Interleukin-6 autocrine signaling mediates melatonin MT1/2 receptor-induced STAT3 Tyr705 phosphorylation
Interleukin-6 autocrine signaling mediates melatonin MT1/2 receptor-induced STAT3 Tyr705 phosphorylation
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DOI：
10.1111/j.1600-079x.2011.00965.x
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发表时间：
2012-05-01
影响因子：
10.3
通讯作者：
Wong, Yung H.
中科院分区：
文献类型：
--
作者：
Lau, Winnie W. I.;Ng, Johnson K. Y.;Wong, Yung H.
褪黑激素受体先前已被证明通过造血特异性G蛋白G16引发细胞信号传导。在本研究中，我们发现这种功能偶联在重组MT1/Ga16细胞和天然Jurkat T细胞（内源性表达MT1和Ga16）中引发STAT3的双相刺激磷酸化，最大Ser727磷酸化发生在15分钟，而标记的Tyr705磷酸化只有在激动剂治疗4小时或更长时间后才能检测到。通过使用转录3 （STAT3）磷酸化抗性突变体（STAT3- y705f和STAT3- s727a），我们进一步发现受体介导的STAT3在Ser727和Tyr705位点的磷酸化是相互独立的。2- imt诱导的细胞分离结果显示，Ser727和Tyr705的磷酸化在空间上是不同的，前者主要位于线粒体和细胞质中，后者主要位于细胞核中。进一步的实验表明，激动剂诱导的STAT3 Tyr705磷酸化被环己亚胺（一种核糖体抑制剂）预处理显著抑制，这表明从头蛋白合成可能在这种反应中起关键作用。使用从2- imt处理的MT1/Ga16细胞中获得的条件培养基，多重免疫分析显示，长期激动剂治疗导致IL-6、GM-CSF和CXCL-8的产生升高。针对IL-6的抗体，而不是针对GM-CSF和CXCL-8的抗体，有效地消除了激动剂诱导的STAT3 Tyr705磷酸化，提示IL-6参与褪黑激素受体介导的STAT3激活。我们的研究结果表明，褪黑激素受体/Ga16偶联能够触发包括IL-6在内的细胞因子的产生，这种自分泌环可能解释了随后STAT3 Tyr705位点的磷酸化。
Melatonin receptors have previously been shown to elicit cellular signaling through the hematopoietic-specific G protein, G16. In the present study, we show that this functional coupling elicited biphasic stimulatory phosphorylation on STAT3 in recombinant MT1/Ga16 cells and native Jurkat T cells (endogenously expressing MT1 and Ga16), with maximal Ser727 phosphorylation occurring at 15 min, while marked Tyr705 phosphorylation became detectable only upon agonist treatment for 4 hr or more. By employing signal transducer and activator of transcription 3 (STAT3) phosphorylation-resistant mutants (STAT3-Y705F and STAT3-S727A), we further showed that the receptor-mediated STAT3 phosphorylations at Ser727 and Tyr705 were independent of each other. Results obtained from fractionation of 2-IMT-induced cells revealed that the Ser727 and Tyr705 phosphorylations were spatially distinct, with the former mainly situated in mitochondria and cytosol, while the latter was predominantly located in the nucleus. Further experiments revealed that the agonist-induced STAT3 phosphorylation at Tyr705 was significantly suppressed by pretreatment with cycloheximide (a ribosome inhibitor), suggesting that de novo protein synthesis might play a critical role for this response. Using conditioned media obtained from 2-IMT-treated MT1/Ga16 cells, multiplex immunoassays revealed that prolonged agonist treatment led to elevated productions of IL-6, GM-CSF and CXCL-8. Antibody against IL-6, but not those for GM-CSF and CXCL-8, effectively abolished the agonist-induced STAT3 Tyr705 phosphorylation, suggesting the involvement of IL-6 in melatonin receptor-mediated STAT3 activation. Our results demonstrate that melatonin receptor/Ga16 coupling is capable of triggering the production of cytokines including IL-6, and this autocrine loop may account for the subsequent STAT3 phosphorylation at Tyr705.