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Effect of calcitonin gene-related peptide on E-cadherin expression in human bronchial epithelial cells
Abstracts / Cell Biology International 32 (2008) S1eS67 proliferation after treated with lactoferrin (10 mmol/L) for 24 hours and 48 hours respectively, and the results of western blot showed that LTF inhibited proliferation of nasopharyngeal carcinoma cells in vitro by modulating the mitogen-activated protein kinase pathway. This work was supported by National Key Project of Scientific Research Program (NO.2006CB910502, NO.2006CB910504); National Natural Sciences Foundation of China, Grant numbers: 30700469; the Special Funds of Science & Technology Departments of Hunan Province, China (05SK1001_1); the national innovative experimental plan for undergraduate (YA07050).
EPITHELIAL FUNCTIONING AND RELATED DISEASES EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON E-CADHERIN EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELLS Hong Bo Bai 1,2, Cha Xiang Guan* 1, Yong Ping Liu 1, Guo Ying Sun 1, Fang Yu 1, Chun Yan Tang 1, Chang Qing Zhang 1, Hui Jun Liu 1 1 Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China 2 Department of Physiology, The Medical College of Guangzhou, Guangdong, China The function of the human bronchial epithelial cells (HBECs) is to act as barriers which can protect the underlying tissue against allergens, irritants, viruses, and microbial pathogens. The effect of the barriers relies on epithelial integrity which in turn is dependent on intercellular adhesion. Epithelial cadherin (E-cd) is an important adhesion molecule leading to adhesion between HBECs. Neuropeptide is constitutively expressed in normal lungs where it localizes to neuroendocrine cells. Among lung neuropeptides, Calcitonin gene-related peptide (CGRP) plays a prominent role for its multiple effects on tissue repair and anti-inflammatory actions. The objective of this study was to investigate the effects of CGRP on E-cd expression of HBECs in vitro. The experiments included following steps. First, the influence of CGRP on E-cd protein and mRNA expression were determined in both unstimulated and O3-stressed HBECs using immunocytochemistry and RT-PCR analysis. Second, the signal transduction pathways of CGRP were observed by using PKC inhibitor H-7, calmodulin inhibitor W-7 and PKA inhibitor H-89. The results showed that E-cd was distributed on HBECs. Furthermore, O3-stress decreased the membranous E-cd expression but increased the cytoplasm E-cd expression. We propose that the change of E-cd expression might mediate HBECs injuries induced by O3-stress and CGRP produced a dose and time dependent increase of membranous E-cd and E-cd mRNA expression on both unstimulated and O3-stressed HBECs probably via PKA, PKC and CaM pathways.
EXPRESSION OF CFTR IN MOUSE OOCYTES AND ITS INVOLVEMENT IN OOCYTE MATURATION Hui Chen 1,2, Min Hui Chen 1,2, Wen Liang Zhou 2, Hsiao Chang Chan 1 1 Epithelial Cell Biology Research Centre, Department of Physiology, Chinese University of Hong Kong, Hong Kong, P.R.China 2 School of Life Science, Sun Yat-Sen University, Guangzhou, P.R.China The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP dependent Cl- channel, is recognized to play an important role in reproduction; however, its exact role in various reproductive events remains obscure. Our previous study has shown that CFTR is expressed in mouse ovary. The present study aimed to investigate the expression of CFTR in mouse oocytes and its involvement in oocyte maturation. The expression of CFTR in oocytes was determined by immunofluorecscence study and the results showed that CFTR was localized to the membrane of the oocyte in follicle. The involvement of CFTR in oocyte maturation was investigated by in vitro maturation (IVM). Cl- channel blocker NPPB (100mM)
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significantly decreased the first polar body extrusion (PB) rate and germinal vesicle breakdown (GVBD) rate of the oocytes by 80.0% (p<0.01) and 46.6% (p<0.05) respectively. CFTR inhibitor Glibenclamide (100mM) also decreased the PB rate by 24.4% (p<0.01), but had no effect on GVBD. IVM result obtained from the occytes of CFTR knockout mice showed that PB rate of knockout homozygous and heterozygous oocytes was 48.5% and 76.1% of that obtained from normal C57BL/6 mouse oocytes (p<0.001), whereas no significant difference was found in GVBD rate among these three genotypes. It is concluded that CFTR is expressed in mouse oocytes and plays an important role in oocyte maturation, especially in first polar body extrusion.
JunD REPRESSES IMPORTIN-a1 TRANSCRIPTION THROUGH ITS PROXIMAL PROMOTER REGION TO REGULATE SUBCELLULAR LOCALIZATION OF HuR IN INTESTINAL EPITHELIAL CELLS Jie Chen 1,2, Tong Tong Zou 1, Jaladanki N. Rao 1, Lan Liu 1, Lan Xiao 1, Emily Bellavance 1, Ting Xi Yu 1, Douglas J. Turner 1, Ting Yu Li 2, Jian Ying Wang 1 1 Cell Biology Group, Departments of Surgery and Pathology, University of Maryland School of Medicine and Baltimore VA Medical Center, Baltimore, MD 2 Children’s Hospital, Chongqing Medical University, Chongqing, China 400014 In intestinal epithelial cells, our recent studies show that increased cytoplasmic levels of HuR stabilize mRNAs of p53, nucleophosmin, and ATF-2, thus inhibiting IEC proliferation. However, the mechanism underlying HuR trafficking between the nucleus and the cytoplasm remains elusive. JunD is an AP-1 transcription factor and is implicated in negatively regulating IEC proliferation. Importin-a1 is an adaptor protein that transports bound cargoes through the nuclear pore complex and is involved in mediating HuR nuclear import. This study determined if the subcellular localization of HuR is regulated by JunD through modulation of importin-a1 expression. Full-length importin-a1 promoter was cloned, and constructs of wild-type and various mutated importin-a1-promoter luciferase reporters were generated. Importin-a1 expression was examined by measuring its promoter activity and mRNA and protein levels. JunD overexpression was induced by transient transfection with the JunD expression vector under control of PCMV promoter in Caco-2 cells. Cytoplasmic and nuclear proteins were isolated for detecting HuR subcellular levels. JunD protein levels were increased by ~15-fold when Caco-2 cells were transfected with the JunD expressing vector for 48 and 72 h. Ectopic expression of JunD repressed importin-a1 gene transcription as indicated by decreased (~70%) importin a1-promoter activity and its mRNA and protein levels, but it did not affect expression of importin-b and transportin-1. Studies using deletionand point-mutations of importin-a1-promoter revealed that JunD repressed importin-a1 transcription through CREB-binding sites that were located at its proximal promoter region. Reduction of importin-a1 by JunD overexpression increased cytoplasmic levels of HuR, although it failed to alter levels in total HuR. Levels of cytoplasmic HuR were ~3-fold the control value after JunD transfection. These findings indicate that JunD represses importin-a1 transcription through its proximal promoter region and that JunD-mediated inhibition of importin-a1 expression is crucial for the subcellular distribution of HuR.
L-ARGININE PROMOTES DNA REPAIR IN CULTURED BRONCHIAL EPITHELIUM WHEN EXPOSED TO OZONE: INVOLVEMENT OF ATM PATHWAY Yan Ru Cui 1,2, Cha Xiang Guan* 1, Fang Yu 1, Guo Ying Sun 1, Chun Yan Tang 1, Chang Qing Zhang 1, Li Ping Zou 1 1 Department of Physiology, Xiangya School of Medicine,,Central South University, Changsha, Hunan, China 2 Department of Physiology, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi, China
EPITHELIAL FUNCTIONING AND RELATED DISEASES EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON E-CADHERIN EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELLS Hong Bo Bai 1,2, Cha Xiang Guan* 1, Yong Ping Liu 1, Guo Ying Sun 1, Fang Yu 1, Chun Yan Tang 1, Chang Qing Zhang 1, Hui Jun Liu 1 1 Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China 2 Department of Physiology, The Medical College of Guangzhou, Guangdong, China The function of the human bronchial epithelial cells (HBECs) is to act as barriers which can protect the underlying tissue against allergens, irritants, viruses, and microbial pathogens. The effect of the barriers relies on epithelial integrity which in turn is dependent on intercellular adhesion. Epithelial cadherin (E-cd) is an important adhesion molecule leading to adhesion between HBECs. Neuropeptide is constitutively expressed in normal lungs where it localizes to neuroendocrine cells. Among lung neuropeptides, Calcitonin gene-related peptide (CGRP) plays a prominent role for its multiple effects on tissue repair and anti-inflammatory actions. The objective of this study was to investigate the effects of CGRP on E-cd expression of HBECs in vitro. The experiments included following steps. First, the influence of CGRP on E-cd protein and mRNA expression were determined in both unstimulated and O3-stressed HBECs using immunocytochemistry and RT-PCR analysis. Second, the signal transduction pathways of CGRP were observed by using PKC inhibitor H-7, calmodulin inhibitor W-7 and PKA inhibitor H-89. The results showed that E-cd was distributed on HBECs. Furthermore, O3-stress decreased the membranous E-cd expression but increased the cytoplasm E-cd expression. We propose that the change of E-cd expression might mediate HBECs injuries induced by O3-stress and CGRP produced a dose and time dependent increase of membranous E-cd and E-cd mRNA expression on both unstimulated and O3-stressed HBECs probably via PKA, PKC and CaM pathways.
EXPRESSION OF CFTR IN MOUSE OOCYTES AND ITS INVOLVEMENT IN OOCYTE MATURATION Hui Chen 1,2, Min Hui Chen 1,2, Wen Liang Zhou 2, Hsiao Chang Chan 1 1 Epithelial Cell Biology Research Centre, Department of Physiology, Chinese University of Hong Kong, Hong Kong, P.R.China 2 School of Life Science, Sun Yat-Sen University, Guangzhou, P.R.China The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP dependent Cl- channel, is recognized to play an important role in reproduction; however, its exact role in various reproductive events remains obscure. Our previous study has shown that CFTR is expressed in mouse ovary. The present study aimed to investigate the expression of CFTR in mouse oocytes and its involvement in oocyte maturation. The expression of CFTR in oocytes was determined by immunofluorecscence study and the results showed that CFTR was localized to the membrane of the oocyte in follicle. The involvement of CFTR in oocyte maturation was investigated by in vitro maturation (IVM). Cl- channel blocker NPPB (100mM)
S41
significantly decreased the first polar body extrusion (PB) rate and germinal vesicle breakdown (GVBD) rate of the oocytes by 80.0% (p<0.01) and 46.6% (p<0.05) respectively. CFTR inhibitor Glibenclamide (100mM) also decreased the PB rate by 24.4% (p<0.01), but had no effect on GVBD. IVM result obtained from the occytes of CFTR knockout mice showed that PB rate of knockout homozygous and heterozygous oocytes was 48.5% and 76.1% of that obtained from normal C57BL/6 mouse oocytes (p<0.001), whereas no significant difference was found in GVBD rate among these three genotypes. It is concluded that CFTR is expressed in mouse oocytes and plays an important role in oocyte maturation, especially in first polar body extrusion.
JunD REPRESSES IMPORTIN-a1 TRANSCRIPTION THROUGH ITS PROXIMAL PROMOTER REGION TO REGULATE SUBCELLULAR LOCALIZATION OF HuR IN INTESTINAL EPITHELIAL CELLS Jie Chen 1,2, Tong Tong Zou 1, Jaladanki N. Rao 1, Lan Liu 1, Lan Xiao 1, Emily Bellavance 1, Ting Xi Yu 1, Douglas J. Turner 1, Ting Yu Li 2, Jian Ying Wang 1 1 Cell Biology Group, Departments of Surgery and Pathology, University of Maryland School of Medicine and Baltimore VA Medical Center, Baltimore, MD 2 Children’s Hospital, Chongqing Medical University, Chongqing, China 400014 In intestinal epithelial cells, our recent studies show that increased cytoplasmic levels of HuR stabilize mRNAs of p53, nucleophosmin, and ATF-2, thus inhibiting IEC proliferation. However, the mechanism underlying HuR trafficking between the nucleus and the cytoplasm remains elusive. JunD is an AP-1 transcription factor and is implicated in negatively regulating IEC proliferation. Importin-a1 is an adaptor protein that transports bound cargoes through the nuclear pore complex and is involved in mediating HuR nuclear import. This study determined if the subcellular localization of HuR is regulated by JunD through modulation of importin-a1 expression. Full-length importin-a1 promoter was cloned, and constructs of wild-type and various mutated importin-a1-promoter luciferase reporters were generated. Importin-a1 expression was examined by measuring its promoter activity and mRNA and protein levels. JunD overexpression was induced by transient transfection with the JunD expression vector under control of PCMV promoter in Caco-2 cells. Cytoplasmic and nuclear proteins were isolated for detecting HuR subcellular levels. JunD protein levels were increased by ~15-fold when Caco-2 cells were transfected with the JunD expressing vector for 48 and 72 h. Ectopic expression of JunD repressed importin-a1 gene transcription as indicated by decreased (~70%) importin a1-promoter activity and its mRNA and protein levels, but it did not affect expression of importin-b and transportin-1. Studies using deletionand point-mutations of importin-a1-promoter revealed that JunD repressed importin-a1 transcription through CREB-binding sites that were located at its proximal promoter region. Reduction of importin-a1 by JunD overexpression increased cytoplasmic levels of HuR, although it failed to alter levels in total HuR. Levels of cytoplasmic HuR were ~3-fold the control value after JunD transfection. These findings indicate that JunD represses importin-a1 transcription through its proximal promoter region and that JunD-mediated inhibition of importin-a1 expression is crucial for the subcellular distribution of HuR.
L-ARGININE PROMOTES DNA REPAIR IN CULTURED BRONCHIAL EPITHELIUM WHEN EXPOSED TO OZONE: INVOLVEMENT OF ATM PATHWAY Yan Ru Cui 1,2, Cha Xiang Guan* 1, Fang Yu 1, Guo Ying Sun 1, Chun Yan Tang 1, Chang Qing Zhang 1, Li Ping Zou 1 1 Department of Physiology, Xiangya School of Medicine,,Central South University, Changsha, Hunan, China 2 Department of Physiology, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi, China