Protein tyrosine phosphatase N2 regulates TNFα- induced signalling and cytokine secretion in human intestinal epithelial cells
Protein tyrosine phosphatase N2 regulates TNFα- induced signalling and cytokine secretion in human intestinal epithelial cells
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DOI：
10.1136/gut.2010.216606
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发表时间：
2011-02-01
期刊：
影响因子：
24.5
通讯作者：
Rogler, Gerhard
中科院分区：
文献类型：
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作者：
Scharl, Michael;McCole, Declan F.;Rogler, Gerhard
目的 克罗恩病（CD）易感基因蛋白酪氨酸磷酸酶N2（PTPN2）调节T - 84肠上皮细胞（IECs）中干扰素γ（IFN - γ）诱导的信号传导和上皮屏障功能。本研究的目的是探讨PTPN2是否也受肿瘤坏死因子α（TNF - α）调节，以及PTPN2是否控制IECs中TNF - α诱导的信号传导和效应。
方法 所有细胞研究均使用T - 84肠上皮细胞。通过蛋白质印迹法评估蛋白质水平，通过逆转录 - 聚合酶链反应（RT - PCR）评估mRNA水平，通过酶联免疫吸附测定（ELISA）评估细胞因子水平。通过小干扰RNA（siRNA）诱导PTPN2敲低。通过免疫组织化学或免疫荧光进行成像。
结果 TNF - α处理提高了PTPN2的mRNA以及细胞核和细胞质中的蛋白质水平，并导致PTPN2在细胞质中积累。活动性CD患者的活检标本在上皮中显示出强烈的PTPN2免疫组织化学染色，而缓解期CD患者的样本中PTPN2水平与无炎症性肠病（IBD）的对照组相似。尽管活动性溃疡性结肠炎（UC）患者的样本比非IBD患者和缓解期UC患者显示出更多的PTPN2蛋白，但其PTPN2表达低于活动性CD。缓解期且对抗TNF治疗有反应的CD患者的样本也显示出与对照患者相似的PTPN2水平。BMS - 345541对核因子 - κB（NF - κB）的药理抑制阻止了TNF - α诱导的PTPN2蛋白升高，且与凋亡事件无关。PTPN2敲低显示该磷酸酶调节TNF - α诱导的细胞外信号调节激酶1/2（ERK1/2）和p38磷酸化，但不影响c - Jun N - 末端激酶（JNK）、κB抑制剂（IκB）或NF - κB磷酸化。PTPN2缺失增强了TNF - α诱导的白细胞介素6（IL - 6）和IL - 8的分泌。在TNF - α和IFN - γ共同处理的细胞中，PTPN2缺失增强了诱导型一氧化氮合酶（iNOS）的蛋白表达。
结论 TNF - α诱导IECs中PTPN2的表达。PTPN2缺失促进TNF - α诱导的丝裂原活化蛋白激酶信号传导和炎症介质的诱导。这些数据表明PTPN2的活性可能在肠道慢性炎症状态（如CD）的形成中起关键作用。
Objective The Crohn's disease (CD) susceptibility gene, protein tyrosine phosphatase N2 (PTPN2), regulates interferon gamma (IFN gamma)-induced signalling and epithelial barrier function in T-84 intestinal epithelial cells (IECs). The aim of this study was to investigate whether PTPN2 is also regulated by tumour necrosis factor a (TNF alpha) and if PTPN2 controls TNF alpha-induced signalling and effects in IECs.Methods T-84 IECs were used for all cell studies. Protein levels were assessed by western blotting, mRNA levels by reverse transcription-PCR (RT-PCR) and cytokine levels by ELISA. PTPN2 knock-down was induced by small interfering RNA (siRNA). Imaging was performed by immunohistochemistry or immunofluorescence.Results TNF alpha treatment elevated PTPN2 mRNA as well as nuclear and cytoplasmic protein levels and caused cytoplasmic accumulation of PTPN2. Biopsy specimens from patients with active CD showed strong immunohistochemical PTPN2 staining in the epithelium, whereas samples from patients with CD in remission featured PTPN2 levels similar to controls without inflammatory bowel disease (IBD). Though samples from patients with active ulcerative colitis (UC) revealed more PTPN2 protein than non-IBD patients and patients with UC in remission, their PTPN2 expression was lower than in active CD. Samples from patients with CD in remission and responding to anti-TNF treatment also showed PTPN2 levels that were similar to those in control patients. Pharmacological inhibition of nuclear factor-kappa B (NF-kappa B) by BMS-345541 prevented the TNF alpha-induced rise in PTPN2 protein, independent of apoptotic events. PTPN2 knock-down revealed that the phosphatase regulates TNF alpha-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation, without affecting c-Jun N-terminal kinase (JNK), inhibitor of kappa B (I kappa B) or NF-kappa B phosphorylation. Loss of PTPN2 potentiated TNF alpha-induced secretion of interleukin 6 (IL-6) and IL-8. In TNF alpha-and IFN gamma-co-treated cells, loss of PTPN2 enhanced protein expression of inducible nitric oxide synthase (iNOS).Conclusions TNF alpha induces PTPN2 expression in IECs. Loss of PTPN2 promotes TNF alpha-induced mitogen-activated protein kinase signalling and the induction of inflammatory mediators. These data indicate that PTPN2 activity could play a crucial role in the establishment of chronic inflammatory conditions in the intestine, such as CD.