microRNA-1271靶向调控白血病细胞CYLD蛋白的表达

目的探讨人白血病细胞中微小RNA(microRNA)-1271对CYLD蛋白表达的调控作用。方法 qRT-PCR检测microRNA-1271在不同的人白血病细胞系、临床初诊未治的几种类型原代白血病细胞、正常人外周血单个核细胞中的表达差异,利用 Targetscan 信息学预测软件预测 microRNA-1271靶向CYLD基因,构建携带靶基因野生型及突变型3’非翻译区(3’UTR)(缺失了整段预测的microRNA-1271结合序列)的双荧光素酶报告基因质粒,采用脂质体 Lipo-fectamine 3000包裹双荧光素酶重组质粒及 microRNA-1271模拟物(mimic)或阴性对照,共转染HEK293A细胞,应用双荧光素酶报告基因检测试剂盒测定荧光素酶活性。 FAM标记的microRNA-1271抑制物和阴性对照分别核转染至人白血病细胞系K562细胞,Western blot法检测CYLD蛋白水平的表达变化。结果 microRNA-1271在不同人白血病细胞系以及临床初诊未治的原代白血病细胞中的表达均明显高于正常人外周血单个核细胞,双荧光素酶报告基因实验验证CYLD是 microRNA-1271的潜在靶基因;K562细胞中转染microRNA-1271抑制物下调 microRNA-1271后, Western blot法检测结果显示CYLD蛋白水平明显上调。结论人白血病细胞中microRNA-1271的表达上调,下调microRNA-1271后可以促进靶基因CYLD蛋白的表达, microRNA-1271有可能成为白血病治疗的新靶点。     

CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes

AIM:To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models.METHODS:Hepatocyte cell death in CYLD knockout mice(CYLD-/-)was analyzed by application of liver injury models for CD95-(Jo2)and tumor necrosis factor(TNF)-α-[D-Gal N/lipopolysaccharide(LPS)]induced apoptosis.Liver injury was assessed by measurement of serum transaminases and histological analysis.Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases.Nuclear factor(NF)-κB,ERK,Akt and jun amino-terminal kinases signaling were assessed.Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-αand with the CD95-ligand Jo2.Cell viability was analyzed by MTT-assay.RESULTS:Livers of CYLD-/-mice showed increased anti-apoptotic NF-κB signaling.In both applied liver injury models CYLD-/-mice showed a significantly reduced apoptosis sensitivity.After D-Gal N/LPS treatment CYLD-/-mice exhibited significantly lower levels of alanine aminotransferase(ALT)(295 U/L vs 859 U/L,P<0.05)and aspartate aminotransferase(AST)(560 U/L vs 1025 U/L,P<0.01).After Jo injection CYLD-/-mice showed 2-fold lower ALT(50 U/L vs 110 U/L,P<0.01)and lower AST(250 U/L vs 435 U/L,P<0.01)serumlevels compared to WT mice.In addition,isolated CYLD-/-primary murine hepatocytes(PMH)were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2,XIAP,c IAP1/2,survivin and c-FLIP expression upon TNF-and CD95-receptor triggering,respectively.Inhibition of NF-κB activation by the inhibitor of NF-κB phosphorylation inhibitor BAY 11-7085 inhibited the expression of antiapoptotic proteins and re-sensitized CYLD-/-PMH towards TNF-and CD95-receptor mediated cell death.CONCLUSION:CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-κB dependent anti-apoptotic signaling.     

The GTPase-activating protein GIT2 protects against colitis by negatively regulating Toll-like receptor signaling

G protein-coupled receptor kinase-interactor 2 (GIT2) regulates thymocyte positive selection, neutrophil-direction sensing, and cell motility during immune responses by regulating the activity of the small GTPases ADP ribosylation factors (Arfs) and Ras-related C3 botulinum toxin substrate 1 (Rac1). Here, we show that Git2-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis, Escherichia coli, or endotoxin-shock challenge, and a dramatic increase in proinflammatory cytokines was observed in Git2 knockout mice and macrophages. GIT2 is a previously unidentified negative regulator of Toll-like receptor (TLR)-induced NF-κB signaling. The ubiquitination of TNF receptor associated factor 6 (TRAF6) is critical for the activation of NF-κB. GIT2 terminates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis to inhibit the ubiquitination of TRAF6. Finally, we show that the susceptibility of Git2-deficient mice to DSS-induced colitis depends on TLR signaling. Thus, we show that GIT2 is an essential terminator of TLR signaling and that loss of GIT2 leads to uncontrolled inflammation and severe organ damage.Inflammatory bowel disease (IBD), which includes the two main forms Crohn’s disease (CD) and ulcerative colitis (UC), is generally thought to develop from an abnormal immune response to the gut luminal microbiota in genetically predisposed individuals (1, 2). The innate immune system, which senses the commensal flora, is critical for intestinal homeostasis and tissue repair after injury (3). Toll-like receptor (TLR) signaling is responsible for the development of colitis (4).Tight control of TLR signaling is crucial for the clearance of pathogens and for avoiding nonresolving inflammation (5, 6). Uncontrolled activation of the TLR-triggered signaling pathway promotes chronic inflammation and autoimmune diseases, such as IBD and obesity (7, 8). TLRs mediate host responses to microorganisms and induce inflammatory responses that are linked to the expression of proinflammatory cytokines and activation of the NF-κB pathway (7). Negative regulation of TLR signaling is crucial to maintain immune homeostasis. The tumor suppressor CYLD is a negative regulator of TLR signaling by deubiquitinating TRAF6 and NEMO (9–11). A deficiency in the catalytic domain of CYLD in mice causes elevated NF-κB activity, and those mice die shortly after birth (12).GIT2 belongs to the group of ADP ribosylation factor (Arf)-directed GTPase activating proteins (GAPs). More than 24 proteins with Arf GAP domains have been identified in humans (13). To our knowledge, none of these proteins have been reported to directly regulate TLR signaling. GIT2 negatively regulates the activation of Arf GTPases through its N-terminal, Arf GTPase activating protein (GAP) domain (14) and of Rac GTPase through its interaction with PIX (p21-activated kinase-interacting exchange factor) (15). Git2 deficiency leads to spontaneous splenomegaly, hypersusceptibility to infection, increased oxygen anion production by neutrophils, and impaired positive selection of CD4 single-positive thymocytes in thymus (16, 17). GIT2 is highly expressed in monocytes and macrophages, especially after stimulation by LPS (18, 19). Combining its known relationships with novel transeQTLs extends the connections of GIT2 to a host of inflammatory mediators (19). We identified that GIT2 interacted with the components of the NF-κB signaling pathway by a large-scale yeast two-hybrid screening (20). Cumulatively, these results show the important roles of GIT2 in innate and adaptive immunity. However, whether GIT2 directly regulates the intestinal immune response remains unknown, and the genetic evidence is lacking to support its physiological roles during intestinal immune responses by directly regulating the TLR signaling pathway.Here, we show that knockout of Git2 induced severe acute and chronic colitis after dextran sulfate sodium (DSS) treatment. Git2-deficient mice were more susceptible to infection by Escherichia coli and endotoxin shock. Git2 deficiency resulted in a greater TLR-induced production of proinflammatory cytokines in vitro and in vivo. GIT2 inhibited the TLR-induced signaling pathway and was critical in inhibiting the activation of TRAF6. Moreover, GIT2 terminated the TLR signaling pathway by recruiting the deubiquitinating enzyme CYLD to the TRAF6 ubiquitin ligase. Thus, GIT2 is a critical terminator of TLR-induced inflammatory responses.     

Multiple trichoepitheliomas – a novel mutation in the CYLD gene

Background Trichoepitheliomas are benign neoplasms with follicular differentiation. They may present as a solitary lesion or as multiple lesions. Multiple trichoepitheliomas are inherited in an autosomal dominant pattern within families, with both variable penetrance and expressivity. Recent investigations support that mutations in CYLD, the gene affected in familial cylindromatosis as well as in Brooke–Spiegler syndrome, are also responsible for multiple trichoepitheliomas. Objective The authors report the case of a 9‐year‐old African girl with multiple facial trichoepitheliomas in whom a mutation in the CYLD gene was hypothesised. Materials and methods After genomic DNA extraction from the peripheral blood, a molecular analysis of the CYLD gene was performed by PCR, DHPLC and automated sequencing. Results A novel heterozygous mutation in exon 18 of the CYLD gene (c.2449delT) was identified, with a deletion of one nucleotide resulting in a premature translational termination codon at amino acid position 831 on the affected allele (p.Cys817Valfs X15). Conclusions The predominating tumours define the classification of these three entities. Nevertheless, studies suggest that they can simply represent phenotypic variations of the same disease spectrum, sharing common genetic mutations.     

Loss of CYLD promotes cell invasion via ALK5 stabilization in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) has a very poor prognosis because of its highly invasive nature, and the 5‐year survival rate has not changed appreciably for the past 30 years. Although cylindromatosis (CYLD), a deubiquitinating enzyme, is thought to be a potent tumour suppressor, its biological and clinical significance in OSCC is largely unknown. This study aimed to clarify the roles of CYLD in OSCC progression. Our immunohistochemical analyses revealed significantly reduced CYLD expression in invasive areas in OSCC tissues, whereas CYLD expression was conserved in normal epithelium and carcinoma in situ. Furthermore, downregulation of CYLD by siRNA led to the acquisition of mesenchymal features and increased migratory and invasive properties in OSCC cells and HaCaT keratinocytes. It is interesting that CYLD knockdown promoted transforming growth factor‐β (TGF‐β) signalling by inducing stabilization of TGF‐β receptor I (ALK5) in a cell autonomous fashion. In addition, the response to exogenous TGF‐β stimulation was enhanced by CYLD downregulation. The invasive phenotypes induced by CYLD knockdown were completely blocked by an ALK5 inhibitor. In addition, lower expression of CYLD was significantly associated with the clinical features of deep invasion and poor overall survival, and also with increased phosphorylation of Smad3, which is an indicator of activation of TGF‐β signalling in invasive OSCC. These findings suggest that downregulation of CYLD promotes invasion with mesenchymal transition via ALK5 stabilization in OSCC cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cloaked in ubiquitin,a killer hides in plain sight: the molecular regulation of RIPK1

In the past decade, studies have shown how instrumental programmed cell death (PCD) can be in innate and adaptive immune responses. PCD can be a means to maintain homeostasis, prevent or promote microbial pathogenesis, and drive autoimmune disease and inflammation. The molecular machinery regulating these cell death programs has been examined in detail, although there is still much to be explored. A master regulator of programmed cell death and innate immunity is receptor-interacting protein kinase 1 (RIPK1), which has been implicated in orchestrating various pathologies via the induction of apoptosis, necroptosis, and nuclear factor-κB-driven inflammation. These and other roles for RIPK1 have been reviewed elsewhere. In a reflection of the ability of tumor necrosis factor (TNF) to induce either survival or death response, this molecule in the TNF pathway can transduce either a survival or a death signal. The intrinsic killing capacity of RIPK1 is usually kept in check by the chains of ubiquitin, enabling it to serve in a prosurvival capacity. In this review, the intricate regulatory mechanisms responsible for restraining RIPK1 from killing are discussed primarily in the context of the TNF signaling pathway and how, when these mechanisms are disrupted, RIPK1 is free to unveil its program of cellular demise.     

氯化钆预处理对ANP肺损伤大鼠肺泡巨噬细胞中CYLD及NF-κB的影响

目的:观察氯化钆(GdCl3)预处理对性坏死性胰腺炎(ANP)大鼠肺泡巨噬细胞(AMs)肿瘤抑制因子CYLD及NF-κB的影响,探讨CYLD在ANP肺损伤中所起的作用。方法:36只SD大鼠随机分成正常对照组,ANP组,GdCl3预处理组。采用5%牛磺胆酸钠逆行性胰胆管注射制备ANP动物模型,GdCl3预处理组在造模前30 min经尾静脉注射GdCl3(10 mg/kg)。术后6 h处死各组动物,经支气管肺泡灌洗获取肺泡AMs,检测支气管肺泡灌洗液(BALF)中TNF-α和IL-1β含量,Western blot检测AMs中NF-κB及CYLD活性水平。检测肺组织髓过氧化物酶(MPO)的水平变化,并行肺组织病理学检查。结果:假手术组肺组织未见病理改变,ANP组和GdCl3预处理组肺组织均出现充血、水肿、炎性渗出等病理改变,但GdCl3预处理组病变程度轻与ANP组;ANP组肺组织的MPO活性,以及BALF的TNF-α,IL-1β含量较正常对照组明显升高(均P<0.05);正常对照组,ANP组,GdCl3预处理组AMs核蛋白NF-κB的表达量分别为0.08±0.03,0.18±0.06及0.11±0.04,3组AMs的CYLD的表达量分别为0.32±0.09,0.15±0.05和0.27±0.07。ANP组,GdCl3预处理组AMs中NF-κB与CYLD的表达活性均呈负相关(r=-0.708,r=-0.571,均P<0.05)。结论:ANP肺损伤中存在CYLD低表达和NF-κB活化,GdCl3预处理能通过增加CYLD表达,减少NF-κB活化,进而减轻肺损伤。