Altered Mucin Gene Expression in Stone-Containing Intrahepatic Bile Ducts and Cholangiocarcinomas

Neoplastic transformation of epithelial cells is commonly associated with alterations in the synthesis and structures of mucin. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aims of this study were to determine the possible alteration of mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas and to try to predict whether or not hepatolithiasis has a predisposition to development of cholangiocarcinoma. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of stone-containing intrahepatic bile ducts, cholangiocarcinomas, and normal controls to identify the expression of MUC2, MUC3, MUC4, MUC5B, and MUC5AC in nonneoplastic and neoplastic biliary epithelium. The findings showed that (1) while multiple diverse mucin genes were expressed in the biliary epithelium, MUC3 and MUC5B mRNA were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls; (2) absent or decreased expression of MUC2, MUC3, and MUC5B of mRNA was found in cholangiocarcinomas in contrast to nonneoplastic biliary epithelium; and (3) increased expression of MUC4 and MU5AC of mRNA was found in cholangiocarcinomas and the biliary epithelium, especially for dysplastic cells of stone-containing intrahepatic bile ducts compared with normal controls. In this study, using in situ hybridization we demonstrated that neoplastic transformation of the biliary epithelium is accompained by alterations in mucin gene expression, the altered mucin gene expression in dysplastic cells of stone-containing intrahepatic bile ducts may reflect a higher potential for malignant transformation in these cells, and it could be a precursor of cholangiocarcinoma in the presence of hepatolithiasis.     

Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor.

Hypersecretory disease associated with Pseudomonas aeruginosa (PA) infections is characterised by increased goblet cells and increased mucin production. Recently, an epidermal growth factor receptor (EGFR) signalling cascade was shown to be a common pathway through which many stimuli induce mucin MUC5AC expression in airways by differentiation to a goblet cell phenotype. This study looked at whether PA products induce EGFR expression and activation and thus result in mucin MUC5AC production. Human airway epithelial (NCI-H292) cells were stimulated with PA culture supernatant (Sup). MUC5AC protein production, MUC5AC and EGFR messenger ribonucleic acid (mRNA) expression, and phosphorylated EGFR and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) were all examined using enzyme-linked immunosorbent assay, by in situ hybridisation and by immunoblotting. PA Sup induced MUC5AC mRNA and subsequent protein expression, EGFR and p44/42 MAPK phosphorylation and EGFR mRNA expression. Induction of MUC5AC mRNA and protein expression and EGFR and p44/42 MAPK phosphorylation were inhibited completely by pretreatment with a selective EGFR tyrosine kinase inhibitor. Pretreatment with a selective inhibitor of MAPK kinase prevented MUC5AC production and p44/42 MAPK phosphorylation but not EGFR phosphorylation. The authors conclude that PA products induce mucin MUC5AC production in human airway epithelial cells via the expression and activation of epidermal growth factor receptor.     

不分型流感嗜血杆菌上调人类MUC5AC黏液素的基因表达

目的 探索不分型流感嗜血杆菌(NTHi)上调人类MUC5AC黏液素基因表达的细胞分子机制.方法 通过反转录套式-PCR及实时荧光定量PCR分析NTHi所诱导的MUC5AC mRNA的表达,免疫沉淀及Western印迹法检测NTHi对表皮生长因子受体(EGFR)及p38丝裂原活化蛋白激酶(p38MAPK)的激活,采用p38 MAPK或EGFR特异性抑制剂,共转染EGFR显性失活质粒,判断在转录水平对NTHi所诱导的MUC5AC表达的影响,并研究EGFR特异性抑制剂对NTHi所诱导p38MAPK激活的影响.结果 在人结肠上皮细胞株(HM3)细胞中,NTHi在mRNA及转录水平上调人类MUC5AC黏液素基因的表达,NTHi能使EGFR或p38MAPK磷酸化.p38MAPK、EGFR特异性抑制剂或共转染EGFR显性失活质粒,可显著抑制NTHi所诱导的MUC5AC的表达,EGFR特异性抑制剂对NTHi所诱导p38MAPK磷酸化有抑制作用.结论 不分型流感嗜血杆菌可通过EGFR-p38MAPK信号通路,上调人类MUC5AC黏液素基因表达.     

表皮生长因子协同上调不分型流感嗜血杆菌诱导的MUC5AC表达及其信号通路

目的 探索表皮生长因子(EGF)协同上调不分型流感嗜血杆菌(NTHi)诱导MUC5AC黏液素基因表达的细胞分子机制.方法 荧光定量PCR及Luciferase分析EGF协同上调NTHi诱导的MUCSAC表达.Western印迹法检测EGF及NTHi对P38有丝分裂原活化蛋白激酶(P38MAPK)、细胞外信号调节激酶(ERK)、P21激活激酶(PAK)4磷酸化的协同作用.采用P38MAPK或EGF特异性抑制剂,共转染P38MAPK、ERK显性失活质粒及PAK4 siRNA,判断对EGF协同上调NTHi诱导MUC5AC表达的影响,并研究PAK4显性失活质粒对EGF及NTHi所致的P38MAPK及ERK协同激活的影响.结果 在HM3、HeLa和HMEEC-1细胞mRNA及转录水平上,EGF协同上调NTHi诱导的MUC5AC基因表达.EGF及NTHi对P38MAPK、ERK、PAK4磷酸化有协同作用;P38MAPK、ERK特异性抑制剂或共转染P38MAPK、ERK显性失活质粒、PAK4siRNA,可显著抑制EGF对NTHi诱导的MUC5AC表达的协同作用,PAK4显性失活质粒抑制EGF和NTHi诱导的P38MAPK和ERK磷酸化的协同作用.结论 EGF通过PAK4依赖的P38MAPK及ERK细胞信号通路协同上调NTHi诱导的MUCSAC黏液素基因表达.     

Melatonin inhibits MUC5AC production via suppression of MAPK signaling in human airway epithelial cells

Mucus acts as a primary defense system in the airway against various stimuli. However, excess mucus production causes a reduction in lung function via limitation of the airflow in the airway of patients suffering from asthma or chronic obstructive pulmonary disease (COPD). In this study, we evaluated the effects of melatonin on the production of MUC5AC, a major constituent of the mucin that is secreted from the airway, using epidermal growth factor (EGF)‐stimulated NCI‐H292 cells, a human mucoepidermoid carcinoma cell line, and an ovalbumin (OVA)‐induced asthma murine model. Melatonin treatment significantly reduced the mRNA and protein levels of MUC5AC and reduced interleukin (IL)‐6 production in EGF‐stimulated H292 cells. Melatonin markedly decreased the phosphorylation of MAPKs, including ERK1/2, JNK, and p‐38, induced by EGF stimulation. These findings were consistent with the results using MAPK inhibitors. Particularly, co‐treatment with melatonin and a MAPK inhibitor more effectively suppressed MAPK phosphorylation than treatment with a MAPK inhibitor alone, which resulted in a reduction in MUC5AC expression. In the asthma murine model, melatonin‐treated mice exhibited a marked reduction in MUC5AC expression in the airway compared with the OVA‐induced mice. These reductions were accompanied by reductions in proinflammatory cytokine production and inflammatory cell infiltration. Collectively, these findings indicate that melatonin effectively inhibits MUC5AC expression. These effects may be closely associated with the inhibition of MAPK phosphorylation. Furthermore, our study suggests that melatonin could represent a potential therapeutic for chronic airway diseases, such as asthma and COPD.     

Prostaglandin E receptors in bile ducts of hepatolithiasis patients and the pathobiological significance for cholangitis.

BACKGROUND & AIMS: In hepatolithiasis, chronic proliferative cholangitis may influence the progression of the disease. Prostaglandin (PG) E(2) experimentally causes morphologic changes to intrahepatic bile ducts, analogous to the changes found in cholangitis. This study was designed to gain an understanding of the involvement of PGE(2) and PGE receptor (EP) subtypes in the development of cholangitis. METHODS: The expression levels of secretory-type group IIA phospholipase A(2) (sPLA(2)-IIA) and cyclooxygenase (COX)-2 as well as EP subtypes were determined in the bile ducts with change of cholangitis. In in vitro experiments, growth promotion and mucin secretagogue properties of biliary epithelial cells in response to EP-selective agonists or antagonists were studied. RESULTS: The messenger RNA (mRNA) level of sPLA(2)-IIA and the protein and mRNA levels of COX-2 were significantly increased in the bile ducts of patients with hepatolithiasis compared with the levels of the bile ducts of control subjects. These changes were associated with a concomitant increase in PGE(2) and total mucin concentrations in the bile. The mRNAs of EP subtypes EP(2), EP(3), and EP(4) but not EP(1) were amplified in the bile ducts. Treatment with an EP(4)-selective agonist (ONO-AE1-329) caused a dose-dependent increase in DNA synthesis, colony number, and mucin secretion in the cells. Conversely, treatment with an EP(4)-selective antagonist (ONO-AE3-208) abolished the biological effects of PGE(2) on the cells. CONCLUSIONS: In hepatolithiasis, an enhanced synthesis of sPLA(2)-/COX-2-derived PGE(2) and its actions mediated via the EP(4) receptor in the bile ducts may be of pathobiological significance for chronic proliferative cholangitis.     

Characterization of intrahepatic cholangiocarcinoma of the intraductal growth-type and its precursor lesions

A cohort of patients with intraductal growth-type intrahepatic cholangiocarcinoma (IG-ICC) and its precursor lesions, collectively termed intraductal papillary neoplasm of the liver (IPNL), was characterized with respect to demographics, clinical manifestations, perioperative management, long-term survival, and molecular features associated with carcinogenesis. A total of 122 patients with IPNL types 1 through 4, 108 patients with non-IG-ICC and 210 patients with hepatolithiasis alone were studied. Expression of CDX2, TFF1, MUC1, MUC2, MUC5AC, EGFR, and p53 was determined by using immunohistochemistry. Females predominated in those with hepatolithiasis alone and IPNL. The mean age of patients with hepatolithiasis alone was 6 to 8 years younger than that of those with IPNL. The association with hepatolithiasis in patients with IPNL types 1 and 2, IPNL types 3 and 4, and non-IG-ICC was 100%, 79%, and 64%, respectively. Mucobilia, anemia, and elevated serum carcinoembryonic antigen levels were helpful in distinguishing IG-ICC and its precursor lesions. The mean survival of patients with IPNL type 3, IPNL type 4, and non-IG-ICC was 55.5 months, 36.9 months, and 15.8 months, respectively. The incidence of expression of CDX2 and TFF1 was maximal in IPNL type 3. Expression and cellular distribution of MUC2 and CDX2 were similar. MUC5AC was strongly expressed in all patients with IPNL; EGFR and p53 were rarely expressed in patients with IPNL. In conclusion, hepatolithiasis appears to be a precipitating factor in the development of IPNL. Signs of mucobilia were specific for the diagnosis of IPNL. Expression of CDX2 and MUC2 are helpful in differentiating IPNL and non-IG-ICC. Significant differences in survival associated with the various lesions studied warrants a more aggressive surgical strategy in their management.     

粘蛋白MUC1、MUC2、MUC4和MUC5AC在胰腺导管腺癌中的表达及意义

目的 研究胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDA)组织中粘蛋白MUC1、MUC2、MUC4和MUC5AC的表达及其与临床病理参数间的关系.方法 应用SP免疫组织化学方法检测26例PDA、4例CP、16例正常胰腺组织、2例导管内乳头状黏液性肿瘤(IPMN)、4例实性假乳头状瘤(SPT)和1例浆液性囊性肿瘤(SCN)组织中MUC1、MUC2、MUC4、MUCSAC的表达.结果 正常胰腺和CP组织仅有MUC1表达,表达率均为100%;PDA中MUC1、MUC4和MUC5AC表达率分别为100%、88.5%(23/26)和76.9%(20/26);2例IPMN均见MUC2和MUC5AC表达;SPT和SCN中4种粘蛋白均无表达.MUC4和MUC5AC表达同PDA的临床病理参数之间无相关性(P＞0.05).结论 PDA时存在多种粘蛋白的表达,联合检测MUC1、MUC2、MUC4和MUC5AC的表达可能有助于对PDA的诊断和鉴别诊断.     

COX-2 expression is correlated with VEGF-C, lymphangiogenesis and lymph node metastasis in human cervical cancer

Lymphangiogenesis has been shown to promote lymph node metastasis in cancers, making it an important target in cancer therapy. Vascular endothelial growth factor (VEGF)-C is upregulated in various tumors/cancers and is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis (LNM). Likewise, cyclooxygenase (COX)-2 plays major roles in carcinogenesis, tumor growth and metastasis via multiple mechanisms including inactivation of host antitumor immunity and promotion of tumor cell migration, tumor cell invasiveness and tumor-associated angiogenesis and lymphangiogenesis. We previously demonstrated an association between COX-2 and VEGF-C in an in vitro model of lung cancer. However, little is known about the regulation of VEGF-C by COX-2 in cervical cancer. In this study, we measured the COX-2 and VEGF-C expressions by immunohistochemistry in 23 LNM-positive and 20 LNM-negative cervical cancer specimens. We then examined the correlations among the expressions and the lymphatic microvessel density (LMVD) and ultrastructural changes to the lymphatic vessel walls by enzyme histochemical staining and electron microscopy. In addition, we used the HeLa cervical cancer cell line to explore the in vitro regulation of VEGF-C by COX-2 and its metabolite, PGE₂, using siRNA-mediated gene silencing and EP receptor blockade. The LNM-positive specimens exhibited significantly higher VEGF-C expression, COX-2 expression and LMVD than the LNM-negative specimens. Furthermore, there were strong correlations between the levels of COX-2 expression and the levels of VEGF-C expression and secretion and a significant positive association between the LMVD and LNM. siRNA-mediated knockdown of COX-2 expression inhibited VEGF-C mRNA expression while EP1 and EP4 receptor antagonists reduced the VEGF-C protein level and tyrosine phosphorylation of Src kinase. Moreover, inhibition of Src kinase with the tyrosine kinase inhibitor PP1 attenuated VEGF-C expression. Collectively, our data provide evidence for a clinical association between COX-2 and VEGF-C expressions in cervical cancer. EP1 and EP4 receptors may be involved in the COX-2-mediated regulation of VEGF-C protein and mRNA expressions. Src may be a downstream mediator of EP1 and EP4 receptors. COX-2 inhibition may diminish LNM by suppressing VEGF-C-mediated lymphangiogenesis.     

Epidermal growth factor increases prostaglandin E2 production via ERK1/2 MAPK and NF-κB pathway in fibroblast like synoviocytes from patients with rheumatoid arthritis

High concentration of epidermal growth factor (EGF) is found in the synovial fluid of rheumatoid arthritis (RA) that might imply the involvement of EGF in the pathogenesis of arthritic diseases. In order to investigate if EGF is involved in the regulation of cyclooxygenase-2 (COX-2) and the prostaglandin E₂ (PGE₂) production in fibroblast like synoviocytes (FLS) from patients with RA. The levels of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and Western blot analysis. Electrophoretic mobility shift assay (EMSA) was performed to investigate EGF mediated DNA binding activity of nuclear factor-κB (NF-κB). PGE₂ levels were analyzed by ELISA. EGF enhanced both COX-2 protein and mRNA expressions. mPGES-1 mRNA level was also increased by EGF treatment. EGF also stimulated ERK1/2 MAPK activity and the inhibition of ERK1/2 by PD098059 (ERK1/2 specific inhibitor) resulted in the suppression of EGF-induced COX-2 expression. The DNA binding activity of NF-κB was remarkably increased by EGF treatment and the pretreatment of PD098059 abolished EGF-stimulated NF-κB activity. We also observed that the level of PGE₂ was significantly elevated with the treatment of EGF in FLS, and the pretreatment of PD098059 abolished this stimulating effect. These results suggest that EGF is involved in the inflammatory process of RA by stimulating COX-2 expression and PGE₂ production. And EGF enhanced PGE₂ production appears to be mediated via ERK1/2 MAPK and NF-κB pathway in FLS.     

Neurophysiological mechanisms of bradykinin-evoked mucosal chloride secretion in guinea pig small intestine

AIM: To investigate the mechanism for bradykinin(BK) to stimulate intestinal secretomotor neurons and intestinal chloride secretion. METHODS: Muscle-stripped guinea pig ileal preparations were mounted in Ussing flux chambers for the recording of short-circuit current(Isc). Basal Isc and Isc stimulated by BK when preincubated with the BK receptors antagonist and other chemicals were recorded using the Ussing chamber system. Prostaglandin E2(PGE2) production in the intestine was determined by enzyme immunologic assay(EIA).RESULTS: Application of BK or B2 receptor(B2R) agonist significantly increased the baseline Isc compared to the control. B2 R antagonist, tetrodotoxin and scopolamine(blockade of muscarinic receptors) significantly suppressed the increase in Isc evoked by BK. The BK-evoked Isc was suppressed by cyclooxygenase(COX)-1 or COX-2 specific inhibitor as well as nonselective COX inhibitors. Preincubation of submucosa/mucosa preparations with BK for 10 min significantly increased PGE2 production and this was abolished by the COX-1 and COX-2 inhibitors.The BK-evoked Isc was suppressed by nonselective EP receptors and EP4 receptor antagonists, but selective EP1 receptor antagonist did not have a significant effect on the BK-evoked Isc. Inhibitors of PLC, PKC, calmodulin or Ca MKⅡ failed to suppress BK-induced PGE2 production. CONCLUSION: The results suggest that BK stimulates neurogenic chloride secretion in the guinea pig ileum by activating B2 R, through COX increasing PGE2 production. The post-receptor transduction cascade includes activation of PLC, PKC, Ca MK, IP3 and MAPK.     

白细胞介素13对小鼠支气管哮喘模型肺组织钙激活的氯通道gob-5和黏蛋白基因MUC5AC表达的作用

目的　研究白细胞介素 13(IL 13)对小鼠支气管哮喘 (简称哮喘 )模型肺组织“钙激活的氯通道”成员gob 5和黏蛋白基因MUC5AC表达的影响 ,了解其在哮喘发病中的作用。方法　 4 5只雄性BALB/c小鼠按随机数字表法分为对照组、哮喘组和IL 13组 ,每组 15只。用逆转录 聚合酶链反应(RT PCR)测定方法和免疫组化法分别检测gob 5mRNA、MUC5ACmRNA和MUC5AC蛋白在肺组织的表达。结果　对照组小鼠肺组织中gob 5mRNA表达为 0 0 0 0± 0 0 0 0 ,哮喘组为 1 136± 0 0 0 7,两组比较差异有统计学意义 (P <0 0 1) ;IL 13组小鼠肺组织中gob 5mRNA为 1 2 4 6± 0 0 0 8,哮喘组为 1 136± 0 0 0 7,两组比较差异有统计学意义 (P <0 0 1) ;对照组小鼠MUC5ACmRNA(0 0 70± 0 0 0 4 )和蛋白 (2只小鼠阳性 )表达与哮喘组 (分别为 0 16 1± 0 0 11和 7只小鼠阳性 )比较 ,差异均有统计学意义 (P分别 <0 0 1、0 0 5 ) ;经IL 13处理后的哮喘小鼠肺组织中MUC5ACmRNA (0 2 5 0± 0 0 14 )和蛋白 (13只小鼠阳性 )的表达与对照组 (分别为 0 0 70±0 0 0 4、2只小鼠阳性 )、哮喘组 (分别为 0 16 1± 0 0 11、7只小鼠阳性 )比较 ,差异均有统计学意义 (P均 <0 0 5 ) ;哮喘组和IL 13组小鼠肺组织中gob 5mRNA表达与MUC5ACm     

Upregulated Cyclooxygenase-2 Inhibits Apoptosis of Human Gastric Epithelial Cells Infected with Helicobacter pylori

Helicobacter pylori induces apoptosis and alters the proliferation of gastric mucosal epithelial cells. Cyclooxygenase-2 (COX-2), the inducible form of prostaglandin (PG) synthesis, is known to cause alteration in epithelial cell growth. The goal of this study was to determine whether COX-2 gene expression by H. pylori infection could influence gastric epithelial cell apoptosis. Expression of COX-2 mRNA and proteins was up-regulated in Hs746T gastric epithelial cell lines infected with H. pylori, when assessed by quantitative RT-PCR and western blot. Inhibition of COX-2 expression using NS-398, a specific COX-2 inhibitor, showed a significant increase of gastric epithelial cell apoptosis and caspase-3 activation in Hs746T cells infected with H. pylori. Moreover, the effect of NS-398 on H. pylori-induced apoptosis was reversed by the addition of PGE₂. These results suggest that up-regulated COX-2 expression by H. pylori infection can inhibit apoptosis of gastric epithelial cells.