睡眠蛋白12与丝氨酸蛋白酶抑制剂B12和去泛素化酶的相互作用驱动人类肠上皮细胞分化。
Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation.
作者信息
Basson Marc D, Wang Qinggang, Chaturvedi Lakshmi S, More Shyam, Vomhof-DeKrey Emilie E, Al-Marsoummi Sarmad, Sun Kelian, Kuhn Leslie A, Kovalenko Pavlo, Kiupel Matti
机构信息
Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, Cambridge, Massachusetts, USA.
Currently at Departments of Pharmaceutical Sciences and Biomedical Sciences-College of Pharmacy, Departments of Basic Sciences and Surgery-College of Medicine, California Northstate University, Cambridge, Massachusetts, USA.
出版信息
Cell Physiol Biochem. 2018;48(3):1274-1290. doi: 10.1159/000492019. Epub 2018 Jul 25.
BACKGROUND/AIMS: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation.
METHODS
We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells.
RESULTS
Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes.
CONCLUSIONS
SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.
背景/目的：人类肠细胞分化在发育、禁食、适应和减肥手术过程中会发生改变，但其细胞内调控机制仍不清楚。我们推测 Schlafen 12（SLFN12）调节肠细胞分化。
方法
我们使用激光捕获显微切割上皮组织、定量逆转录聚合酶链反应（qRT-PCR）和免疫组织化学来评估 SLFN12 在对照和禁食人类十二指肠黏膜活检组织中的表达，并利用病毒过表达和小干扰 RNA（siRNA）来追踪 SLFN12 在人 Caco-2 和 HIEC6 肠上皮细胞中的信号通路。
结果
禁食的人类十二指肠黏膜中 SLFN12 的 mRNA 和蛋白质表达减少，同时伴随着蔗糖酶-异麦芽糖酶等肠细胞标志物的减少。SLFN12 的过表达独立于增殖作用增加了 Caco-2 细胞中蔗糖酶-异麦芽糖酶的启动子活性、mRNA 和蛋白质水平，并激活了 SLFN12 的假定启动子。SLFN12 与丝氨酸蛋白酶抑制剂 B12（SERPB12）共沉淀。一种失活的 SLFN12 点突变既阻止了 SERPB12 的结合，也阻止了蔗糖酶-异麦芽糖酶的诱导。SERPB12 的过表达也诱导了蔗糖酶-异麦芽糖酶，而降低 SERPB12 的表达则阻止了 SLFN12 对蔗糖酶-异麦芽糖酶的作用。通过降低泛素羧基末端水解酶 L5（UCHL5）或泛素特异性蛋白酶 14（USP14），SLFN12 和 SERPB12 对蔗糖酶-异麦芽糖酶的诱导作用均减弱，同时降低两者则阻断诱导作用。SERPB12 刺激 USP14 的活性，但不刺激 UCHL5 的活性。SERPB12 与 USP14 共沉淀，但不与 UCHL5 共沉淀。此外，SLFN12 增加了蔗糖酶-异麦芽糖酶启动子结合转录因子 Cdx2 的蛋白质水平，而不改变 Cdx2 的 mRNA 水平。降低 UCHL5 和 USP14 可阻止这种情况发生。我们在体外和体内进一步验证了这一信号通路。SLFN12 或 SERPB12 的过表达在人非恶性 HIEC-6 肠细胞中诱导了蔗糖酶-异麦芽糖酶。
结论
SLFN12 通过一条涉及 SERPB12、去泛素化酶和 Cdx2 的信号通路调节人类肠细胞分化。该信号通路可能成为在黏膜萎缩、短肠或肥胖情况下调控人类肠细胞分化的靶点。
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