Background&Aims

PAK1 belongs to a family of serine-threonine kinases and contributes to cellular pathways like NFκB, MAPK, PI3K/AKT and Wnt/ β-catenin all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to IBD as well as colitis-associated cancer (CAC) and similar was observed in IL-10 KO mice, a model of colitis and CAC. Here we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL-10 KO mice.

Methods

IL-10/PAK1 double knockout mice (DKO) were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and PAS. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480 and normal human colon epithelial cells (HCEC-1CT).

Results

When compared to IL-10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyper-proliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated upregulation of the Notch1 target HES1 and the stem cell receptor LGR5 in DKO mice. Interestingly, the stem cell marker OLFM4 was present in colonic tissue. Increased β-catenin mRNA and cytoplasmic accumulation indicated an aberrant Wnt-signaling. A co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation.

Conclusion

PAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of IBD and CAC.

中文翻译：

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一种新的 PAK1-Notch1 轴调节肠道炎症中的隐窝稳态

背景&目标

PAK1 属于丝氨酸-苏氨酸激酶家族，参与细胞通路，如 NFκB、MAPK、PI3K/AKT 和 Wnt/β-catenin，所有这些通路都参与肠道稳态。PAK1 的过度表达与 IBD 以及结肠炎相关癌症 (CAC) 相关，并且在 IL-10 KO 小鼠（一种结肠炎和 CAC 模型）中观察到类似的情况。在这里，我们测试了 PAK1 缺失对 IL-10 KO 小鼠肠道炎症和致癌作用的影响。

方法

产生 IL-10/PAK1 双基因敲除小鼠 (DKO) 并分析结肠炎和 CAC 的发展。测量大肠并准备用于组织学或 RNA 分离。瑞士卷被 H&E 和 PAS 染色。使用肠道类器官、SW480 和正常人结肠上皮细胞 (HCEC-1CT) 进行免疫共沉淀和免疫荧光。

结果

与 IL-10 KO 小鼠相比，DKO 显示出更长的结肠和更长的隐窝，尽管具有更高的炎症和发育不良的数量。隐窝过度增殖与 Notch1 激活和隐窝分化减少有关，这表明杯状细胞减少。基因表达分析表明，在 DKO 小鼠中，Notch1 靶标 HES1 和干细胞受体 LGR5 上调。有趣的是，干细胞标志物 OLFM4 存在于结肠组织中。β-连环蛋白 mRNA 和细胞质积累的增加表明 Wnt 信号传导异常。在结肠上皮细胞中发现了 Notch1 和 PAK1 的共定位和直接相互作用。Notch1 激活取消了这种效果，而 PAK1 的沉默导致 Notch1 激活。

结论

PAK1 通过控制 Notch1 有助于调节炎症条件下的隐窝稳态。这在 IBD 和 CAC 的肠道病理生理学中确定了一个新的 PAK1-Notch1 轴。