Division of Gastroenterology and Hepatology, Zurich Center for Integrative Human Physiology, University Hospital Zurich, Zurich, Switzerland.
Gut. 2011 Feb;60(2):189-97. doi: 10.1136/gut.2010.216606. Epub 2010 Nov 29.
The Crohn's disease (CD) susceptibility gene, protein tyrosine phosphatase N2 (PTPN2), regulates interferon γ (IFNγ)-induced signalling and epithelial barrier function in T₈₄ intestinal epithelial cells (IECs). The aim of this study was to investigate whether PTPN2 is also regulated by tumour necrosis factor α (TNFα) and if PTPN2 controls TNFα-induced signalling and effects in IECs.
T₈₄ 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.
TNFα 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-κB (NF-κB) by BMS-345541 prevented the TNFα-induced rise in PTPN2 protein, independent of apoptotic events. PTPN2 knock-down revealed that the phosphatase regulates TNFα-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation, without affecting c-Jun N-terminal kinase (JNK), inhibitor of κB (IκB) or NF-κB phosphorylation. Loss of PTPN2 potentiated TNFα-induced secretion of interleukin 6 (IL-6) and IL-8. In TNFα- and IFNγ-co-treated cells, loss of PTPN2 enhanced protein expression of inducible nitric oxide synthase (iNOS).
TNFα induces PTPN2 expression in IECs. Loss of PTPN2 promotes TNFα-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.
克罗恩病（CD）易感基因蛋白酪氨酸磷酸酶 N2（PTPN2）调节 T₈₄肠上皮细胞（IECs）中干扰素 γ（IFNγ）诱导的信号传导和上皮屏障功能。本研究旨在探讨 PTPN2 是否也受肿瘤坏死因子 α（TNFα）调节，以及 PTPN2 是否控制 IEC 中 TNFα 诱导的信号传导和作用。
使用 T₈₄ IEC 进行所有细胞研究。通过 Western blot 评估蛋白水平，通过逆转录-PCR（RT-PCR）评估 mRNA 水平，通过 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。
TNFα 处理可提高 PTPN2 mRNA 以及核和细胞质蛋白水平，并导致 PTPN2 在细胞质中积累。来自活动性 CD 患者的活检标本在肠上皮中显示出强烈的 PTPN2 免疫染色，而处于缓解期的 CD 患者样本的 PTPN2 水平与无炎症性肠病（IBD）的对照相似。尽管来自活动性溃疡性结肠炎（UC）患者的样本显示出比非 IBD 患者和缓解期 UC 患者更多的 PTPN2 蛋白，但它们的 PTPN2 表达低于活动性 CD。对 TNF 治疗有反应的缓解期 CD 患者的样本也显示出与对照患者相似的 PTPN2 水平。通过 BMS-345541 抑制核因子-κB（NF-κB）可防止 TNFα 诱导的 PTPN2 蛋白升高，而与凋亡事件无关。PTPN2 敲低表明该磷酸酶调节 TNFα 诱导的细胞外信号调节激酶 1/2（ERK1/2）和 p38 磷酸化，而不影响 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 TNFα 诱导的白细胞介素 6（IL-6）和白细胞介素 8（IL-8）的分泌。在 TNFα 和 IFNγ 共同处理的细胞中，PTPN2 的缺失增强了诱导型一氧化氮合酶（iNOS）的蛋白表达。
TNFα 在 IECs 中诱导 PTPN2 表达。PTPN2 的缺失促进了 TNFα 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 活性可能在肠道中慢性炎症状态的建立中发挥关键作用，例如 CD。
