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Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第9期274卷 5443-5453页

作者： Wong, V Ching, DX McCrea, PD Firestone, GL Univ Calif Berkeley Dept Mol & Cell Biol Berkeley CA 94720 USA Univ Calif Berkeley Canc Res Lab Berkeley CA 94720 USA Univ Texas MD Anderson Cancer Ctr Dept Biochem & Mol Biol Houston TX 77030 USA

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin, Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton, Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments, However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.

关键词：肌动蛋白类/代谢载体蛋白质类/代谢细胞间通讯细胞支架蛋白质类/代谢地塞米松/药理学减量调节/药物作用上皮细胞/细胞学上皮细胞/药物作用上皮细胞/代谢乳房肿瘤实验性/代谢乳房肿瘤实验性/病理学膜电位膜蛋白质类/代谢微丝蛋白质类/代谢反式激活因子类肿瘤细胞培养的 β连环素动物小鼠大鼠

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Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly

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MOLECULAR AND CELLULAR BIOLOGY 2000年第17期20卷 6354-6363页

作者： Zhao, ZS Manser, E Loo, TH Lim, L Natl Univ Singapore Inst Mol & Cell Biol Glaxo IMCB Grp Singapore 117609 Singapore Inst Neurol London WC1N 1PJ England

The p21-activated kinase PAK is targeted to focal complexes (FCs) through interactions with the SH3 domains of the PAK-interacting exchange factor PIX and Nck PIX is a Rac GTP exchange factor that also binds the G-protein coupled receptor kinase-interacting protein known as GIT1, Overexpression of GIT1 in fibroblasts or epithelial cells causes a loss of paxillin from FCs and stimulates cell motility. This is due to the direct interaction of a C-terminal 125-residue domain of GIT1 with paxillin, under the regulation of PM. In its activated state, GIT1 can promote FC disassembly independent of actin-myosin contractile events. Additionally, GIT directly couples to a key component of FCs, focal adhesion kinase (FAK), via a conserved Spa2 homology domain. We propose that GIT1 and FAK cooperate to promote motility both by directly regulating focal complex dynamics and by the activation of Rac.

关键词：衔接蛋白质类信号转导 COS细胞细胞周期蛋白质类/化学细胞周期蛋白质类/代谢细胞运动鸡细胞支架蛋白质类/代谢细胞骨架/代谢 DNA 互补/代谢上皮细胞/代谢成纤维细胞/代谢真菌蛋白质类/代谢 GTP酶激活蛋白质类/化学 GTP酶激活蛋白质类/代谢 GTP酶激活蛋白质类/生理学谷胱甘肽转移酶/代谢鸟嘌呤核苷酸交换因子类/代谢 HeLa细胞显微镜检查相差模型生物学癌基因蛋白质类/代谢桩蛋白磷蛋白类/代谢质粒/代谢沉淀素试验蛋白质结合蛋白质结构三级蛋白质丝氨酸苏氨酸激酶/代谢酿酒酵母蛋白质类信号传导转染 p21活化激酶类 racGTP结合蛋白质类/代谢 src同源域动物人类

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Transforming growth factor-β1 is up-regulated by podocytes in response to excess intraglomerular passage of proteins -: A central pathway in progressive glomerulosclerosis

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AMERICAN JOURNAL OF PATHOLOGY 2002年第6期161卷 2179-2193页

作者： Abbate, M Zoja, C Morigi, M Rottoli, D Angioletti, S Tomasoni, S Zanchi, C Longaretti, L Donadelli, R Remuzzi, G Osped Riuniti Bergamo Azienda Ospedaliera Unit Nephrol & Dialysis I-24100 Bergamo Italy Mario Negri Inst Pharmacol Res I-24100 Bergamo Italy

Chronic diseases of the kidney have a progressive course toward organ failure. Common pathway mechanisms of progressive injury, irrespectively of the etiology of the underlying diseases, include glomerular capillary hypertension and enhanced passage of plasma proteins across the glomerular capillary barrier because of impaired permselective function. These changes are associated with podocyte injury and glomerular sclerosis. Direct evidence for causal roles is lacking, particularly for the link between intraglomerular protein deposition and sclerosing reaction. Because transforming growth factor-beta1 (TGF-beta1) is the putative central mediator of scarring, we hypothesized that TGF-beta1 can be up-regulated by protein overload of podocytes thereby contributing to sclerosis. in rats with renal mass reduction, protein accumulation in podocytes as a consequence of enhanced transcapillary passage preceded podocyte dedifferentiation and injury, increase in TGF-beta1 expression in podocytes, and TGF-beta1-dependent activation of mesangial cells. Angiotensin-converting enzyme inhibitor prevented both accumulation of plasma proteins and TGF-beta1 overexpression in podocytes and sclerosis. Albumin load on podocytes in vitro caused loss of the synaptopodin differentiation marker and enhanced TGF-beta1 mRNA and protein. Conditioned medium of albumin-stimulated podocytes induced a sclerosing phenotype in mesangial cells, an effect mimicked by TGF-beta1 and blocked by anti-TGF-beta1 antibodies. Thus, the passage of excess plasma proteins across the glomerular capillary wall is the trigger of podocyte dysfunction and of a TGFbeta1-mediated mechanism underlying sclerosis. Agents to reduce TGF-beta1, possibly combined with angiotensin blockade, should have priority in novel approaches to treatment of progressive nephropathies.

关键词：血管紧张素转换酶抑制药/药理学血蛋白质类/代谢细胞系结蛋白/代谢疾病模型动物疾病恶化上皮细胞/细胞学上皮细胞/药物作用上皮细胞/代谢免疫组织化学原位杂交肾/外科学肾小球/细胞学肾小球/药物作用肾小球/代谢肾小球/病理学赖诺普利/药理学巨噬细胞/代谢微丝蛋白质类/代谢大鼠 Sprague-Dawley 硬化/病理学转化生长因子β/遗传学转化生长因子β/代谢转化生长因子β1 增量调节/生理学动物人类男(雄)性小鼠大鼠

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Amplification of extracellular matrix and oncogenes in tat-transfected human salivary gland cell lines with expression of laminin, fibronectin, collagens I, III, IV, c-myc and p53

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ARCHIVES OF ORAL BIOLOGY 2001年第6期46卷 545-555页

作者： McArthur, CP Wang, Y Heruth, D Gustafson, S Univ Missouri Sch Dent Dept Oral Biol Kansas City MO 64108 USA Truman Med Ctr Dept Pathol Kansas City MO 64134 USA William Jewel Coll Liberty MO USA

Considerable progress has been made in the transfer of foreign genes into salivary glands in vivo using adenovirus vectors in rats. In an attempt to avoid the transient expression inherent, when using these vectors, retroviral vectors and human cell lines where used here in attempt to develop an in vitro model of HIV-associated salivary gland disease. The HIV-1-tat protein is increasingly implicated in the pathogenesis of the AIDS through altering the expression of strategic cellular genes. The purpose of this study was to transfect human salivary gland (HSG) cell lines in vitro, with the pHIV-1/LTR-tat plasmid, and examine the effect of tar on expression of matrix and basement membrane genes known to be important in the pathogenesis of salivary gland disease. HSG cells were transfected with HIV-1-tat plasmid by the lipofection method. Transfection was confirmed by polymerase chain reaction (PCR) and Southern blot, which verified that tat-specific DNA was present. Tat-mRNA was analysed by Northern blotting and quantified by reverse transcriptase polymerase chain reaction (RT-PCR) to demonstrate its expression. Numerous clones were found to contain integrated tat DNA sequences and analysis of mRNA showed stable expression of tat-specific RNA. Further analysis of mRNA expression for various marker proteins important in HIV pathogenesis showed that the HSG cell line transfected with HIV-1-tat, was associated with significant induction of mRNA expression for extracellular matrix protein. rat-amplified transcription of the major basement membrane protein laminin, as well as of fibronectin, collagen I and III, and c-myc oncogene was demonstrated. Conversely, expression of p53 suppressor gene mRNA was reduced. Post-transfection expression of collagen IV was erratic and inconclusive. It was concluded that the presence of HIV-tat in this in vitro model of salivary ductal epithelial cell model alters the mRNA expression of several matrix, basement membrane and oncoproteins kn

关键词：基膜/代谢印迹法 DNA 细胞系胶原/遗传学 DNA 病毒/分析上皮细胞/代谢细胞外基质/代谢细胞外基质蛋白质类/遗传学纤连蛋白类/遗传学基因扩增基因表达调控病毒基因 myc/遗传学基因 p53/遗传学基因 tat/遗传学 HIV/遗传学 HIV感染/遗传学层黏连蛋白/遗传学癌基因/遗传学质粒 RNA 信使/分析逆转录聚合酶链反应涎腺导管/代谢涎液疾病/遗传学涎液疾病/病毒学涎腺/代谢转录遗传转染人类

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Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells

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AMERICAN JOURNAL OF PATHOLOGY 2003年第3期163卷 1157-1166页

作者： Wyatt, TA Forgèt, MA Sisson, JH Univ Nebraska Med Ctr Dept Internal Med Pulm & Crit Care Med SectNebraska Med Ctr 985300 Omaha NE 68198 USA Res Serv Med Ctr Dept Vet Affairs Omaha NE USA

Previously, we have shown that ethanol (ROH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both I nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (greater than or equal to100 mumol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).

关键词：支气管/药物作用支气管/生理学细胞培养的纤毛/药物作用纤毛/生理学环AMP/代谢环AMP依赖性蛋白激酶类/代谢环GMP/类似物和衍生物环GMP/代谢环GMP/药理学环GMP依赖性蛋白激酶类/代谢酶激活酶抑制剂/药理学上皮细胞/药物作用上皮细胞/代谢上皮细胞/生理学乙醇/药理学鸟苷酸环化酶/拮抗剂和抑制剂一氧化氮/代谢恶二唑类/药理学喹恶啉类/药理学硫代核苷酸类/药理学动物牛

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Effect of topical nasal pharmaceuticals on sodium and chloride transport by human airway epithelia

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AMERICAN JOURNAL OF RHINOLOGY 2000年第6期14卷 405-409页

作者： Jepsen, M Graham, S Karp, PH Zabner, J Univ Iowa Coll Med Dept Otolaryngol Head & Neck Surg Iowa City IA 52242 USA

The human airway epithelium lines the respiratory tract from the nasal mucosa to the bronchioles. Electrolyte transport by these epithelia is crucial in maintaining the appropriate volume and salt composition of the airway surface fluid When this epithelium becomes functionally impaired, the airways are more prone to respiratory infections. We studied the effect of six common topical agents that are commonly used to treat rhinorrhea and nasal inflammation on the transepithelial resistance, sodium, and chloride transport of primary cultures of human airway epithelia grown at the air-liquid interface. The pharmaceuticals fluticasone propionate, cromolyn sodium, ipratropium bromide, azelastine, oxymetazoline, and normal saline were used and the electrical function of the epithelia was studied in Ussing chambers. Azelastin and ipratropium bromide-treated epithelia were found to have a significant decrease in transepithelial resistance. Both normal saline and fluticasone propionate resulted in significant increases in amiloride-sensitive shout circuit currents that reflect sodium transport. Finally, normal saline resulted in a significant increase in bumetanide-sensitive short circuit current that reflects chloride transport across the epithelia. The data presented may explain a mechanism by which some topical pharmaceuticals help reduce rhinorrhea, and may point to some unwanted side effects of some pharmaceuticals on the electrolyte transport of the airway epithelia. In summary, several of the common topical nasal agents alter the electrolyte transport of the nasal airway epithelia. The in vivo significance of these findings is to he determined.

关键词：投药局部抗哮喘药/药理学消炎药/药理学生物转运/药物作用支气管扩张药/药理学细胞培养的氯化物/代谢上皮细胞/药物作用上皮细胞/代谢糖皮质激素类鼻充血减轻剂/药理学呼吸系统/细胞学呼吸系统/药物作用呼吸系统/代谢钠/代谢人类

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Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium

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AMERICAN JOURNAL OF PATHOLOGY 2003年第1期163卷 295-301页

作者： Tanaka, M Kyo, S Kanaya, T Yatabe, N Nakamura, M Maida, Y Okabe, M Inoue, M Kanazawa Univ Sch Med Dept Obstet & Gynecol Kanazawa Ishikawa 9208641 Japan

The endometrium. is a highly regenerative tissue that plays a crucial role in implantation. We examined the clonal. constitution of glandular cells as well as the luminal epithelium of this unique tissue. Using collagenase-based digestion techniques with microscopic manipulation, we isolated individual human endometrial glands and examined their clonality using a polymerase chain reaction-based assay for nonrandom X chromosome inactivation with an X-linked androgen receptor gene. Most of the glands analyzed were composed of monoclonal populations of epithelial cells and one of the glands exhibited a loss of heterogeneity in the androgen receptor gene. In addition, adjacent glands within a 1-mm(2) area shared clonality, suggesting that clonality of the luminal epithelium is regionally defined. The clonality of endometrium was further confirmed in a study of female mice that harbor the green fluorescent protein gene on either the maternal or paternal X chromosome. Fluorescent microscopy of uterine sections revealed that individual endometrial glands consisted completely of either fluorescent or nonfluorescent cells and that the surface epithelium exhibited a clear boundary between these cell types. These findings suggest that single or multiple stem cells with uniform clonality exist on the bottom of each endometrial gland and genetic alterations occurring in such cells may play a critical role in endometrial carcinogenesis. The possible association between area-specific X inactivation of the endometrial surface and the endometrial receptivity of embryo implantation remains to be clarified.

关键词：剂量补偿作用遗传学子宫内膜/细胞学上皮细胞/细胞学上皮细胞/代谢外分泌腺/细胞学外分泌腺/代谢绿色荧光蛋白质类发光蛋白质类/遗传学发光蛋白质类/代谢小鼠转基因镶嵌现象受体雄激素/遗传学干细胞/生理学动物女(雌)性人类小鼠

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Gram positive bacteria induce IL-6 and IL-8 production in human alveolar macrophages and epithelial cells

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INFLAMMATION 1999年第3期23卷 217-230页

作者： Larsson, BM Larsson, K Malmberg, P Palmberg, L Natl Inst Working Life Dept Occupat Med S-17184 Solna Sweden

Background and Objective: Inhalation of dust from swine confinement buildings results in an acute inflammatory reaction in the respiratory tract. The dust has a high microbial content, dominated by Gram positive bacteria. The aim of the present study was to evaluate the significance of bacteria in the induction of IL-6 and IL-8 release from respiratory epithelial cells and alveolar macrophages. The results would give an indication to what extent the bacteria contribute to the toxic inflammation following exposure to swine dust. Methods: Epithelial cells from a human lung carcinoma cell line (A549) and human alveolar macrophages obtained from healthy subjects by bronchoalveolar lavage, were stimulated with swine dust, LPS, one Gram negative and four Gram positive bacteria strains. The dose-response release of IL-6 and IL-8 were studied. In addition, a bacteria-free supernatant was prepared from each strain and used for stimulation. Results: With a few exceptions, a dose-dependent IL-6 and IL-8 release was demonstrated from both cell types after stimulation with bacteria. In epithelial cells, Escherichia coli was the most potent bacteria at the highest concentration of 400 bacteria/cell regarding secretion of both IL-6 and IL-8 (P < 0.001), followed by Staphylococcus hominis and Staphylococcus lentus, In alveolar macrophages, S. lentus was the most potent strain (P < 0.001) in inducing cytokine release(P < 0.001), followed by S. hominis and E. coli concerning IL-6 secretion or Micrococcus luteus and E. coli with respect to IL-8 secretion (P < 0.001). Differences in potency between the various bacteria could be demonstrated, both within the two cell types as well as between the epithelial cells and macrophages. Bacteria-free supernatants were also able to induce cytokine release in both cell types. In macrophages the supernatants were even more potent stimuli than whole bacteria. Conclusions: The results indicate that bacteria or bacterial products could be an importan

关键词：上皮细胞/免疫学上皮细胞/代谢上皮细胞/微生物学革兰氏阳性菌/免疫学白细胞介素6/生物合成白细胞介素8/生物合成巨噬细胞肺泡/免疫学巨噬细胞肺泡/代谢巨噬细胞肺泡/微生物学猪肿瘤细胞培养的成年人动物女(雌)性人类

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Inhibition of extracellular signal-regulated kinase suppresses endotoxin-induced nitric oxide synthesis in mouse macrophages and in human colon epithelial cells

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第3期294卷 1188-1194页

作者： Lahti, A Lähde, M Kankaanranta, H Moilanen, E Univ Tampere Sch Med Dept Pharmacol FIN-33014 Tampere Finland Tampere Univ Hosp Dept Resp Med Tampere Finland Tampere Univ Hosp Dept Clin Chem FIN-33521 Tampere Finland

Macrophages produce large amounts of nitric oxide (NO) in response to proinflammatory cytokines and lipopolysaccharide (LPS) by expressing inducible isoform of NO synthase (iNOS). We examined the role of extracellular signal-regulated kinase p42/44(MAPK) (Erk1/2) in signal transduction pathways leading to induction of NO synthesis in response to LPS in J774 mouse macrophages and T-84 human colon epithelial cells. LPS activated Erk1/2 and induced iNOS and subsequent NO production. Erk1/2 activation was inhibited by PD 98059, a specific inhibitor of mitogen-activated protein kinase kinase (Mek) that is an upstream activator of Erk1/2. At corresponding concentrations PD 98059 reduced LPS-induced NO formation by 40 to 50% by inhibiting iNOS expression in J774 and T-84 cells. Inhibition of iNOS expression was not mediated by nuclear factor-kappa B because PD 98059 had no effect on nuclear factor-kappa B activity in J774 macrophages, In addition, PD 98059 reduced LPS-induced L-arginine transport into the cells as measured in J774 macrophages, whereas the availability of tetrahydrobiopterin was not a limiting factor in NO production after PD 98059. Our results indicate that Erk1/2 activation mediates up-regulation but is not essential for LPS-induced iNOS expression.

关键词：精氨酸/代谢生物转运生物蝶呤/类似物和衍生物生物蝶呤/代谢细胞培养的结肠/细胞学结肠/药物作用结肠/酶学结肠/代谢酶抑制剂/药理学上皮细胞/药物作用上皮细胞/酶学上皮细胞/代谢类黄酮物质/药理学脂多糖类/药理学巨噬细胞/药物作用巨噬细胞/酶学巨噬细胞/代谢丝裂原活化蛋白激酶1/拮抗剂和抑制剂丝裂原活化蛋白激酶3 丝裂原激活蛋白激酶类/拮抗剂和抑制剂一氧化氮/拮抗剂和抑制剂一氧化氮/生物合成一氧化氮合酶/拮抗剂和抑制剂一氧化氮合酶/代谢一氧化氮合酶Ⅱ型动物人类小鼠

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Experimental induction of AGEs in fetal L132 lung cells changes the level of intracellular cathepsin D

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第1期261卷 175-182页

作者： Kasper, M Schinzel, R Niwa, T Münch, G Witt, M Fehrenbach, H Wilsch-Bräuninger, M Pehlke, K Hofer, A Funk, RHW Tech Univ Dresden Fac Med Dept Anat D-01307 Dresden Germany Tech Univ Dresden Fac Med Dept Pathol D-01307 Dresden Germany Univ Wurzburg Inst Physiol Chem D-8700 Wurzburg Germany Nagoya Univ Med Ctr Nagoya Aichi Japan

The effect of the carbonyl compound glyoxal on the induction of advanced glycation end products (AGEs) in the fetal epithelial lung cells L132 was investigated using immunohistochemical, immunoelectron microscopic, and biochemical methods. It was found that glyoxal treatment resulted in morphological changes of the cells and in the membranous and cytosolic localization of AGEs such as methyl-glyoxal-derived compounds, N-(carboxymethyllysine) (CML) and imidazolone. The formation of AG;Es was accompanied with a change in the intracellular expression of cathepsin D and a loss of enzymatic activity. (C) 1999 Academic Press.

关键词：抗体组织蛋白酶D/遗传学组织蛋白酶D/代谢细胞系细胞膜/药物作用细胞膜/代谢细胞膜/超微结构细胞核/药物作用细胞核/代谢细胞核/超微结构细胞大小/药物作用胞质溶胶/药物作用胞质溶胶/代谢胞质溶胶/超微结构上皮细胞/药物作用上皮细胞/酶学上皮细胞/代谢上皮细胞/超微结构基因表达调控酶学/药物作用糖基化终产物高级/代谢乙二醛/药理学咪唑类/代谢免疫组织化学肺/细胞学肺/药物作用肺/胚胎学肺/代谢赖氨酸/类似物和衍生物赖氨酸/代谢溶酶体/药物作用溶酶体/代谢显微镜检查电子肺纤维化/酶学肺纤维化/代谢 RNA 信使/遗传学 RNA 信使/代谢人类

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