Tumor Necrosis Factor-α Converting Enzyme Research Articles

Inhibitory effects of purpurogallin on the endothelial protein C receptor shedding in vitro and in vivo

Endothelial cell protein C receptor (EPCR) plays important roles in the regulation of blood coagulation and inflammation. Activity of EPCR is markedly changed by ectodomain cleavage and released as soluble protein (sEPCR). EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). Purpurogallin (PPG) plays an important role in inhibiting glutathione S-transferase and xanthine oxidase as well as effective in the cell protection of several cell types. Here, we investigated the effects of PPG on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Human umbilical vein endothelial cells pretreated with PPG (0, 5, 10, 20 or 50 μg/mL) for 6 h and exposed to PMA (1 μM) for 1 h, and CLP-operated mice were administrated with PPG. Data showed that treatment with PPG resulted in potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. In addition, PPG reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential for use of PPG as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

Enhancement of long-term depression by soluble amyloid β protein in rat hippocampus is mediated by metabotropic glutamate receptor and involves activation of p38MAPK, STEP and caspase-3

It is reported that the amyloid-β protein (Aβ)-induced impairments in synaptic plasticity coincide with memory decline and dementia. Although Aβ-induced inhibition of hippocampal long-term potentiation has been intensively investigated, the underlying mechanism of Aβ-enhanced long-term depression (LTD) is not clear. Here, we report that acute exposure of rat hippocampal slices to soluble Aβ-enhanced LTD induced by weak low-frequency stimulation (wLFS; 1Hz for 3min, 180 pulses) in granule cells of the dentate gyrus. Application of LY341495 (a non-selective Group I/II metrabotropic glumate receptor (mGluR) antagonist) completely blocked Aβ-enhanced LTD, whereas D-AP5 (a not selective N-methyl-d-aspartate receptor (NMDAR) antagonist) had no effect on Aβ-enhanced LTD compared with controls. In addition, Aβ-enhanced LTD was occluded by pre-application of 3,5-dihydroxyphenylglycine, a Group1 mGluR (mGluR1/5) agonist, suggesting Aβ-enhanced LTD depends on mGluR1/5 but not NMDAR. We also report here that p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580 and postsynaptic protein tyrosine phosphatase inhibitors phenylarsine oxide and sodium orthovanadate prevented the facilitatory effect of Aβ on LTD. Application of striatal-enriched protein tyrosine phosphatase (STEP) activator MG132 facilitated induction of LTD by wLFS, but did not block following Aβ-enhanced LTD induced by another wLFS. On the other hand, Aβ-enhanced LTD blocked following MG132-LTD by wLFS, suggesting Aβ-enhanced hippocampal LTD involves STEP activation. Application of either non-selective caspase inhibitor Z-VAD-FMK or caspase-3 selective inhibitor Z-DEVD-FMK prevented Aβ-enhanced LTD. However, neither the tumor necrosis factor-α converting enzyme inhibitor TAPI-2 nor the mammalian target of rapamycin inhibitor rapamycin prevented the enhancement of Aβ on LTD. Therefore, we conclude that soluble Aβ enhances LTD in the hippocampal dentate gyrus region, and the facilitatory effect of Aβ on LTD involves mGluR1/5, p38MAPK, STEP and caspase-3 activation.

PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases

α-secretase-mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-β (Aβ) peptides, and α-cleavage of cellular prion protein (PrP(C)) prevents its conversion into misfolded, pathogenic prions (PrP(Sc)). The mechanisms leading to decreased α-secretase activity in Alzheimer's and prion disease remain unclear. Here, we find that tumor necrosis factor-α-converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrP(Sc) or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide-dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aβ deposition and in the brains of individuals with Alzheimer's disease. PDK1 induces phosphorylation and caveolin-1-mediated internalization of TACE. This dysregulation of TACE increases PrP(Sc) and Aβ accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrP(C) and TNFR1, and attenuates PrP(Sc)- and Aβ-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrP(Sc) infection and in APP-transgenic mice reduces Alzheimer's disease-like pathology and memory impairment.

Dietary α-linolenic acid increases the platelet count in ApoE−/− mice by reducing clearance

AbstractPreviously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE−/−) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbβ3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α–converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.

Strain-induced Differentiation of Fetal Type II Epithelial Cells Is Mediated via the Integrin α6β1-ADAM17/Tumor Necrosis Factor-α-converting Enzyme (TACE) Signaling Pathway

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and β1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and β1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6β1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.

Generation of antitumor peptides by connection of matrix metalloproteinase-9 peptide inhibitor to an endostatin fragment

Previous applications of matrix metalloproteinase (MMP) inhibitors in cancer treatment have resulted in disappointing outcomes. Therefore, it is necessary to develop more active or better targeted MMP inhibitors. In this study, Inhibitor2, a heptapeptide MMP inhibitor, was connected to the N-terminus or C-terminus of ES-2, an 11-amino-acid antiangiogenic peptide, and two designed peptides, P2 and P4, were generated. P2 inhibited MMP-2, MMP-8, MMP-9, and tumor necrosis factor-α converting enzyme (TACE) activity with IC50 values of 1.40, 0.35, 1.36, and 1.95 μmol/l, whereas those for P4 were 19, 20, 18, and 18 μmol/l. P2 showed a higher affinity with integrin α5β1 in a human umbilical vein endothelial cell (HUVEC) adhesion assay than P4. In the HUVEC migration assay, P2 showed a better inhibitory effect on HUVEC migration than P4 and Inhibitor2 and the inhibitory ratio at 2 μg/ml was 77%. In a chorioallantoic membrane assay, at a concentration of 3.28 μg/ml, P2 and P4 showed 69.8 and 56.8% inhibition of formation of new blood vessels on the embryo membrane. Furthermore, P2 significantly inhibited B16F10 growth in a syngeneic mouse model with an inhibition ratio of 57.92% by tumor weight at a dose of 20 mg/kg/day, whereas P4 and Inhibitor2 exerted no such in-vivo effect. The antiangiogenic effect of P2 was confirmed by CD31 staining of the tumor tissue sections. The Inhibitor2 part of P2 may function both as an MMP inhibitor and as a targeting motif. These studies represented an example of how to better apply the potent and peptidomimetic MMP inhibitors in cancer treatment.

Inhibitory effects of epi-sesamin on endothelial protein C receptor shedding in vitro and in vivo

Endothelial protein C receptor (EPCR) plays a pivotal role in augmenting Protein C activation by the thrombin-thrombomodulin complex. The activity of EPCR is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). The EPCR shedding is mediated by the tumor necrosis factor-α converting enzyme (TACE). Epi-sesamin (ESM), from the roots of Asarum siebodlii, is known to exhibit anti-allergic and anti-fungal activities. However, little is known about the effects of ESM on EPCR shedding. We investigated this issue by monitoring the effects of ESM on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-mediated EPCR shedding. Data showed that ESM induced potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding, likely through suppression of TACE expression. In addition, treatment with ESM resulted in a reduction of PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, ESM should be viewed as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of EPCR shedding.

Selective inhibition of tumor necrosis factor-α converting enzyme attenuates liver toxicity in a murine model of concanavalin A induced auto-immune hepatitis

Emerging evidence suggest that tumor necrosis factor (TNF)-α plays a major role in pathogenesis of auto-immune hepatitis (AIH) induced liver injury. Blockade of TNF-α synthesis or bio-activity protects against experimental AIH. TNF-α converting enzyme (TACE) is a member of the ADAM (a disintegrin and metalloproteinase) family which processes precursor TNF-α to release soluble TNF-α. We hypothesized that selective inhibition of TACE might protect AIH. To investigate this, we studied the effects of a selective TACE inhibitor DPC-333 on murine model of liver injury and fibrosis induced with concanavalin A (Con A). Pre-treatment with DPC-333 significantly suppressed plasma alanine transaminase, aspartate transaminase and cytokines such as TNF-α, interferon (IFN)-γ, interleukin (IL)-2 and IL-6 levels due to acute Con A challenge. Interestingly; DPC-333 inhibited liver poly (ADP-ribose) polymerase (PARP)-1 activity which was associated with reduced number of necrotic hepatocytes in histological examination and mortality associated with Con A. In fibrosis study, repeated Con A administration significantly up-regulated liver collagen deposition as assessed by measurement of hydroxyproline content which was further confirmed in liver histology with Masson's trichrome staining. Treatment with 30mg/kg of DPC-333 was able to suppress liver hydroxyproline and fibrous tissue proliferation which corroborated well with inhibition in expression of pro-fibrotic genes such as tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-β1. These observations suggest that selective TACE inhibition is an effective approach for the treatment of both immune mediated hepatic inflammation and fibrosis.

Rosmarinic acid down-regulates endothelial protein C receptor shedding in vitro and in vivo

The endothelial protein C receptor (EPCR) plays pivotal roles in coagulation and inflammation, however, its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). According to previous studies, there are approximately 100ng/ml sEPCR in human plasma and the levels increase in inflammatory diseases. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). We recently reported on the anti-inflammatory and barrier protective activities of rosmarinic acid (RA), an important component of the leaves of Perilla frutescens. However, little is known about the effects of RA on EPCR shedding. Here, we investigated this issue by monitoring the effects of RA on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Data showed that treatment with RA resulted in potent inhibition of PMA, TNF-α, IL-induced EPCR shedding by suppression of TACE expression. In addition, RA reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results suggest the potential for use of RA as an anti-sEPCR shedding reagent against PMA, TNF-α, IL-1β and CLP-mediated EPCR shedding.

Down-regulation of endothelial protein C receptor shedding by persicarin and isorhamnetin-3-O-galactoside

Increasing evidence has shown that beyond its role in coagulation, endothelial protein C receptor (EPCR) plays an important role in the cytoprotective pathway. Previous reports have shown that EPCR can be shed from the cell surface, and that this is mediated by tumor necrosis factor-α converting enzyme (TACE) and that sEPCR levels are increased in patients with systemic inflammatory diseases. Persicarin and isorhamnetin-3-O-galactoside (I3G) are active compounds from Oenanthe javanica, which has been widely studied for its neuroprotective, antioxidant, and barrier protective activities. However, little is known of the effects of persicarin on EPCR shedding. Here, we investigated this issue by monitoring the effects of persicarin and I3G on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. According to the results, persicarin and I3G induced potent inhibition of PMA and CLP-induced EPCR shedding by suppressing expression of TACE. In addition, persicarin and I3G reduced PMA-stimulated phosphorylation of p38MAPK, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, persicarin and I3G could be used as a candidate therapeutic for treatment of severe vascular inflammatory diseases.

Emodin-6-O-β-d-glucoside down-regulates endothelial protein C receptor shedding

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway and in the cytoprotective pathway. Previously, EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). Soluble EPCR levels are increased in patients with systemic inflammatory diseases. Recently, we reported that a new active compound, emodin-6-O-β-D-glucoside (EG) from Reynoutria japonica, has anti-inflammatory activities. However, little is known of the effects of EG on EPCR shedding. Here, we investigated this issue by monitoring the effects of EG on the phorbol-12-myristate 13-acetate (PMA) or the cecal ligation and puncture (CLP)-mediated EPCR shedding and its underlying mechanisms. Data showed that EG potently inhibited the PMA and CLP-induced EPCR shedding by suppressing TACE expression. Given these results, EG could be used as a candidate therapeutic for the treatment of vascular inflammatory diseases.

MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands

The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and check-point kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR.

Human neutrophil elastase-mediated goblet cell metaplasia is attenuated in TACE-deficient mice

Neutrophilic inflammation is associated with chronic airway diseases. It has been observed that human neutrophil elastase (HNE), which is secreted by active neutrophils during inflammation, induces both mucin overproduction and goblet cell metaplasia. Several in vitro studies suggest that tumor necrosis factor-α converting enzyme (TACE) regulates the signaling axis that mediates HNE-induced mucin overproduction; however, it is unknown whether TACE performs a similar function in HNE-induced goblet cell metaplasia in vivo. We conducted this study to determine whether the inactivation of Tace gene expression attenuates HNE-induced goblet cell metaplasia in mice. Deletion of Tace is lethal shortly after birth in mice; therefore, we utilized Tace(flox/flox)R26CreER(+/-) mice and induced conditional deletion of Tace using a tamoxifen injection. Wild-type mice were given tamoxifen to control for its effect. Tace conditional deletion mice and wild-type mice were exposed to HNE via nasal instillation three times at 3-day intervals, and the lungs were harvested on day 11 after initial HNE exposure. Using periodic acid-Schiff staining and MUC5AC immunohistochemical staining to visualize goblet cells in the lungs, we found that HNE induced goblet cell metaplasia in the wild-type mice and that HNE-induced goblet cell metaplasia was significantly attenuated in the Tace conditional deletion mice. These findings suggest that TACE could be a potential target in the treatment of goblet cell metaplasia in patients with chronic airway diseases.

Differential chemokine induction by 1-nitropyrene and 1-aminopyrene in bronchial epithelial cells: Importance of the TACE/TGF-α/EGFR-pathway

1-nitropyrene (1-NP), a common PAH in diesel exhaust, and its amine metabolite 1-aminopyrene (1-AP) induce distinctly different chemokine-responses in bronchial epithelial cells (BEAS-2B) characterized by increases in CXCL8 and CCL5, respectively. Tumor necrosis factor-α converting enzyme (TACE), which cleaves membrane-bound transforming growth factor (TGF)-α, activating the epidermal growth factor receptor (EGFR), may regulate pro-inflammatory responses induced by a variety of endogenous and exogenous agents. The present results suggest that CXCL8, but not CCL5 responses in 1-NP- or 1-AP-exposed cells required TACE/TGF-α/EGFR-signaling. The findings strengthen the notion that TACE/TGF-α/EGFR-signaling is central in epithelial CXCL8-regulation upon exposure to multiple airborne pollutants.

Three-dimensional quantitative structure–activity relationship CoMFA/CoMSIA on pyrrolidine-based tartrate diamides as TACE inhibitors

A series of pyrrolidine-based tartrate diamides having selective tumor necrosis factor-α converting enzyme (TACE) inhibitory activity was selected for the three-dimensional quantitative structure–activity relationship (3D-QSAR) studies. Total 76 compounds were selected by considering a high deviation in the biological activity and structural variations. The quality and predictive power of 3D-QSAR, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models for the atom-based, centroid/atom-based, data-based alignments were performed. Various models were developed with the help of these alignments. The best model was developed with data-based alignment. The optimal predictive CoMFA model was obtained with cross-validated r2 = 0.53 with six component, non-cross-validated r2 = 0.94, standard error of estimates 0.23, F-value = 121.98 and optimal CoMSIA model was obtained with cross-validated r2 = 0.53 with five components, non-cross-validated r2 = 0.93, standard error of estimates = 0.24 and F-value = 138.83. These models also showed the best test set prediction with predictive r2 value of 0.65 and 0.73, respectively. Thus, on the basis of predictive power COMSIA model appeared to be the best one. The statistical parameters from these models indicate that the data are being well fitted and also have high predictive ability. Moreover, the resulting 3D-CoMFA/CoMSIA contour maps provide useful guidance for designing of highly active TACE inhibitors.

Chemical–physical analysis of a tartrate model compound for TACE inhibition

We have synthesized and done an extensive chemical-physical analysis of the behavior of a new compound, named MBET306, a synthetic precursor of the recently discovered tartrate-based inhibitors of the protein Tumor Necrosis factor-α Converting Enzyme (TACE). Experimental and theoretical data have shown that in water solution MBET306 is overwhelmingly found as a monoanion at physiological pH, in a conformation that differs substantially from that detected in the known co-crystal structures of MBET306 derivatives bound to TACE. The body of collected experimental and theoretical data indicates that the monoanionic species binds Zn(ii) inducing a strong stabilization of the crystal-like arrangement of the central tartrate zinc-binding group, lending support for a two step TACE docking mechanism via a zinc-bound intermediate. The thorough chemical-physical characterization of the conformational behavior of free and zinc-bound MBET306 in water bulk solution opens new avenues for the rational drug design of tartrate-based highly specific TACE inhibitors.

Molecular Descriptors in Modelling the Tumour Necrosis Factor-α Converting Enzyme Inhibition Activity of Novel Tartrate-Based Analogues

The tumour necrosis factor-α converting enzyme inhibition activity of a series comprising of novel tartrate-based analogues has been quantitatively analysed in terms of molecular descriptors. The statistically validated quantitative structure-activity relationship models provided rationales to explain the inhibition activity of these congeners. The descriptors identified through combinatorial protocol in multiple linear regression analysis have highlighted the role of Moran autocorrelation of lag 7, weighted by atomic van der Waals volume, presence of both prime and nonprime amide carbonyl oxygen in the tartrate moiety and occurrence of five membered ring bearing substituents at varying sites. A few potential novel tartrate-based analogues have been suggested for further investigation.

LPS Increases MUC5AC by TACE/TGF-α/EGFR Pathway in Human Intrahepatic Biliary Epithelial Cell

Background. Mucin 5AC (MUC5AC) overproduction plays important roles in stone formation and recurrence of hepatolithiasis. We aim to investigate the involved mechanism and the potential target to block this process. Methods. 42 bile duct samples from hepatolithiasis and 15 normal bile duct samples from hemangioma patients were collected for detecting MUC5AC expression by immunohistochemistry. MUC5AC and phosphoepidermal growth factor receptor (pEGFR) expressions in human intrahepatic biliary epithelial cells (HIBECs) cultured with or without lipopolysaccharide (LPS) were detected by real-time PCR and western blot analysis. Transforming growth factor-α (TGF-α) secretion in HIBECs was detected by ELISA. Results. MUC5AC was overexpressed in bile ducts of hepatolithiasis samples compared with bile ducts from hemangioma samples. LPS upregulated MUC5AC expression in HIBECs. LPS promoted EGFR activation, and inhibiting EGFR activation by AG1478 significantly decreased LPS-induced MUC5AC overexpression in HIBECs. Moreover, LPS increased TGF-α secretion, and inhibiting tumor necrosis factor-α converting enzyme (TACE), which has been implicated in ectodomain cleavage of TGF-α, significantly inhibited LPS-induced EGFR activation and subsequent MUC5AC overexpression in HIBECs. Conclusion. Our results suggested that LPS increases MUC5AC expression through the TACE/TGF-α/EGFR pathway in HIBECs. This new finding might give light to the prevention of stone formation and recurrence of hepatolithiasis.

Blockade of tumor necrosis factor-α converting enzyme (TACE) enhances IL-1β and IFN-γ via caspase-1 activation: A probable cause for loss of efficacy of TACE inhibitors in humans?

TNF-α converting enzyme (TACE) is a member of the ADAM (a disintegrin and metalloproteinase) family and is known as ADAM17, which processes precursor TNF-α in order to release soluble TNF-α (sTNF-α). Inhibition of TACE has been effective as a strategy to inhibit arthritis in animal models; however, it has not been translated in the clinic due to lack of efficacy or toxicity. We hypothesized that inhibition of TACE may activate a different pro-inflammatory pathway in human. To investigate this, we studied the effect of TACE inhibitor DPC-333 on cytokine levels in concanavalin A (Con A) activated human peripheral blood mononuclear cells (hPBMC). We have also studied the effects of DPC-333 on Con A induced cytokine levels in mice in vivo or in vitro in whole blood assay. DPC-333 treatment significantly up-regulated IL-1β and IFN-γ in Con A activated hPBMC. In contrast, pre-treatment with DPC-333 effectively suppressed IL-1β and IFN-γ in mice in vivo or in vitro. Interestingly, DPC-333 was found to up-regulate mRNA expression of caspase-1 in hPBMC in a dose dependent fashion and selective caspase-1 inhibitor completely restored DPC-333 induced IL-1β and IFN-γ. Furthermore, selective IL-1β receptor antagonist (anakinra) prevented DPC-333 induced IFN-γ. In conclusion, our data demonstrates that blockade of TACE enhances IL-1β in a caspase-1 dependent manner in vitro in hPBMC and the elevation of IFN-γ is secondarily mediated via IL-1β. This novel finding might explain the possible cause behind the loss of efficacy of TACE inhibitors in human.

Insights into Ectodomain Shedding and Processing of Protein-tyrosine Pseudokinase 7 (PTK7)

The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP(621)↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.