Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.     

Characterization of three pro-inflammatory cytokines,TNFα1, TNFα2 and IL-1β, in cage-reared Atlantic bluefin tuna Thunnus thynnus

Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1β, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses.     

Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-α converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 Å resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.     

Development of a prodrug of hydantoin based TACE inhibitor

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue. The pivalate prodrug 3 can be formulated as stable neutral form and demonstrated improved DMPK properties when compared with parent compound.     

TACE与手术治疗原发性大肝癌的临床疗效比较

目的:比较经导管肝动脉化疗栓塞(transcatheter arterial chemoembolization,TACE)和手术治疗原发性大肝癌(肿瘤最大直径≥5 cm)的临床疗效和安全性。方法:回顾性分析48例经TACE或手术(Operation)治疗的原发性大肝癌患者,其中TACE组25例,Operation组23例,治疗后随访24个月,评价和比较其治疗效果、生存曲线、镇痛剂的日平均用量及不良反应的发生情况。结果:治疗后3个月,TACE组患者的Karnofsky评分(P=0.033)显著高于Operation组,而血清甲胎蛋白(AFP)水平(P=0.022)显著低于Operation组。此外,TACE组的镇痛药杜冷丁的日均使用量显著低于Operation组(P=0.031),便秘的日均发生次数(P=0.045)显著少于Operation组。治疗后24个月,TACE组和Operation组的生存率分别为48.0%和17.4%,TACE组显著高于Operation组(P=0.0415,95%CI of ratio:1.006 to 1.994)。结论:TACE治疗原发性大肝癌的临床疗效可能优于手术治疗,且安全性高。     

The human cathelicidin LL-37 enhances airway mucus production in chronic obstructive pulmonary disease

Airway mucus overproduction is a distinguishing feature of chronic obstructive pulmonary disease (COPD). LL-37 is the only member of human cathelicidins family of antimicrobial peptides and plays a central role in many immune and inflammatory reactions. Increasing evidence suggests the involvement of LL-37 in the pathogenesis of COPD. Here, we investigated the effects of LL-37 on airway mucus overproduction in COPD. We observed overexpression of both LL-37 and MUC5AC mucin (a major mucin component of mucus) in airways of COPD patients and found a correlation between them. We showed in vitro that LL-37 induces MUC5AC mucin production by airway epithelial NCI-H292 cells in the absence and presence of cigarette smoke extract, with TNF-α converting enzyme (TACE)–EGFR–ERK1/2 pathway and IL-8 required for the induction. Therefore, we concluded that LL-37 enhances the mucus production in COPD airways, thus contributing to the progression of COPD.     

索拉非尼联合化疗栓塞治疗晚期原发性肝癌的疗效分析

目的:原发性肝癌(Primary hepatocellular carcinoma,PHC)是最常见的消化系统恶性肿瘤之一,严重威胁人类的健康。目前,治疗晚期肝癌的首选方法是肝动脉化疗栓塞(Transcatheter arterial chemoembolization,TACE),配合抗癌药物使用,治疗效果明显。索拉非尼因具有抑制肿瘤生长的作用而被越来越广泛的用于治疗肝癌。本研究针对索拉非尼的靶向性,探讨该药与化疗栓塞联合治疗晚期原发性肝癌的疗效,旨在为肝癌的临床治疗提供可参考的依据。方法:选取我院2008年10月-2012年6月收治的晚期原发性肝癌患者96例,随机分为对照组和观察组,每组各48例。对照组患者采用肝动脉化疗栓塞单独治疗,观察组患者采用索拉非尼联合化疗栓塞治疗。比较两组患者的客观有效率、临床获益率、治疗一年生存率、两年以上生存率及甲胎蛋白(AFP)水平的变化情况。结果:观察组患者的客观有效率为52.1%,临床获益率为89.6%,均高于对照组患者的对应值33.3%和47.9%,差异有统计学意义(P0.05);观察组患者一年生存率为89.6%,两年生存率为72.9%,均高于对照组患者的对应值58.3%和35.4%,差异显著(P0.05);两组患者治疗后的AFP水平均比治疗前降低,观察组患者AFP水平的改善情况显著优于对照组,差异具有统计学意义(P0.05)。结论:索拉非尼联合化疗栓塞治疗晚期原发性肝癌具有明显的效果,且安全性高,值得临床推广使用。     

The regulation of TACE catalytic function by its prodomain

Aim To study the function of the prodomain of ADAM17 (TACE) and to develop an approach for interfering with inflammation processes. Method The expression plasmids of the TACE ectodomain (T1300), prodomain (T591), signal peptide and prodomain (T648), full length (T2472), and the turncated TACE without prodomain (T57-T1824) were constructed and designated as pET-28a-T300, pET-28a-T591, pIRES2-EGFP-648, pEGFP-N1-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824, respectively. After Ni²⁺-NTA resin-affinity chromatography, the recombinant T591 and T1300 proteins were obtained and assayed by western blotting and circular dichroism. The experiment was carried out on THP1 cell lines stimulated by LPS in vitro. The inhibition of recombinant protein T591 to TACE activity was detected by ELISA and immunohistochemical detection. The expression plasmids (pIRES2-EGFP-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824) were used to transfect the U937 cells. HeLa cells were also transfected with pEGFP-N1-T648. The transfected U937 cells were then stimulated by LPS and the effect of expression plasmids on TNF-α secretion was detected by ELISA and flow cytometry (FCM). Results The recombinant prodomain protein inhibited 57% of the TNF-α secretion and mediated an accumulation of TNF-α on the surface of THP1 cells. An intense green fluorescence was seen in the membranes of HeLa cells transfected with pEGFP-N1-T648. The plasmid pIRES2-EGFP-T648 inhibited TNF-α secretion by 61.09% and mediated an accumulation of mTNF-α on the surface of the U937 cells. The secretion of sTNF-α and the level of the mTNF-α in the pIRES2-EGFP-T57-T1824 transfected cells gave no difference when compared with the pIRES2-EGFP transfected cells. Also the secretion of sTNF-α from the cells transfected by the plasmid pIRES2-EGFP-T2472 increased, while the level of mTNF-α decreased, compared with the pIRES2-EGFP-transfected cells. Conclusion The prodomain has dual effects and might be useful in the molecular design of an anti-inflammatory drug.     

Multiple shRNA-mediated knockdown of TACE reduces the malignancy of HeLa cells

Tumor necrosis factor-alpha (TNF-α) converting enzyme (TACE) is a key enzyme involved in the proteolytic shedding of the ectodomain of several membrane-bound growth factors, cytokines and receptors. Here, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against TACE. We found that in HeLa cells our multiple shRNAs vector produced a higher level of TACE knockdown than any single shRNA vector containing only one TACE shRNA. Silencing TACE expression in HeLa cells decreased their malignancy by decreasing the proliferation, adhesion and migration, as well as inducing apoptosis in these cells. Furthermore, our data suggest that the effects of TACE on the malignancy of HeLa cells may be mediated via activation of the EGFR (epidermal growth factor receptor) signaling pathway. Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy for tumors.