A Lymphocyte Serine Protease Granzyme A Causes Detachment of a Small-Intestinal Epithelial Cell Line (IEC-6)

Granzyme A (GzmA) is a serine protease (trypsin-like specificity) produced in cytotoxic lymphocytes. This enzyme is believed to enter virus-infected cells and growing tumors and induce apoptosis, but the roles of GzmA expressed in lymphocytes scattered through the epithelial layer of the normal small intestine are unknown. In the present study, recombinant rat GzmA (rGzmA) was found to cause morphological changes and detachment of a non-transformed rat small-intestinal epithelial cell line IEC-6, although the rGzmA-treated cells detached as aggregates with no changes characteristic of apoptosis. rGzmA-induced deformation and detachment occurred in IEC-6 cells plated with collagen type IV and fibronectin, but not in those plated with laminin. These findings suggest that GzmA in the normal small intestine participates in the reduction of adhesion between epithelial cells and basement membranes, through its ability to cleave extracellular matrix components.     

A lymphocyte serine protease granzyme A causes detachment of a small-intestinal epithelial cell line (IEC-6)

Granzyme A (GzmA) is a serine protease (trypsin-like specificity) produced in cytotoxic lymphocytes. This enzyme is believed to enter virus-infected cells and growing tumors and induce apoptosis, but the roles of GzmA expressed in lymphocytes scattered through the epithelial layer of the normal small intestine are unknown. In the present study, recombinant rat GzmA (rGzmA) was found to cause morphological changes and detachment of a non-transformed rat small-intestinal epithelial cell line IEC-6, although the rGzmA-treated cells detached as aggregates with no changes characteristic of apoptosis. rGzmA-induced deformation and detachment occurred in IEC-6 cells plated with collagen type IV and fibronectin, but not in those plated with laminin. These findings suggest that GzmA in the normal small intestine participates in the reduction of adhesion between epithelial cells and basement membranes, through its ability to cleave extracellular matrix components.     

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-alpha in IEC-6 cells

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-alpha, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with alpha-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-alpha/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-alpha-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.     

Giardipain-1, a protease secreted by Giardia duodenalis trophozoites,causes junctional,barrier and apoptotic damage in epithelial cell monolayers

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25?kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell–cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell–cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell–cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.     

IL-6 protects enterocytes from hypoxia-induced apoptosis by induction of bcl-2 mRNA and reduction of fas mRNA

Interleukin-6 (IL-6) has been shown to rescue enterocytes from hypoxia-induced apoptosis when given orally following hemorrhagic shock. In vitro models using an intestinal epithelial cell line (IEC-6) cultured with lipopolysaccharide (LPS) under low O2 conditions, to mimic intestinal conditions, show that these cells also undergo apoptosis, which can be reduced by subsequent culture with IL-6. To examine further the mechanisms of rescue, we cultured normal rat intestinal epithelial cells (IEC-6) under both normoxic and hypoxic conditions and analyzed their responses to LPS and IL-6. We showed that IEC-6 expressed IL-6 receptor on its surface. Further, IEC-6 cells could be rescued from hypoxia-induced apoptosis by co-culture with IL-6. RNase protection assay (RPA) examination revealed that under hypoxic conditions, IEC-6 cells that were resistant to apoptosis showed reduced fas expression and increased bcl-2 expression after co-culture with LPS+IL-6.     

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.     

Interaction exists between matriptase inhibitors and intestinal epithelial cells

The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.     

Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.     

Differential glucocorticoid effects on repair mechanisms and NF-kappaB activity in the intestinal epithelium

Glucocorticoids are effective agents in the management of inflammatory bowel diseases. However, information about their effects on repair mechanisms of the intestinal epithelium is incomplete.Therefore, the aim was to analyse in vitro effects of glucocorticoids on proliferation, restitution, and apoptosis as well as their effects on activity and expression of nuclear factor (NF)-kappaB, a known regulator of apoptosis and inflammation, in intestinal epithelial cells.Non-transformed rat jejunum epithelial cells (IEC-6) were cultured in the presence and absence of various concentrations of prednisolone and budesonide. IEC-6 cell proliferation was assessed by [3H]-thymidine incorporation. Restitution was analysed by an IEC-6 in vitro assay. Apoptosis was evaluated by ELISA and fluorescence microscopy. DNA binding activity and nuclear expression of NF-kappaB was determined by electrophoretic mobility shift assays and Western blotting, respectively.Prednisolone and budesonide stimulated IEC-6 cell proliferation at low to medium pharmacologic concentrations (prednisolone: 10(-9) to 10(-6) M; budesonide: 10(-11) to 10(-8) M). In contrast, high concentrations (>5 x 10(-5) M) had inhibitory effects on proliferation. 10(-7) M prednisolone and 10(-8) M budesonide increased restitution of IEC-6 cells, whereas high concentrations (10(-4) M) of prednisolone and budesonide decreased restitution. Apoptosis of IEC-6 cells was substantially enhanced by 10(-4) M budesonide; apoptosis was slightly increased by the highest prednisolone concentration used (5 x 10(-4) M). Furthermore, both glucocorticoids inhibited DNA binding activity and nuclear NF-kappaB expression in IEC-6 cells in a dose- and time-dependent fashion.In conclusion, prednisolone and budesonide modulate repair mechanisms of intestinal epithelial cells in vitro in a dose-dependent manner and profoundly modulate the inflammatory regulator NF-kappaB.     

NF-kappaB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion

Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-kappaB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-kappaB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-kappaB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine not only activated NF-kappaB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing IkappaBalpha superrepressor (AdIkappaBSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-kappaB through AdIkappaBSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-alpha and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-kappaB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.     

Involvement of the cytoplasmic juxtamembrane region of matriptase in its exclusive localization to the basolateral membrane domain of Madin–Darby canine kidney epithelial cells

Matriptase is a type II transmembrane serine protease. This protease is strongly expressed in simple epithelial cells such as enterocytes and kidney tubular cells in which the plasma membranes are separated into apical and basolateral domains. Although matriptase was found previously to occur exclusively on the basolateral membrane of enterocytes, the underlying mechanism of localization is unclear. In the present study, a full-length rat matriptase and a chimera consisting of the cytoplasmic and transmembrane regions of the protease and green fluorescent protein (designated as 1–86GFP) were found to localize exclusively to the basolateral membrane domain when expressed in Madin–Darby canine kidney epithelial cells. Mutagenesis analysis of 1–86GFP revealed that the matriptase cytoplasmic juxtamembrane amino acid residues (Lys45, Val47, and Arg50) play a role in mediating the localization in the cells. This study provides the first evidence that matriptase carries information for its localization in simple epithelia.     

Carrageenan inhibits granzyme A-induced detachment of and interleukin-8 release from alveolar epithelial A549 cells

Granzyme A (GrA) is a lymphocyte serine protease that is believed to enter virus-infected cells and growing tumors and induce apoptosis. We found recently that recombinant rat GrA (rGrA) promotes detachment of and interleukin (IL)-8 release from alveolar epithelial A549 cells and suggested that this protease is involved in the pathogenesis of certain inflammatory lung diseases. In the present study, we found that λ-carrageenan (a sulfated oligosaccharide constituting the cell walls of seaweeds) potently inhibits rGrA-induced detachment and IL-8 release of A549 cells. This sulfated oligosaccharide might be useful for suppressing the development of inflammatory lung diseases in which GrA is thought to be involved.     

Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.     

Polyamine depletion inhibits irradiation-induced apoptosis in intestinal epithelia

Our group has previously shown that polyamine depletion delays apoptosis in rat intestinal epithelial (IEC-6) cells (Ray RM, Viar MJ, Yuan Q, and Johnson LR, Am J Physiol Cell Physiol 278: C480-C489, 2000). Here, we demonstrate that polyamine depletion inhibits gamma-irradiation-induced apoptosis in vitro and in vivo. Pretreatment of IEC-6 cells with 5 mM alpha-difluoromethylornithine (DFMO) for 4 days significantly reduced radiation-induced caspase-3 activity and DNA fragmentation. This protective effect was prevented by the addition of 10 muM exogenous putrescine. Radiation exposure to mice resulted in a high frequency of apoptosis over cells positioned fourth to seventh in crypt-villus units. Pretreatment of mice with 2% DFMO in drinking water significantly reduced apoptotic cells from approximately 2.75 to 1.61 per crypt-villus unit, accompanied by significant decreases in caspase-3 levels. Further examination showed that DFMO pretreatment inhibited the radiation-induced increase in the proapoptotic protein Bax. Moreover, DFMO pretreatment significantly enhanced the intestinal crypt survival rate by 2.1-fold subsequent to radiation and ameliorated mucosal structural damage. We conclude that polyamine depletion by DFMO inhibits gamma-irradiation-induced apoptosis of intestinal epithelial cells both in vitro and in vivo through inhibition of Bax and caspase-3 activity, which leads to attenuation of radiation-inflicted intestinal injury. These data indicate that DFMO may be therapeutically useful to counteract the gastrointestinal toxicity caused by chemoradiotherapy. This is the first demonstration that polyamines are required for apoptosis in vivo.     

PI3K激活NF-κB信号通路保护中子辐射致IEC-6细胞损伤

中子属于高传能线密度电离辐射,能产生比κ射线更为严重的放射损伤,肠上皮对中子辐射高度敏感,迄今未见有关中子辐射致肠上皮细胞损伤中PI3K对NF-κB信号通路调控的研究报道.本研究旨在探讨中子照射后肠上皮细胞中PI3K对NF-κB信号通路的调控及其在中子辐射致肠上皮细胞损伤中的作用.选取肠上皮细胞系-6（intestinal epithelial cell No.6,IEC-6）进行传代培养,随机分为对照组、4Gy中子照射组和4Gy中子照射＋LY294002处理组,照射组和LY294002处理组细胞采用4Gy中子均匀照射,LY294002处理组细胞在照前24h给予终浓度为10κmol/L的LY294002,各组于照射后6和24h采用MTT比色法、流式细胞术和免疫印迹（Western blot）方法检测IEC-6细胞增殖活力、凋亡与坏死率以及NF-κB信号通路相关分子NF-κB（p65）,IKKκ和IκBκ的表达变化.研究发现,4Gy中子照射后6和24h,IEC-6细胞增殖活力下降,凋亡和坏死率增加;应用LY294002后IEC-6细胞增殖活力较照射组明显下降,IEC-6细胞凋亡和坏死率较照射组增加.4Gy中子照射后6和24h,IEC-6细胞NF-κB（p65）和IKKκ表达升高,IκBκ表达降低;应用LY294002后NF-κB（p65）和IKKκ表达降低,IκBκ表达升高,表明4Gy中子照射可引起IEC-6细胞增殖活力下降,凋亡和坏死率增加;PI3K可激活NF-κB信号通路,对中子辐射IEC-6细胞损伤发挥保护作用.     

Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro

Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.     

The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation (N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin–Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD-Myc(His)₆] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD-Myc(His)₆ exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.     

Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor.

Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acid-sensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the two-chain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.