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. 2009 Jul;46(11-12):2256-66.
doi: 10.1016/j.molimm.2009.04.008. Epub 2009 May 23.

Signaling Pathways Mediating beta3-adrenergic Receptor-Induced Production of interleukin-6 in Adipocytes

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Free PMC article

Signaling Pathways Mediating beta3-adrenergic Receptor-Induced Production of interleukin-6 in Adipocytes

Inna E Tchivileva et al. Mol Immunol. .
Free PMC article

Abstract

The beta(3)-adrenergic receptor (beta(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta(3)AR activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the beta(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.

Figures

Fig. 1
Fig. 1
β3AR stimulation up-regulates IL-6 production in adipocytes. 3T3-L1 cells treated with the β3AR-agonist CL316243 (1 μM for 45 min) showed a significant increase in IL-6 mRNA. The CL316243-induced increase in cytokine transcripts was completely blocked by pre-treatment with SR59230A (1 μM for 1 h) but not with betaxolol (0.5 μM for 1 h) or ICI118,551 (0.5 μM for 1 h) (a). 3T3-L1 cells treated with the β3AR-agonist CL316243 (1 μM for 24 and 48 h) showed a significant increase in IL-6 protein measured in cell supernatant (b). ***P<0.001 different from untreated.
Fig. 2
Fig. 2
β3AR-induced IL-6 expression is PKA- and Epac-independent. C3H10T1/2 cells were pretreated with H89 (10 μM for 1 h) prior to stimulation with CL316243 (1 μM for 2 h). The CL316243-induced activation of the CRE reporter was not inhibited by H89 in the homogenous reporter assay (a). 3T3-L1 cells were treated with KT5720 (10 μM) or H89 (5-10 μM) for 1 h prior to treatment with CL316243 (1 μM for 45 min). The CL316243-induced up-regulation of IL-6 mRNA was not inhibited by KT5720 or H89 (b). Treatment of 3T3-L1 with CTX (100 ng/ml for 4 h) led to increases in IL-6 transcript, while treatment with the Epac agonist 8CPT-2’-O-Me-cAMP (CPTOMe) (10 μM for 45 min) did not enhance IL-6 expression (c). ***P<0.001 and **P<0.01 different from untreated.
Fig. 3
Fig. 3
β3AR-induced IL-6 expression is not mediated by NF-κB pathway. C3H10T1/2 cells were treated with CL316243 (1 μM) and TNFα (10 ng/ml) for 2 h. In the homogenous reporter assay, CL316243 didn’t activate the NF-κB reporter, while TNFα did (a). 3T3-L1 cells were pre-treated with NBD (100 μM) or MG-132 (25-50 μM) for 1 h prior to treatment with CL316243 (1 μM for 45 min). The CL316243-induced up-regulation of IL-6 mRNA was not inhibited by NBD or MG-132 (b). SEAP activity was assayed from the culture media of HEK 293 cells co-transfected with the pNF-κB-SEAP reporter construct and β3AR expression vector. No significant increase in SEAP activity was observed at 6, 12 and 24 h post-treatment with CL316243 (1 μM) (c). ***P<0.001 and **P<0.01 different from untreated.
Fig. 4
Fig. 4
β3AR-induced IL-6 expression is Gi- and ERK1/2-independent and mediated by p38 signaling pathway. 3T3-L1 cells were pretreated with PTX (100 ng/ml and 200 ng/ml for 1 h) prior to stimulation with CL316243 (1 μM for 45 min). The CL316243-induced up-regulation of IL-6 transcripts was not blocked by PTX (a). 3T3-L1 cells were pretreated with either U0126 (10 μM) or SB203580 (5-10 μM) for 1 h prior to stimulation with CL316243 (1 μM for 45 min). The CL316243-induced up-regulation of IL-6 transcripts was blocked by SB203580, but not U0126 (b). C3H10T1/2 cells were pretreated with SB203580 (10 μM for 1 h) prior to stimulation with CL316243 (1 μM for 45 min). The CL316243-induced activation of CRE reporter was inhibited by SB203580 in homogenous reporter assay (c). The cells lysates were also subjected to Western blot analyses with antibodies to p-p38. Increased phosphorylation of p38 was observed at 3, 30 and 60 min post-treatment with CL316243. β-actin was used as the loading control (d). ***P<0.001 and **P<0.01 different from untreated. #P<0.05 different from CL316243-treated.
Fig. 5
Fig. 5
β3AR-induced IL-6 expression is dependent on the PKC signaling pathway. 3T3-L1 cells were pretreated with RO31-8220 (1-10 μM) for 1 h prior to stimulation with CL316243 (1 μM for 45 min). The CL316243-induced up-regulation of IL-6 transcripts was blocked by RO31-8220 (a). C3H10T1/2 cells were pretreated with RO31-8220 (10 μM for 1 h) prior to stimulation with CL316243 (1 μM for 2 h). The CL316243-induced activation of CRE reporter was inhibited by RO31-8220 in the homogenous reporter assay (b). The cells lysates were also subjected to Western blot analyses with antibodies to pPKCpan (βII Ser660), pPKCα/βII (Thr638/641), pPKCθ (Thr538), and pPKCε (Ser729). Increased phosphorylation of PKCα/βII was observed at 30 sec and PKCpan, PKCθ and PKCε 45 min post-treatment with CL316243. β-actin was used as the loading control (c). ***P<0.001 and **P<0.01 different from untreated. #P<0.05 different from CL316243-treated.
Fig. 6
Fig. 6
Stimulation of β3ARs results in up-regulation of the reporter construct containing the CRE site. HEK 293 cells were transiently co-transfected with pCRE-SEAP (a), pAP1-SEAP (b), pC/EBPβ-SEAP (c), or pETS-SEAP (d) reporter constructs along with the β3AR expression vector. 24 h after transfection, media was changed, and cells were stimulated with CL316243 (1 μM) for 6, 12 and 24 h. Significant increase in SEAP activity was observed with pCRE-SEAP construct only. ***P<0.001 different from untreated.
Fig. 7
Fig. 7
Effect of β3AR agonist stimulation on phosphorylation of CREB, ATF-1, and ATF-2. 3T3-L1 cells were stimulated with CL316243 (1 μM) for 3, 30, and 60 min, then cell lysates were subjected to Western blot analyses with antibodies to pCREB, pATF-1, and pATF-2. CL316243 stimulation increased phosphorylation of pCREB, pATF-1, and pATF-2 (a). 3T3-L1 cells were pretreated with SB203580 (10 μM for 1h) or RO31-8220 (10 μM for 1 h) before stimulation with CL316243 (1 μM for 45 min). CL316243-induced phosphorylation of CREB and ATF-2 was attenuated in the presence of SB203580, while ATF-2 phosphorylation was also decreased in the presence of RO31-8220, although non-specific phosphorylation of ATF-2 also has been observed in presence of inhibitors alone. ATF-1 phosphorylation was not affected by inhibitor treatment. β-actin was used as the loading control (b).
Fig. 8
Fig. 8
Schematic diagram of the proposed signaling pathways for activation of IL-6 gene by β3AR stimulation in white adipocytes.

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