AIM： To investigate the correlation between the expression levels of interleukin （IL）-6 and proteins in tight junctions （TJs） in the esophageal mucosa of rats modeling different types of reflux esophagitis （RE）...AIM： To investigate the correlation between the expression levels of interleukin （IL）-6 and proteins in tight junctions （TJs） in the esophageal mucosa of rats modeling different types of reflux esophagitis （RE）, and the ability of aluminum phosphate to protect against RE-induced mucosal damage via these proteins. METHODS： Male SPF Wistar rats aged 56 d were divided randomly into acid RE, alkaline RE, mixed RE, and control groups. Various surgical procedures were performed to establish rat models of acid RE. At 14 d after the procedure, some of the rats started aluminum phosphate treatment. Transmission electron microscopy （TEM） was used to observe the morphological features of TJs and desmosomes in the esophageal epithelium. Immunohistochemical methods and Western blotting were used to measure expression of claudin 1, occludin, ZO-1, JAM-l, DSG-1 and IL-6; reverse transcription polymerase chain reaction （RT- PCR） was used to measure expression of mRNA of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6. RESULTS： At day 14 alter the procedures, an RE model was established in all subsequently sacrificed rats of groups A, B and C. By both gross and microscopic observation, the mucosa was damaged and thickened as the disease progressed. With TEM observation, a widened intercellular space was noticed, with significantly fewer desmosomes. Immunohistochemistry showed significantly higher levels of all proteins in all RE models compared to control rats at 3 d after operation （65.5% ± 25.6% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. At 14 d after operation, along with continuing hyperplasia in the basal layer, the expression of TJ proteins in individual cells gradually decreased （12.4% ± 2.1% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. Western blottings and RT-PCR showed a directly proportional increase in IL-6 levels in relation to TJ proteins, as compared to controls （0.878 ± 0.024 vs 0.205 ± 0.021 and 0.898±0.022 vs 0.205 ± 0.021, P 〈 0.05, respectively）. Upon treatment with aluminum phosphate, however, these protein levels were restored to normal gradually over 30-60 d in rats with acid RE （30.4% ± 2.1% vs 20.5% ± 2.1%, P 〉 0.05, treated vs untreated, respectively）. These levels increased in the rat with alkaline RE, and this increase was accompanied by continued hyperplasia in comparison with controls （85.5% ± 25.6% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. Furthermore, the expression of TJ proteins was not correlated significantly with that of IL-6 in this group. CONCLUSION： These findings indicate that TJ proteins are highly expressed as an early molecular event involved in RE development, and that IL-6 is an inflammatory factor in this process,展开更多
Objective： To study the expression of extracellular matrix （ECM） proteins including Collagen Ⅳ （Co Ⅳ）, Fibronectin, Laminin in human non-small cell lung cancer （NSCLC） specimens and the relationship between E...Objective： To study the expression of extracellular matrix （ECM） proteins including Collagen Ⅳ （Co Ⅳ）, Fibronectin, Laminin in human non-small cell lung cancer （NSCLC） specimens and the relationship between ECM and cell adhesion, proliferation, apoptosis and drug sensitivity in NSCLC cell line. And to investigate the role of phosphatidylinositol 3-kinase （PI3-K） in signal transduction of Co Ⅳ in NSCLC. Methods： The expression of ECM proteins was detected by using immunohistochemical staining （Envision＇s）. Adherent cells were stained with 1% methylene blue. Cell proliferation and cytotoxic effects were monitored by MTT assay. Cell apoptosis was analyzed by FITC-Annexin V/PI double staining variables flow cytometry （FCM）. Results： The expression rate of Co Ⅳ （93%） was the highest compared to others in NSCLC stroma. After treated with Co Ⅳ, the adhesion of H1299 cells was increased and the cytotoxicity of cis-platinum （DDP） against H1299 cells was decreased compared to the control （P〈0.05）. After treated with Co Ⅳ both survival and proliferation rates were higher and apoptosis rate was lower than without Co Ⅳ （P〈0.05）. PI3-K inhibitor LY294002 decreased both survival and proliferation rates （82.7%±2.0% and 75.2%±6.8%, respectively）, even on Co Ⅳ-coated surface （92.2%±2.8% and 84.6%±9.2%, respectively）. And it also helped DDP increase apoptosis. Conclusion： ECM remodeling existed in NSCLC. Co Ⅳ protected NSCLC cells from DDP-induced apoptosis and weakened the cytotoxicity of DDP. PI3-K pathway might be the crucial mechanism of apoptosis impairment and drug resistance.展开更多
Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is...Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.展开更多
基金Supported by A grant from the Doctoral Program of China Medical University
文摘AIM： To investigate the correlation between the expression levels of interleukin （IL）-6 and proteins in tight junctions （TJs） in the esophageal mucosa of rats modeling different types of reflux esophagitis （RE）, and the ability of aluminum phosphate to protect against RE-induced mucosal damage via these proteins. METHODS： Male SPF Wistar rats aged 56 d were divided randomly into acid RE, alkaline RE, mixed RE, and control groups. Various surgical procedures were performed to establish rat models of acid RE. At 14 d after the procedure, some of the rats started aluminum phosphate treatment. Transmission electron microscopy （TEM） was used to observe the morphological features of TJs and desmosomes in the esophageal epithelium. Immunohistochemical methods and Western blotting were used to measure expression of claudin 1, occludin, ZO-1, JAM-l, DSG-1 and IL-6; reverse transcription polymerase chain reaction （RT- PCR） was used to measure expression of mRNA of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6. RESULTS： At day 14 alter the procedures, an RE model was established in all subsequently sacrificed rats of groups A, B and C. By both gross and microscopic observation, the mucosa was damaged and thickened as the disease progressed. With TEM observation, a widened intercellular space was noticed, with significantly fewer desmosomes. Immunohistochemistry showed significantly higher levels of all proteins in all RE models compared to control rats at 3 d after operation （65.5% ± 25.6% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. At 14 d after operation, along with continuing hyperplasia in the basal layer, the expression of TJ proteins in individual cells gradually decreased （12.4% ± 2.1% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. Western blottings and RT-PCR showed a directly proportional increase in IL-6 levels in relation to TJ proteins, as compared to controls （0.878 ± 0.024 vs 0.205 ± 0.021 and 0.898±0.022 vs 0.205 ± 0.021, P 〈 0.05, respectively）. Upon treatment with aluminum phosphate, however, these protein levels were restored to normal gradually over 30-60 d in rats with acid RE （30.4% ± 2.1% vs 20.5% ± 2.1%, P 〉 0.05, treated vs untreated, respectively）. These levels increased in the rat with alkaline RE, and this increase was accompanied by continued hyperplasia in comparison with controls （85.5% ± 25.6% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. Furthermore, the expression of TJ proteins was not correlated significantly with that of IL-6 in this group. CONCLUSION： These findings indicate that TJ proteins are highly expressed as an early molecular event involved in RE development, and that IL-6 is an inflammatory factor in this process,
基金This project was supported by the Science Foundation of Shanghai Municipal Commission of Science and Technology (034119953).
文摘Objective： To study the expression of extracellular matrix （ECM） proteins including Collagen Ⅳ （Co Ⅳ）, Fibronectin, Laminin in human non-small cell lung cancer （NSCLC） specimens and the relationship between ECM and cell adhesion, proliferation, apoptosis and drug sensitivity in NSCLC cell line. And to investigate the role of phosphatidylinositol 3-kinase （PI3-K） in signal transduction of Co Ⅳ in NSCLC. Methods： The expression of ECM proteins was detected by using immunohistochemical staining （Envision＇s）. Adherent cells were stained with 1% methylene blue. Cell proliferation and cytotoxic effects were monitored by MTT assay. Cell apoptosis was analyzed by FITC-Annexin V/PI double staining variables flow cytometry （FCM）. Results： The expression rate of Co Ⅳ （93%） was the highest compared to others in NSCLC stroma. After treated with Co Ⅳ, the adhesion of H1299 cells was increased and the cytotoxicity of cis-platinum （DDP） against H1299 cells was decreased compared to the control （P〈0.05）. After treated with Co Ⅳ both survival and proliferation rates were higher and apoptosis rate was lower than without Co Ⅳ （P〈0.05）. PI3-K inhibitor LY294002 decreased both survival and proliferation rates （82.7%±2.0% and 75.2%±6.8%, respectively）, even on Co Ⅳ-coated surface （92.2%±2.8% and 84.6%±9.2%, respectively）. And it also helped DDP increase apoptosis. Conclusion： ECM remodeling existed in NSCLC. Co Ⅳ protected NSCLC cells from DDP-induced apoptosis and weakened the cytotoxicity of DDP. PI3-K pathway might be the crucial mechanism of apoptosis impairment and drug resistance.
基金Acknowledgments We thank M Hamaguchi （Nagoya Univ., Japan） and T Iwahara （Osaka Bioscience Institute, Japan） forJak null MEFs, and N Gotoh （Tokyo Univ., Japan）, H Higashi （Hokkaido Univ., Japan）, N Mochizuki （National Cardiovascular Cent. Res. Inst., Japan）, H Hanafusa （Prof. emeritus, The Rockefeller Univ., USA and Direc- tor em., OBI, Japan）, SK Hanks （Vanderbilt Univ., USA）, and M Matsuda （Kyoto Univ., Japan） for plasmids. We also thank K Sasai （Hokkaido Univ., Japan） and Y Ohba （Hokkaido Univ., Japan） for valuable discussion. This work was supported in part by grants-in- aid from the Ministry of Education, Science, Culture, and Sports, and the Ministry of Health, Labor, and Welfare, Japan, as well as Suhara Memorial Foundation （Sapporo, Japan）, and the Mochida Medical Science Foundation （Tokyo, Japan）. The work of SF and TK is supported by grants from Cancer Research UK and the Brit- ish Cancer Charity ＂Heads Up＂. We dedicate this work to our great mentor Hidesaburo Hana- fusa, professor emeritus of the Rockefeller University, who passed away on March 15, 2009 at the age of 79. He devoted his life to science and in particular to creating the oncogene research field, to teaching and to providing profound affection to his students and postdocs. All of the alumni of Saburo＇s laboratory pride them- selves in having been his apprentices and we would like to hereby express our deeply felt gratitude to Saburo.
文摘Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.
