changes that occurred were not reproducible and did not reach significance, but we cannot completely rule out a minor effect of TSH on these cells. Overall, these responses to the bTSH preparations are predominantly TSHR-dependent. To address this issue further, we repeated the stimulation studies, this time using the rhTSH analog TR1401 [31]. The same TSHR-dependent pattern of signaling, i.e. significant and large responses only occurring in the CHO-hTSHR cells, was observed as previously noted with C-bTSH or S-bTSH stimulation (Fig. 1). Therefore, all three TSH reagents stimulate significant increases in phosphorylation of CREB and IKKβ in a TSHRdependent fashion. We compared the signaling responses activated by C-bTSH versus S-bTSH in human abdominal subcutaneous differentiated adipocytes that express TSHR endogenously. Each TSH preparation stimulated CREB phosphorylation to the same extent (Fig. 2), as was observed when comparing these agonists using the CHOhTSHR cells (Fig. 1A). Total CREB levels did not show any consistent change. However, only C-bTSH was competent to increase IKKβ phosphorylation and the consequent degradation of IκB. C-bTSH was used to examine upstream regulators of IKKβ phosphorylation and IκBα degradation in human abdominal subcutaneous differentiated adipocytes. GPCRs increase reactive oxygen species (ROS) in different cell types via activation of NADPH oxidase [25]. Following stimulation of human differentiated adipocytes with 50 mU/ml of C-bTSH, NADPH oxidase activity increased significantly by 21% (Fig. 3A). We examined whether inhibiting formation of ROS, or accelerating their clearance, would inhibit the IKKβ response to TSH. The level of IKKβ phosphorylation in response to C-bTSH (50 mU/ml) was reduced by 45% in the presence of diphenyleneiodonium (DPI;
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C-bTSH (50 mU/ml) was observed at 30 min (Fig. 4A), assessed by Thr505 phosphorylation [34]. We investigated whether blocking PKCδ would reduce IKKβ and IκB signaling in response to C-bTSH. An inhibitor of PKCδ, rottlerin (10 μmol/L) [35,36], reduced C-bTSH-stimulated IKKβ phosphorylation by 55%, and IκB degradation by 35% (Fig. 4B). Given that C-bTSH signals via IKKβ to increase IL-6 protein release from human abdominal subcutaneous differentiated adipocytes [20], we examined whether it might regulate other cytokines. C-bTSH (50 mU/ml) strongly increased MCP-1 mRNA expression by 12-fold, with no effect on either RBP4 or adiponectin, two other adipokines known to be perturbed in the low-grade inflammatory state of obesity (Fig. 5A). We investigated whether the TSH-stimulated MCP-1 response in differentiated adipocytes required signaling via IKKβ and/or PKA, using inhibitors as previously reported [20]. The IKKβ inhibitor sc-514 (100 μmol/L) reduced C-bTSHstimulated MCP-1 mRNA expression by 59% (Fig. 5B). C-bTSH increased MCP-1 protein release by 3-fold, and this was completely inhibited by sc-514 (Fig. 5C). PKA inhibitor H89 (20 μmol/L) reduced C-bTSH-stimulated MCP-1 mRNA expression and MCP-1 protein release by 54% and 58%, respectively (Fig. 5D and E). Rottlerin, which inhibited IKKβ phosphorylation and IκB degradation (Fig. 4B), also reduced the C-bTSHstimulated mRNA expression of MCP-1 mRNA by 62% (Fig. 5F).
4.
Fig. 1 – TSH stimulates IKKβ and CREB phosphorylation in CHO-hTSHR cells and human differentiated adipocytes. Confluent CHO-hTSHR and parental (CHO) cells were stimulated with 50 mU/ml of either C-bTSH, S-bTSH, rhTSH analog TR1401 (TR), or with vehicle control for 30 min. Equal amounts of solubilized cellular protein were immunoblotted with the indicated antibodies. Immunoblots from one experiment are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 separate experiments are graphically presented as mean ± S.E.M. ** indicates P < 0.01, and * indicates P < 0.05 compared to control condition, as assessed by 2-way ANOVA with Tukey post-hoc tests.
10 μmol/L), an oxidase inhibitor [32]. The accompanying IκB degradation response was also inhibited by 45%, but did not reach significance (Fig. 3B). N-acetyl cysteine (NAC; 10 mmol/L), a ROS scavenger [32], reduced the C-bTSH (50 mU/ml)-stimulated IKKβ phosphorylation response by 53% (Fig. 3C). There was a trend towards attenuation of IκB degradation of 18% that did not reach significance. Taken together, DPI or NAC reduces TSHstimulated IKKβ phosphorylation in human differentiated adipocytes. The effects of these agents on IκB degradation only showed non-significant trends. The p47phox subunit of NADPH oxidase is phosphorylated by PKCδ in response to some GPCRs [33]. Activation of PKCδ by
Discussion
Our studies on human abdominal differentiated adipocytes reveal MCP-1 is a novel TSH-stimulated adipokine, and suggest signaling via IKKβ and PKA pathways is involved in mediating this effect. Little is known about how TSH leads to IKKβ activation, and our results point to PKCδ and NADPH oxidase being positioned upstream of IKKβ in this TSH response. Adipose tissue is an extrathyroidal TSH target. Adipocytes express TSHR [5–8]. However, the way TSH signals to produce its catalogue of responses in these cells is not well understood. It may act on adipocytes in the neonatal period to release energy during acclimation to the environment [18]. TSH activates PKA and this is required for lipolysis, and for phosphorylation of hormone-sensitive lipase and perilipin, in differentiated human adipocytes [11]. Subsequent work suggested TSH could activate conventional PKC, and this was required for TSH to fully stimulate perilipin phosphorylation and lipolysis [19]. TSH also upregulates IL-6 mRNA expression and IL-6 protein release in human differentiated adipocytes, and this depends on IKKβ, but not PKA [20]. We have identified MCP-1 as a novel TSH-stimulated adipokine. Consistent with this observation, levels of MCP-1 are elevated in patients with chronic autoimmune thyroiditis with mild hypothyroidism characterized by elevated TSH serum concentrations [37]. The increase in MCP-1 mRNA expression and MCP-1 protein release by TSH was blocked by either the IKKβ inhibitor sc-514 or the PKA inhibitor H89. This contrasts to what we previously observed with IL-6, which was only inhibited by sc-514 [20]. Our studies were performed with a single dose of TSH and one time point based on previous work with IL-6 [11,20]; future studies examining a range of doses and time points may provide more information on the MCP-1 response.
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Fig. 2 – IKKβ phosphorylation and IκBα degradation in response to TSH in human abdominal subcutaneous differentiated adipocytes. Human differentiated adipocytes were stimulated with 50 mU/ml C-bTSH, S-bTSH, or with vehicle control for 30 min. Equal amounts of solubilized cellular protein were immunoblotted with indicated antibodies. Immunoblots from one experiment are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 patient samples are graphically presented as mean ± S.E.M. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, assessed by one-way ANOVA with Student–Newman–Keul post-hoc tests.
TSH signaling to PKA is thought to be due to TSHR interacting with Gs to activate adenylate cyclase, generating a rise in cAMP. However, little is known about how TSH leads to IKKβ phosphorylation. Studies on other GPCR ligands in other cell systems have indicated that NADPH oxidase and ROS generation lie upstream of IKKβ phosphorylation [25,26]. In human adipose cells, Nox4 is the isoform that is predominantly expressed [38]. We found that TSH was able to increase NADPH oxidase activity in differentiated human adipocytes. To determine if this was required for IKKβ phosphorylation, we used DPI, an oxidase inhibitor to prevent ROS formation, or NAC, to accelerate the clearance of ROS. Either strategy reduced TSH-stimulated IKKβ phosphorylation by ~ 50%, indicating the importance of this event. The inhibition was not total, possibly due to other TSH-stimulated molecular pathways regulating IKKβ phosphorylation, or to technical limitations of the inhibitors used. PKCδ, a novel PKC isoform, has been implicated in phosphorylating and activating NADPH oxidase [33]. We have previously observed that TSH can activate PKC in human differentiated adipocytes based on an increase in
phosphorylated proteins detected by a phospho-PKC substrate antibody specific for conventional PKC [19]. Our data here indicate that TSH is also able to activate PKCδ, indicated by its phosphorylation [34]. Rottlerin, a PKCδ selective inhibitor [35,36], inhibited TSH-stimulated IKKβ phosphorylation and IκBα degradation, consistent with a role for PKCδ in this TSH-stimulated pathway. Additional investigations will be needed to identify proximal events linking TSHR activation to this signaling pathway. The dose of TSH for our in vitro studies is higher than physiological, but it approximates doses used in thyrocyte studies [39]; it is also important to note that corresponding data related to cytokine release have been observed in clinical studies [9,10,12,14,15]. Our data, based on pharmacological inhibitors, suggest that the activation of PKCδ and NADPH oxidase by TSH lies upstream of IKKβ in human differentiated adipocytes. Limitations of inhibitors include off-target effects. In particular, the specificity of rotttlerin is debated, but it continues to be used as an investigational agent [35,36]. Future studies using RNAi to reduce PKCδ or NADPH oxidase
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Fig. 3 – NADPH oxidase and TSH-stimulated IKKβ phosphorylation and IκBα degradation depend on NADPH oxidase activity. (A) Human subcutaneous differentiated adipocytes were stimulated for 15 min with 50 mU/ml C-bTSH. NADPH-dependent oxidation of lucigenin was measured. Results are the mean ± S.E.M. of 3 patient samples, each performed in duplicate. (B and C) Human abdominal subcutaneous differentiated adipocytes were pretreated as indicated, with 10 μmol/L DPI for 30 min (B), 10 mmol/L NAC for 2 h (C), or appropriate vehicle, prior to stimulation with 50 mU/ml C-bTSH or vehicle for another 30 min. Equal amounts of solubilized cellular protein were separated by SDS-PAGE and immunoblotted with indicated antibodies. Immunoblots from one patient sample are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 (B) or 4 (C) separate patient samples is graphically presented as mean ± S.E.M. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, as assessed by paired T-test (A) or two-way ANOVA with Tukey post-hoc tests (B and C).
expression are needed to add further support for their role in TSH signaling. However, differentiated human adipocytes are resistant to RNA silencing methods, and even recent
approaches, using lipid-based siRNA transfection with suspended cells, are not entirely effective at knocking down protein expression [40].
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Fig. 4 – PKCδ and TSH-stimulated IKK phosphorylation and IκBα degradation. (A and B) Human abdominal subcutaneous differentiated adipocytes were stimulated directly (A) or pretreated (B) with 10 μmol/L rottlerin or vehicle for 30 min prior to stimulation with, 50 mU/ml C-bTSH or vehicle for 30 min. Equal amounts of solubilized cellular protein were separated by SDS-PAGE and immunoblotted with indicated antibodies. Immunoblots from one patient sample are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 separate patient samples are graphically presented as mean ± S.E.M. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, as assessed by paired t-test (A) or two-way ANOVA with Tukey post-hoc tests (B).
In examining how TSH acts in differentiated adipocytes, we also performed methodical comparisons of TSH signaling using C-bTSH versus S-bTSH, as well as a recombinant human TSH analogue. All three, at the same dose and for the same time duration, activated PKA and IKKβ specifically in CHOhTSHR cells. To demonstrate that these TSH-stimulated signaling responses were not a function of the artificial expression of hTSHR in CHO cells, we undertook studies on human subcutaneous differentiated adipocytes. C-bTSH activated both pathways in differentiated adipocytes, but surprisingly, S-bTSH only activated the PKA pathway in those cells under the same conditions. More studies will be needed to address the reason why S-bTSH activates IKKβ in CHOTSHR cells but not differentiated adipocytes. Variation in glycosylation of TSH can lead to selective activation of TSHR signaling, and differences in how C-bTSH versus S-bTSH is purified may result in TSH heterogeneity with respect to posttranslational modifications such as glycosylation [41]. Selective TSHR signaling as a function of ligand has also been observed with TSHR stimulating antibodies [3].
In summary, we have identified a new pro-inflammatory cytokine that is stimulated by TSH in human differentiated adipocytes. Future studies will have to address whether the MCP1 released under the influence of TSH can mediate recruitment of monocytes, and to determine if this might lead to monocyte infiltration in vivo. Learning more about the actions of TSH on adipocytes may help provide insights into how TSH may modulate inflammatory responses in conditions such as subclinical hypothyroidism and its associated risk of cardiovascular disease.
Author contributions A. Sorisky and A Gagnon designed the study. Experimental work and data analysis were performed by A. Gagnon, M. Langille, S. Chaker, T Antunes, and J. Durand. A. Sorisky wrote the manuscript, and all authors were involved in reviewing and editing the manuscript.
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Fig. 5 – TSH increases MCP-1 mRNA expression and protein release. Human subcutaneous differentiated adipocytes were stimulated directly, or pretreated with 100 μmol/L sc-514 (1 h), 20 μmol/L H89 (1 h), 10 μmol/L rottlerin (30 min) or vehicle prior to stimulation with 50 mU/ml C-bTSH or vehicle for 2 h (A, B, D, and F) or 4 h (C and E). (A, B, D, and F) RNA was isolated and quantified by real time PCR, using indicated primers. Levels were normalized to endogenous 18S RNA, and expressed as a function of the control condition. Results are the mean ± S.E.M. of separate patient samples (A, B, and D, n = 4-5), or mean ± range (F, n = 2). (C and E) MCP-1 protein release in the medium was quantified by ELISA. Results were normalized to protein content and are the mean ± S.E.M. of 3 separate patient samples. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, as assessed by paired t-test (A) or two-way ANOVA with Tukey post-hoc tests (B and C).
Funding This work was supported by the Canadian Institutes of Health Research (operating grant MOP-102585 to AS). MLL
was the recipient of an Ontario Graduate Scholarship in Science and Technology Award. TTA was the recipient of a Heart and Stroke Foundation of Canada Doctoral Research Award.
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Acknowledgments [14]
We thank the patients and surgeons of The Ottawa Hospital for human adipose tissue samples. [15]
Conflicts of interests [16]
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research presented.
[17]
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TSH signaling pathways that regulate MCP-1 in human differentiated adipocytes AnneMarie Gagnon, Melanie L. Langille, Seham Chaker, Tayze T. Antunes, Jason Durand, Alexander Sorisky⁎ Chronic Disease Program, Ottawa Hospital Research Institute, Departments of Medicine and of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa, Ontario, Canada Chronic Disease Program, Ottawa Hospital Research Institute, Department of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa, Ontario, Canada
A R T I C LE I N FO Article history:
AB S T R A C T Objective. Adipose tissue is an extra-thyroidal thyroid-stimulating hormone (TSH) target.
Received 11 November 2013
Increases in lipolysis and in expression and release of interleukin-6 (IL-6) occur in TSH-
Accepted 25 February 2014
stimulated adipocytes, and levels of circulating free fatty acids and IL-6 rise following TSH administration to patients with previous thyroidectomy and radioablation for thyroid
Keywords:
cancer. Our first objective was to compare how TSH stimulates protein kinase A (PKA) and
Thyroid-stimulating hormone
inhibitor of κB (IκB) kinase (IKK)-β. Our second objective was to investigate whether TSH
Monocyte chemoattractant protein-1
induces other cytokines besides IL-6.
Inhibitor of κB kinase-β Protein kinase C-δ NADPH oxidase
Methods. TSH stimulation of either CHO cells expressing human TSH receptor or human abdominal subcutaneous differentiated adipocytes. Results. Signaling studies showed TSH increased NADPH oxidase activity, and either diphenyleneiodonium (oxidase inhibitor) or N-acetyl cysteine (scavenger of reactive oxygen species) reduced IKKβ phosphorylation. Phosphorylation of protein kinase C-δ, an upstream regulator of NADPH oxidase, was increased by TSH, and rottlerin (PKCδ inhibitor) reduced TSH-stimulated IKKβ phosphorylation. TSH upregulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression and the release of MCP-1 protein in human abdominal differentiated adipocytes. H89 (PKA inhibitor) and sc-514 (IKKβ inhibitor) each blocked TSH-stimulated MCP-1 mRNA expression and protein release, suggesting PKA and IKKβ participate in this pathway. Conclusions. These data provide new information about TSH signaling in human differentiated adipocytes, and add to the evidence that TSH is a pro-inflammatory stimulus of adipocytes. © 2014 Elsevier Inc. All rights reserved.
Abbreviations: BSA, bovine serum albumin; CHO, Chinese hamster ovarian; CREB, cAMP response element-binding protein; DMEM, Modified Eagle’s Medium; DPI, diphenyleneiodonium; FBS, fetal bovine serum; FFA, free fatty acids; GPCR, G protein-coupled receptor; IκB, inhibitor of κB; IKK, inhibitor of IκB kinase; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; PKA, protein kinase A; PKC, protein kinase C; RBP4, retinol binding protein 4; rh, recombinant human; RT, reverse transcriptase; TSH, thyroidstimulating hormone; TSHR, TSH receptor. ⁎ Corresponding author at: Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada. E-mail address: [email protected] (A. Sorisky). http://dx.doi.org/10.1016/j.metabol.2014.02.015 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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1.
Introduction
In thyrocytes, thyroid-stimulating hormone (TSH) binds to its receptor (TSHR), a G protein-coupled receptor (GPCR) that can communicate with several G proteins, including Gs and G q . A variety of downstream signaling pathways are consequently activated, regulating thyrocyte growth and hormone production [1–4]. Extra-thyroidal expression of TSHR in adipocytes has been documented [5–8], but little is known about how TSH influences adipocyte signaling and cellular responses. In vivo, TSH stimulates the release of pro-inflammatory molecules and free fatty acids (FFA) when administered acutely to patients previously treated for thyroid cancer by total thyroidectomy and radioablation [9–11]. Furthermore, serum levels of interleukin-6 (IL-6), tumor necrosis factor-α, C-reactive protein, and FFA are elevated in patients with subclinical hypothyroidism, a condition whereby chronically elevated TSH serum levels compensate for mild thyroid failure to maintain normal thyroid hormone levels [12–15]. These inflammatory and metabolic disturbances might explain the epidemiological association of accelerated cardiovascular disease with subclinical hypothyroidism [16,17]. Studies on adipocyte cell cultures reveal similar TSHstimulated responses to the ones described above for in vivo studies. TSH stimulates release of FFA in human differentiated adipocytes and neonatal adipocytes in culture, and requires protein kinase A (PKA) as well as protein kinase C (PKC) [11,18,19]. TSH also dose-dependently stimulates IL-6 production and release in human abdominal differentiated adipocytes, and this depends on the inhibitor of κB kinase (IKK) β/nuclear factor (NF) κB pathway [9,20]. This pathway is also activated by TSH in orbital adipose cells and other cell types [21–24]. The downstream activation of IKKβ by other GPCR may occur via the activation of NADPH oxidase [25]. The generation of reactive oxygen species by NADPH oxidase results in the activation of IKKβ [26]. Our objectives were to investigate how TSHR activates the IKKβ pathway, and to evaluate whether adipokines other than IL-6 are regulated by TSH. We examined if there is a role for NADPH oxidase and PKC-δ upstream of TSH-stimulated IKKβ, and also whether monocyte chemoattractant protein-1 (MCP-1) is a TSH-induced adipokine.
2.
Material and methods
2.1.
Chinese hamster ovarian (CHO) cell culture
Parental CHO cells (CHO-JP02) and human TSHR overexpressing CHO cells (CHO-JP2626, or CHO-hTSHR) were kindly provided by J. E. Dumont, Erasme University Hospital, Free University of Brussels, Brussels, Belgium [27]. Cells were plated and grown in Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS), antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and 400 μg/ml G418 (all from Life Technologies, Burlington, ON, Canada). Medium was changed every 2 days until confluence.
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2.2. Isolation, culture, and differentiation of human abdominal subcutaneous stromal cells Human abdominal subcutaneous adipose tissue was obtained from 22 patients (19 female, 3 male), with mean age 49 ± 9 years (± S.D.) and mean body mass index 27 ± 6 (±S.D.), undergoing elective abdominal surgery (approved by the Ottawa Hospital Research Ethics Board, protocol 1995023-01H). They were weight-stable, did not have diabetes, and were not on steroid therapy. The stromal preadipocytes were isolated as previously described [28]. Briefly, blood vessels and fibrous tissue were carefully removed, followed by collagenase digestion (CLS type I; 600 U/g of tissue; Worthington Biochemical Corporation, Lakewood, NJ, USA), and sequential centrifugation, size filtration, and treatment with erythrocyte lysis buffer. Preadipocytes were cultured in growth medium which was Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies) supplemented with 10% FBS and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 U/ml nystatin; EMD Millipore, Billerica, MA, USA). Upon reaching 80%–90% confluence, cells were either re-plated for a maximum of three passages or cryopreserved until needed. The presence of macrophages (CD14) or endothelial cells (CD31) is < 2% in our culture conditions. For differentiation, preadipocytes were seeded at a density of 3 × 104 cells/cm2 and allowed to adhere overnight. Adipogenesis was induced with growth medium supplemented with 0.85 μmol/L insulin, 0.25 mmol/L isobutylmethylxanthine, 100 μmol/L indomethacin, and 0.5 μmol/L dexamethasone for 14 days. Cells were then placed in growth medium for 2 days prior to stimulation studies.
2.3.
Acute stimulation
CHO cells, CHO-hTSHR cells, or human differentiated adipocytes, in DMEM supplemented with 4% fatty acid-free bovine serum albumin (BSA) or 1% calf serum (Life Technologies), were stimulated with 50 mU/ml Calbiochem bovine TSH (C-bTSH; EMD Millipore), Sigma bovine TSH (S-bTSH; Sigma-Aldrich, Oakville, ON, Canada), an analog of recombinant human (rh) TSH (TR1401, a kind gift from MW Szkudlinski,Trophogen, Rockville, MD), or vehicle (H2O) for 0 to 4 h, as shown. Where indicated, cells were pre-treated for 30 min with 10 μmol/L diphenyleneiodonium (DPI; Sigma-Aldrich), 10 μmol/L rottlerin (EMD Millipore), or vehicle (0.1% DMSO); for 1 h with 100 μmol/L sc-514, 20 μmol/L H-89 (both from EMD Millipore), or vehicle (0.1% DMSO); or for 2 h with 10 mmol/L N-acetylcysteine (NAC; SigmaAldrich) or vehicle (H2O), prior to stimulation with TSH. Cells were then lysed and processed for immunoblot analysis, NADPH oxidase activity, or RNA isolation and qPCR. Conditioned medium was collected and assessed for cytokine content by ELISA.
2.4.
Immunoblot analysis
Cells were lysed in Laemmli buffer [29] containing 50 mmol/L sodium fluoride, 5 mmol/L sodium pyrophostate, 5 mmol/L EGTA, and 1 mmol/L sodium orthovanadate. Protein was quantified by the modified Lowry assay (BioRad; Bio-Rad, Hercules, CA, USA), with BSA as standard. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose. Non-specific antigenic sites were blocked and
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membranes were probed with the indicated primary antibodies: anti-phospho-cAMP response element-binding protein (CREB; Ser133; 1:1000), anti-CREB (1:1000), anti-phospho-IKK α Ser180/IKKβ Ser181 (1:1000), anti-inhibitor of κBα (1:1000), antiIKKβ (1:500), anti-phospho-PKC δ (Thr505; 1:500), anti-PKCδ (1:1000, all from Cell Signaling; Danvers, MA, USA), or anti-actin (serves as a loading control, Santa Cruz Biotech., Santa Cruz, CA, USA). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch, Laboratories, West Grove, PA, USA), and immunoreactivity was visualized by enhanced chemiluminescence (EMD Millipore). Densitometric analysis of immunoreactive bands was performed with AlphaEaseFC software (Alpha Innotec Co, San Leandro, CA, USA), and data were expressed as integrated optical density (IOD) units.
2.5.
NADPH oxidase activity
Following stimulation of cells with TSH as described, cells were lysed in 20 mmol/L KH2PO4, 1 mmol/L EGTA pH 7.4, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mmol/L PMSF. Reaction tubes included 175 μl of assay buffer (20 mmol/L KH2PO4, 1 mmol/L EGTA, 150 mmol/L sucrose pH 7.4), 25 μL of cellular lysates, and 5 μmol/L lucigenin. Lucigenin-enhanced chemiluminescence measurements were performed in the presence versus absence of 1 mmol/L NAPDH, over 3 min in a FluoStar Optima reader (BMG Labtech, Cary, NC, USA). After subtraction of a buffer blank, the results were expressed as foldstimulation of control conditions.
2.6.
qPCR
Cells were lysed in Qiazol (Qiagen, Toronto, ON, Canada), and RNA was isolated and treated with DNA-free (Life Technologies), as per manufacturer’s instructions. qPCR was performed as previously described [30]. Briefly, total RNA (1 μg per sample) was heat-denatured, then reverse-transcribed using the Retroscript kit with random primers (Life Tehcnologies). Control reactions, without transcriptase, were performed for all samples. Real-time qPCR assays were performed using the QuantiTect SYBR Green RT-PCR kit (for target genes) or the QuantiTect Probe RT-PCR kit (for 18S rRNA; both from Qiagen), according to manufacturer’s instructions. Reactions were run on the Roche Light Cycler RealTime PCR System (Roche, Laval, QC, Canada), and data were analyzed with Light Cycler Software, version 3.0 (Roche). Primer sequences were: adiponectin: forward: 5’-GCAGAGATGG CACCCCTG-3’, reverse: 5’-GGTTTCACCGATGTCTCCCTTA-3’; MCP-1: forward: 5’-CAGCCAGATGCAATCAATGC-3’, reverse: 5’GTGGTCCATGGAATCCTGAA-3’; and retinol-binding protein 4 (RBP4): forward: 5’-GCCTCTTTCTGCAGGACAAC-3’, reverse: 5’-GCACACGTCCCAGTTATTCA-3’.
2.7.
ELISA
Conditioned medium was collected and centrifuged at 500 ×g, 5 min at 4 °C, to remove any cellular debris. Supernatant was collected and MCP-1 protein was quantified using a Quantikine ELISA kit (MCP-1; R&D systems, Minneapolis, MN, USA). The cell monolayer was lysed in Laemmli buffer [29] and protein was quantified using the modified Lowry assay, with BSA as
standard. MCP-1 release into the medium was normalized to cellular protein content.
2.8.
Statistical analysis
Statistical analysis was performed with Student’s t-test for paired samples (two means compared), one-way ANOVA followed by Student–Newman–Keuls post hoc test (three or more means with one variable compared), or with two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was defined as P < 0.05.
3.
Results
To clarify the activation of the PKA and IKKβ signaling pathways by TSH, we compared bovine TSH (bTSH) from Calbiochem (C-bTSH) versus Sigma (S-bTSH). Since the CbTSH and S-bTSH preparations are not completely purified, other factors present in the reagents could possibly be responsible for the activation of these signaling molecules. The JP2626 CHO-hTSHR and JP02 CHO control cell lines stably transfected with either human TSHR [27] or control vector, respectively, were used. CREB is a direct substrate of PKA and its phosphorylation indicates PKA activation [20]. Each TSH preparation (50 mU/ml) significantly increased the levels of phospho-CREB and phospho-IKKβ in the CHO-hTSHR cells (Fig. 1). There was no consistent change in total CREB levels. In the CHO controls, the small changes that occurred were not reproducible and did not reach significance, but we cannot completely rule out a minor effect of TSH on these cells. Overall, these responses to the bTSH preparations are predominantly TSHR-dependent. To address this issue further, we repeated the stimulation studies, this time using the rhTSH analog TR1401 [31]. The same TSHR-dependent pattern of signaling, i.e. significant and large responses only occurring in the CHO-hTSHR cells, was observed as previously noted with C-bTSH or S-bTSH stimulation (Fig. 1). Therefore, all three TSH reagents stimulate significant increases in phosphorylation of CREB and IKKβ in a TSHRdependent fashion. We compared the signaling responses activated by C-bTSH versus S-bTSH in human abdominal subcutaneous differentiated adipocytes that express TSHR endogenously. Each TSH preparation stimulated CREB phosphorylation to the same extent (Fig. 2), as was observed when comparing these agonists using the CHOhTSHR cells (Fig. 1A). Total CREB levels did not show any consistent change. However, only C-bTSH was competent to increase IKKβ phosphorylation and the consequent degradation of IκB. C-bTSH was used to examine upstream regulators of IKKβ phosphorylation and IκBα degradation in human abdominal subcutaneous differentiated adipocytes. GPCRs increase reactive oxygen species (ROS) in different cell types via activation of NADPH oxidase [25]. Following stimulation of human differentiated adipocytes with 50 mU/ml of C-bTSH, NADPH oxidase activity increased significantly by 21% (Fig. 3A). We examined whether inhibiting formation of ROS, or accelerating their clearance, would inhibit the IKKβ response to TSH. The level of IKKβ phosphorylation in response to C-bTSH (50 mU/ml) was reduced by 45% in the presence of diphenyleneiodonium (DPI;
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C-bTSH (50 mU/ml) was observed at 30 min (Fig. 4A), assessed by Thr505 phosphorylation [34]. We investigated whether blocking PKCδ would reduce IKKβ and IκB signaling in response to C-bTSH. An inhibitor of PKCδ, rottlerin (10 μmol/L) [35,36], reduced C-bTSH-stimulated IKKβ phosphorylation by 55%, and IκB degradation by 35% (Fig. 4B). Given that C-bTSH signals via IKKβ to increase IL-6 protein release from human abdominal subcutaneous differentiated adipocytes [20], we examined whether it might regulate other cytokines. C-bTSH (50 mU/ml) strongly increased MCP-1 mRNA expression by 12-fold, with no effect on either RBP4 or adiponectin, two other adipokines known to be perturbed in the low-grade inflammatory state of obesity (Fig. 5A). We investigated whether the TSH-stimulated MCP-1 response in differentiated adipocytes required signaling via IKKβ and/or PKA, using inhibitors as previously reported [20]. The IKKβ inhibitor sc-514 (100 μmol/L) reduced C-bTSHstimulated MCP-1 mRNA expression by 59% (Fig. 5B). C-bTSH increased MCP-1 protein release by 3-fold, and this was completely inhibited by sc-514 (Fig. 5C). PKA inhibitor H89 (20 μmol/L) reduced C-bTSH-stimulated MCP-1 mRNA expression and MCP-1 protein release by 54% and 58%, respectively (Fig. 5D and E). Rottlerin, which inhibited IKKβ phosphorylation and IκB degradation (Fig. 4B), also reduced the C-bTSHstimulated mRNA expression of MCP-1 mRNA by 62% (Fig. 5F).
4.
Fig. 1 – TSH stimulates IKKβ and CREB phosphorylation in CHO-hTSHR cells and human differentiated adipocytes. Confluent CHO-hTSHR and parental (CHO) cells were stimulated with 50 mU/ml of either C-bTSH, S-bTSH, rhTSH analog TR1401 (TR), or with vehicle control for 30 min. Equal amounts of solubilized cellular protein were immunoblotted with the indicated antibodies. Immunoblots from one experiment are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 separate experiments are graphically presented as mean ± S.E.M. ** indicates P < 0.01, and * indicates P < 0.05 compared to control condition, as assessed by 2-way ANOVA with Tukey post-hoc tests.
10 μmol/L), an oxidase inhibitor [32]. The accompanying IκB degradation response was also inhibited by 45%, but did not reach significance (Fig. 3B). N-acetyl cysteine (NAC; 10 mmol/L), a ROS scavenger [32], reduced the C-bTSH (50 mU/ml)-stimulated IKKβ phosphorylation response by 53% (Fig. 3C). There was a trend towards attenuation of IκB degradation of 18% that did not reach significance. Taken together, DPI or NAC reduces TSHstimulated IKKβ phosphorylation in human differentiated adipocytes. The effects of these agents on IκB degradation only showed non-significant trends. The p47phox subunit of NADPH oxidase is phosphorylated by PKCδ in response to some GPCRs [33]. Activation of PKCδ by
Discussion
Our studies on human abdominal differentiated adipocytes reveal MCP-1 is a novel TSH-stimulated adipokine, and suggest signaling via IKKβ and PKA pathways is involved in mediating this effect. Little is known about how TSH leads to IKKβ activation, and our results point to PKCδ and NADPH oxidase being positioned upstream of IKKβ in this TSH response. Adipose tissue is an extrathyroidal TSH target. Adipocytes express TSHR [5–8]. However, the way TSH signals to produce its catalogue of responses in these cells is not well understood. It may act on adipocytes in the neonatal period to release energy during acclimation to the environment [18]. TSH activates PKA and this is required for lipolysis, and for phosphorylation of hormone-sensitive lipase and perilipin, in differentiated human adipocytes [11]. Subsequent work suggested TSH could activate conventional PKC, and this was required for TSH to fully stimulate perilipin phosphorylation and lipolysis [19]. TSH also upregulates IL-6 mRNA expression and IL-6 protein release in human differentiated adipocytes, and this depends on IKKβ, but not PKA [20]. We have identified MCP-1 as a novel TSH-stimulated adipokine. Consistent with this observation, levels of MCP-1 are elevated in patients with chronic autoimmune thyroiditis with mild hypothyroidism characterized by elevated TSH serum concentrations [37]. The increase in MCP-1 mRNA expression and MCP-1 protein release by TSH was blocked by either the IKKβ inhibitor sc-514 or the PKA inhibitor H89. This contrasts to what we previously observed with IL-6, which was only inhibited by sc-514 [20]. Our studies were performed with a single dose of TSH and one time point based on previous work with IL-6 [11,20]; future studies examining a range of doses and time points may provide more information on the MCP-1 response.
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Fig. 2 – IKKβ phosphorylation and IκBα degradation in response to TSH in human abdominal subcutaneous differentiated adipocytes. Human differentiated adipocytes were stimulated with 50 mU/ml C-bTSH, S-bTSH, or with vehicle control for 30 min. Equal amounts of solubilized cellular protein were immunoblotted with indicated antibodies. Immunoblots from one experiment are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 patient samples are graphically presented as mean ± S.E.M. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, assessed by one-way ANOVA with Student–Newman–Keul post-hoc tests.
TSH signaling to PKA is thought to be due to TSHR interacting with Gs to activate adenylate cyclase, generating a rise in cAMP. However, little is known about how TSH leads to IKKβ phosphorylation. Studies on other GPCR ligands in other cell systems have indicated that NADPH oxidase and ROS generation lie upstream of IKKβ phosphorylation [25,26]. In human adipose cells, Nox4 is the isoform that is predominantly expressed [38]. We found that TSH was able to increase NADPH oxidase activity in differentiated human adipocytes. To determine if this was required for IKKβ phosphorylation, we used DPI, an oxidase inhibitor to prevent ROS formation, or NAC, to accelerate the clearance of ROS. Either strategy reduced TSH-stimulated IKKβ phosphorylation by ~ 50%, indicating the importance of this event. The inhibition was not total, possibly due to other TSH-stimulated molecular pathways regulating IKKβ phosphorylation, or to technical limitations of the inhibitors used. PKCδ, a novel PKC isoform, has been implicated in phosphorylating and activating NADPH oxidase [33]. We have previously observed that TSH can activate PKC in human differentiated adipocytes based on an increase in
phosphorylated proteins detected by a phospho-PKC substrate antibody specific for conventional PKC [19]. Our data here indicate that TSH is also able to activate PKCδ, indicated by its phosphorylation [34]. Rottlerin, a PKCδ selective inhibitor [35,36], inhibited TSH-stimulated IKKβ phosphorylation and IκBα degradation, consistent with a role for PKCδ in this TSH-stimulated pathway. Additional investigations will be needed to identify proximal events linking TSHR activation to this signaling pathway. The dose of TSH for our in vitro studies is higher than physiological, but it approximates doses used in thyrocyte studies [39]; it is also important to note that corresponding data related to cytokine release have been observed in clinical studies [9,10,12,14,15]. Our data, based on pharmacological inhibitors, suggest that the activation of PKCδ and NADPH oxidase by TSH lies upstream of IKKβ in human differentiated adipocytes. Limitations of inhibitors include off-target effects. In particular, the specificity of rotttlerin is debated, but it continues to be used as an investigational agent [35,36]. Future studies using RNAi to reduce PKCδ or NADPH oxidase
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Fig. 3 – NADPH oxidase and TSH-stimulated IKKβ phosphorylation and IκBα degradation depend on NADPH oxidase activity. (A) Human subcutaneous differentiated adipocytes were stimulated for 15 min with 50 mU/ml C-bTSH. NADPH-dependent oxidation of lucigenin was measured. Results are the mean ± S.E.M. of 3 patient samples, each performed in duplicate. (B and C) Human abdominal subcutaneous differentiated adipocytes were pretreated as indicated, with 10 μmol/L DPI for 30 min (B), 10 mmol/L NAC for 2 h (C), or appropriate vehicle, prior to stimulation with 50 mU/ml C-bTSH or vehicle for another 30 min. Equal amounts of solubilized cellular protein were separated by SDS-PAGE and immunoblotted with indicated antibodies. Immunoblots from one patient sample are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 (B) or 4 (C) separate patient samples is graphically presented as mean ± S.E.M. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, as assessed by paired T-test (A) or two-way ANOVA with Tukey post-hoc tests (B and C).
expression are needed to add further support for their role in TSH signaling. However, differentiated human adipocytes are resistant to RNA silencing methods, and even recent
approaches, using lipid-based siRNA transfection with suspended cells, are not entirely effective at knocking down protein expression [40].
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Fig. 4 – PKCδ and TSH-stimulated IKK phosphorylation and IκBα degradation. (A and B) Human abdominal subcutaneous differentiated adipocytes were stimulated directly (A) or pretreated (B) with 10 μmol/L rottlerin or vehicle for 30 min prior to stimulation with, 50 mU/ml C-bTSH or vehicle for 30 min. Equal amounts of solubilized cellular protein were separated by SDS-PAGE and immunoblotted with indicated antibodies. Immunoblots from one patient sample are shown; images from a single immunoblot were cropped to remove irrelevant lanes. Densitometric analysis of 3 separate patient samples are graphically presented as mean ± S.E.M. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, as assessed by paired t-test (A) or two-way ANOVA with Tukey post-hoc tests (B).
In examining how TSH acts in differentiated adipocytes, we also performed methodical comparisons of TSH signaling using C-bTSH versus S-bTSH, as well as a recombinant human TSH analogue. All three, at the same dose and for the same time duration, activated PKA and IKKβ specifically in CHOhTSHR cells. To demonstrate that these TSH-stimulated signaling responses were not a function of the artificial expression of hTSHR in CHO cells, we undertook studies on human subcutaneous differentiated adipocytes. C-bTSH activated both pathways in differentiated adipocytes, but surprisingly, S-bTSH only activated the PKA pathway in those cells under the same conditions. More studies will be needed to address the reason why S-bTSH activates IKKβ in CHOTSHR cells but not differentiated adipocytes. Variation in glycosylation of TSH can lead to selective activation of TSHR signaling, and differences in how C-bTSH versus S-bTSH is purified may result in TSH heterogeneity with respect to posttranslational modifications such as glycosylation [41]. Selective TSHR signaling as a function of ligand has also been observed with TSHR stimulating antibodies [3].
In summary, we have identified a new pro-inflammatory cytokine that is stimulated by TSH in human differentiated adipocytes. Future studies will have to address whether the MCP1 released under the influence of TSH can mediate recruitment of monocytes, and to determine if this might lead to monocyte infiltration in vivo. Learning more about the actions of TSH on adipocytes may help provide insights into how TSH may modulate inflammatory responses in conditions such as subclinical hypothyroidism and its associated risk of cardiovascular disease.
Author contributions A. Sorisky and A Gagnon designed the study. Experimental work and data analysis were performed by A. Gagnon, M. Langille, S. Chaker, T Antunes, and J. Durand. A. Sorisky wrote the manuscript, and all authors were involved in reviewing and editing the manuscript.
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Fig. 5 – TSH increases MCP-1 mRNA expression and protein release. Human subcutaneous differentiated adipocytes were stimulated directly, or pretreated with 100 μmol/L sc-514 (1 h), 20 μmol/L H89 (1 h), 10 μmol/L rottlerin (30 min) or vehicle prior to stimulation with 50 mU/ml C-bTSH or vehicle for 2 h (A, B, D, and F) or 4 h (C and E). (A, B, D, and F) RNA was isolated and quantified by real time PCR, using indicated primers. Levels were normalized to endogenous 18S RNA, and expressed as a function of the control condition. Results are the mean ± S.E.M. of separate patient samples (A, B, and D, n = 4-5), or mean ± range (F, n = 2). (C and E) MCP-1 protein release in the medium was quantified by ELISA. Results were normalized to protein content and are the mean ± S.E.M. of 3 separate patient samples. ** denotes P < 0.01, and * denotes P < 0.05 between indicated pairs, as assessed by paired t-test (A) or two-way ANOVA with Tukey post-hoc tests (B and C).
Funding This work was supported by the Canadian Institutes of Health Research (operating grant MOP-102585 to AS). MLL
was the recipient of an Ontario Graduate Scholarship in Science and Technology Award. TTA was the recipient of a Heart and Stroke Foundation of Canada Doctoral Research Award.
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Acknowledgments [14]
We thank the patients and surgeons of The Ottawa Hospital for human adipose tissue samples. [15]
Conflicts of interests [16]
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research presented.
[17]
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