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T 细胞受体下游激酶的转录调控:糖皮质激素的另一种免疫调节机制。

Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids.

机构信息

Department of Medicine, University of Perugia, Perugia, Italy.

出版信息

BMC Pharmacol Toxicol. 2014 Jul 3;15:35. doi: 10.1186/2050-6511-15-35.

Abstract

BACKGROUND

Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR.

METHODS

Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student's t-test was employed for statistical evaluation.

RESULTS

We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk and Lck genes. To investigate the function associated with Itk up-regulation, dexamethasone-induced apoptosis of thymocytes from Itk-deficient mice was evaluated. Our results demonstrated that Itk deficiency causes increased sensitivity to dexamethasone but not to other pro-apoptotic stimuli.

CONCLUSIONS

Modulation of Itk, Txk, and Lck in thymocytes and mature lymphocytes is another mechanism by which glucocorticoids modulate T-cell activation and differentiation. Itk up-regulation plays a protective role in dexamethasone-treated thymocytes.

摘要

背景

糖皮质激素会影响外周免疫反应,包括 T 细胞的激活、分化和凋亡的调节。T 细胞受体 (TCR) 触发的细胞内信号的数量和质量调节 T 细胞功能。因此,糖皮质激素可能通过干扰 TCR 信号级联来影响 T 细胞。本研究的目的是寻找 TCR 下游受糖皮质激素调节的激酶。

方法

使用 RNA 保护测定法、实时 PCR 和 Western 印迹研究了用糖皮质激素处理或来自糖皮质激素处理的小鼠的淋巴样细胞中的基因调节。通过进行低渗碘化丙啶染色和流式细胞术研究了遗传修饰的胸腺细胞对糖皮质激素诱导的细胞凋亡的敏感性。采用学生 t 检验进行统计学评估。

结果

我们发现,在小鼠 T 细胞杂交瘤中,合成糖皮质激素地塞米松可上调非受体酪氨酸激酶 Tec 家族的 Itk 的转录。相比之下,地塞米松下调了 Tec 激酶 Txk 的表达,该激酶与 Itk 功能冗余,以及 TCR 下游的Src 激酶 Lck。我们研究了在不同时间点和剂量下体外和体内地塞米松处理后胸腺细胞和成熟淋巴细胞中 Itk、Txk 和 Lck 的表达。激酶表达被不同程度地调节并遵循不同的动力学。在所有测试的细胞类型和条件下均上调了 Itk。在所有测试的细胞类型和条件下均上调了 Itk。在体外或体内,Txk 在成熟淋巴细胞中被强烈上调,但在胸腺细胞中被弱上调或不被调节。相反,Lck 在胸腺细胞中被下调,但在不同的实验条件下在成熟淋巴细胞中没有被调节或上调。这种复杂的行为与 Itk、Txk 和 Lck 基因中存在的正糖皮质激素反应元件 (GRE) 和负糖皮质激素反应元件 (nGRE) 相关。为了研究与 Itk 上调相关的功能,评估了缺乏 Itk 的小鼠的胸腺细胞对地塞米松诱导的凋亡。我们的结果表明,Itk 缺乏导致对地塞米松但不是其他促凋亡刺激物的敏感性增加。

结论

在胸腺细胞和成熟淋巴细胞中调节 Itk、Txk 和 Lck 是糖皮质激素调节 T 细胞激活和分化的另一种机制。Itk 的上调在接受地塞米松治疗的胸腺细胞中发挥保护作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51fc/4105561/cb27aa96b803/2050-6511-15-35-1.jpg

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