Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1^−/−/TNFR2^−/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.     

2-(2-Aminothiazol-4-yl)pyrrolidine-based tartrate diamides as potent, selective and orally bioavailable TACE inhibitors

TNF-α converting enzyme (TACE) inhibitors are promising agents to treat inflammatory disorders and cancer. We have investigated novel tartrate diamide TACE inhibitors where the tartrate core binds to zinc in a unique tridentate fashion. Incorporating (R)-2-(2-N-alkylaminothiazol-4-yl)pyrrolidines into the left hand side amide of the tartrate scaffold led to the discovery of potent and selective TACE inhibitors, some of which exhibited good rat oral bioavailability.     

影响肝切除术后行TACE 患者预后因素的分析

目的:对原发性肝细胞肝癌(HCC)肝部分切除术后行经肝动脉化疗栓塞(TACE)的病人和未行TACE病人影响其预后的多种因素进行分析和评价,为肝切除术后是否行TACE治疗寻找筛选条件。方法:对我院2003～2008年期间在我院肝胆外科行原发性肝细胞肝癌手术治疗221例(术后介入治疗103例,术后非介入治疗118例)患者进行全面随访了解患者的预后情况,分别对术后接受介入治疗和非介入治疗两组通过Kaplan-Meier及COX回归分析影响预后的因素,包括:年龄、性别、血清HBsAg、肿瘤直径、肿瘤大体分型、有无癌栓形成,肿瘤分期(TNM)共7项指标。结果:在1年生存期内介入治疗组中的性别、年龄、血清HBsAg、肿瘤直径、肿瘤大体分型无统计学意义(p>0.05),有无癌栓形成及肿瘤分期有意义(p<0.05);非介入组内年龄、性别、血清HBsAg无统计学意义,肿瘤直径、肿瘤大体分型、有无癌栓形成,肿瘤分期有意义;在3年生存期内介入治疗组中的以上指标无统计学意义,而非介入组在肿瘤直径、有癌栓形成及肿瘤分期方面与统计学意义。结论:对于肿瘤直径>5cm及术后病理证实为低分化的患者给予积极TACE治疗可明显提高近期生存率。     

影响肝切除术后行TACE患者预后因素的分析

目的：对原发性肝细胞肝癌（HCC）肝部分切除术后行经肝动脉化疗栓塞（TACE）的病人和未行TACE病人影响其预后的多种因素进行分析和评价,为肝切除术后是否行TACE治疗寻找筛选条件。方法：对我院2003～2008年期间在我院肝胆外科行原发性肝细胞肝癌手术治疗221例（术后介入治疗103例,术后非介入治疗118例）患者进行全面随访了解患者的预后情况,分别对术后接受介入治疗和非介入治疗两组通过Kaplan-Meier及COX回归分析影响预后的因素,包括：年龄、性别、血清HBsAg、肿瘤直径、肿瘤大体分型、有无癌栓形成,肿瘤分期（TNM）共7项指标。结果：在1年生存期内介入治疗组中的性别、年龄、血清HBsAg、肿瘤直径、肿瘤大体分型无统计学意义（p〉0.05）,有无癌栓形成及肿瘤分期有意义（p〈0.05）;非介入组内年龄、性别、血清HBsAg无统计学意义,肿瘤直径、肿瘤大体分型、有无癌栓形成,肿瘤分期有意义;在3年生存期内介入治疗组中的以上指标无统计学意义,而非介入组在肿瘤直径、有癌栓形成及肿瘤分期方面与统计学意义。结论：对于肿瘤直径〉5cm及术后病理证实为低分化的患者给予积极TACE治疗可明显提高近期生存率。     

重组人血管内皮抑制素(恩度)联合肝动脉栓塞化疗术(TACE)治疗中晚期肝癌的临床疗效评价

目的:探讨重组人血管内皮抑制素(恩度)联合肝动脉栓塞化疗术(TACE)治疗中晚期肝癌的临床疗效,为中晚期肝癌的临床治疗提供参考依据。方法:选择我院2012年6月至2013年7月收治的中晚期肝癌患者82例,根据治疗方法随机分为观察组(43例)和对照组(39例),对照组给予TACE治疗;观察组在给予TACE治疗的基础上,联合恩度治疗。治疗结束后,观察和评价两组患者的临床疗效和不良反应的发生情况。结果:治疗后,观察组的有效率为46.51%(20/43),显著高于对照组30.77%(12/39)(P0.05);观察组的AFP水平为(412.58±10.66)μg/m L,明显低于对照组(445.27±11.39)μg/m L(P0.05);两组并发症的发生率比较,差异无统计学意义(P0.05)。结论:恩度联合TACE治疗可以有效提高中晚期肝癌的临床疗效,且不增加不良反应。     

Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2–protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2–protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.     

TACE术联合胸腺肽α1治疗原发性肝癌并发门静脉癌栓的疗效及对免疫功能的影响

目的:研究经肝动脉化疗栓塞术(transcatheter arterial chemoembolization,TACE)联合胸腺肽α1治疗原发性肝癌并发门静脉癌栓的疗效及对免疫功能的影响。方法:选择2018年9月~2020年2月我院的55例原发性肝癌并发门静脉癌栓患者,根据治疗方法分为两组。两组均开展TACE术,观察组联用胸腺肽α1。比较两组的白蛋白(Albumin,ALB)、谷氨酸转氨酶(glutamate aminotransferase,ALT)、总胆红素(total bilirubin,TBIL)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、血清白介素(interleukin,IL)-4、IL-2和IL-6水平。结果:观察组的有效率明显高于对照组(35.71%vs 18.52%,P<0.05);治疗后,两组的ALB、CD4⁺/CD8⁺、CD3⁺、CD4⁺水平明显升高(P<0.05),ALT、TBIL、AST、血清IL-4、IL-2、IL-6水平、CD8⁺明显降低(P<0.05),且观察组上述指标明显优于对照组(P<0.05)。结论:TACE术联合胸腺肽α1对于原发性肝癌并发门静脉癌栓有显著的疗效,能改善其肝功能和免疫功能,降低血清IL-4、IL-2和IL-6水平。     

Structural determinants involved in the regulation of CXCL14/BRAK expression by the 26 S proteasome

The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells; however, in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation.     

Effect of tumor necrosis factor-alpha converting enzyme (TACE) and metalloprotease inhibitor on amyloid precursor protein metabolism in human neurons

Tumor necrosis factor-alpha (TNF-alpha) is implicated in inflammatory processes and much effort is being directed at inhibiting the release of TNF-alpha for treatment of inflammatory conditions. In this context, the drug CP-661,631 has been developed to inhibit the TNF-alpha converting enzyme (TACE). However, TACE is also implicated in amyloid precursor protein secretion. Amyloid precursor protein (APP) undergoes constitutive and regulated secretion by alpha-secretase endoproteolytic cleavage within the amyloid beta peptide (Abeta) domain. Alternative cleavage at the N- and C-terminus of the Abeta domain by beta- and gamma-secretases results in the production of Abeta. In many cellular and in vivo animal models, increased secretion of APP results in a concomitant decrease in the production of Abeta suggesting that the two pathways are intricately linked. However, in human primary neuron cultures, increased APP secretion is not associated with a decrease in total Abeta production. To determine if the use of CP-661,631 may enhance amyloidogenic processing in human brain, we have assessed the effect of CP-661,631 on APP metabolism in primary cultures of human neurons. Our results show that CP-661,631 effectively prevents regulated APP secretion but does not increase total Abeta levels in human primary neuron cultures.     

Identifying lysophosphatidic acid receptor subtype 1 (LPA1) as a novel factor to modulate microglial activation and their TNF-α production by activating ERK1/2

Microglia regulate immune responses in the brain, and their activation is key to the pathogenesis of diverse neurological diseases. Receptor-mediated lysophosphatidic acid (LPA) signaling has been known to regulate microglial biology, but it is still unclear which receptor subtypes guide the biology, particularly, microglial activation. Here, we investigated the pathogenic aspects of LPA receptor subtype 1 (LPA₁) in microglial activation using a systemic lipopolysaccharide (LPS) administration-induced septic mouse model in vivo and LPS-stimulated rat primary microglia in vitro. LPA₁ knockdown in the brain with its specific shRNA lentivirus attenuated the sepsis-induced microglia activation, morphological transformation, and proliferation. LPA₁ knockdown also resulted in the downregulation of TNF-α, at both mRNA and protein levels in septic brains, but not IL-1β or IL-6. In rat primary microglia, genetic or pharmacological blockade of LPA₁ attenuated gene upregulation and secretion of TNF-α in LPS-stimulated cells. In particular, the latter was associated with the suppressed TNF-α converting enzyme (TACE) activity. We reaffirmed these biological aspects using a BV2 microglial cell line in which LPA₁ expression was negligible. LPA₁ overexpression in BV2 cells led to significant increments in TNF-α production upon stimulation with LPS, whereas inhibiting LPA₁ reversed the production. We further identified ERK1/2, but not p38 MAPK or Akt, as the underlying effector pathway after LPA₁ activation in both septic brains and stimulated microglia. The current findings of the novel role of LPA₁ in microglial activation along with its mechanistic aspects could be applied to understanding the pathogenesis of diverse neurological diseases that involve microglial activation.