Cell Culture Techniques: Corticosteroid Treatment in A549 Human Lung Epithelial Cell

Marcos-Vadillo, Elena; Garcı́a-Sánchez, Asunción

doi:10.1007/978-1-4939-3652-6_12

Cited by 2 publications

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References 7 publications

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“…A549 cells (human adenocarcinoma alveolar basal epithelial cells, type II) were kindly provided by Dr Odero (CIMA, University of Navarra) and maintained in RPMI with 10% Fetal Bovine Serum (FBS) (Gibco, Invitrogen-Life Technologies, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 . To perform luciferase and expression experiments, cells were grown in RPMI supplemented with 10% Charcoal Stripped FBS (Gibco, Invitrogen-Life Technologies, MA, USA) to avoid possible interference with serum steroids [ 23 , 24 ].…”

Section: Methodsmentioning

confidence: 99%

PTGDR gene expression and response to dexamethasone treatment in an in vitro model

et al. 2017

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Asthma is a multifactorial pathology influenced by environmental and genetic factors. Glucocorticoid treatment decreases symptoms by regulating genes involved in the inflammatory process through binding to specific DNA sequences. Polymorphisms located in the promoter region of the Prostaglandin D Receptor (PTGDR) gene have been related to asthma. We aimed to analyze the effect of PTGDR promoter haplotypes on gene expression and response to corticosteroid therapy. A549 lung epithelial cells were transfected with vectors carrying four different PTGDR haplotypes (CTCT, CCCC, CCCT and TCCT), and treated with dexamethasone. Different approaches to study the promoter activity (Dual Luciferase Reporter System), gene expression levels (qPCR) and cytokine secretion (Multiplexed Bead-based Flow Cytometric) were used. In addition, in silico analysis was also performed. Cells carrying the TCCT haplotype showed the lowest promoter activity (p-value<0.05) and mRNA expression levels in basal conditions. After dexamethasone treatment, cells carrying the wild-type variant CTCT showed the highest response, and those carrying the TCCT variant the lowest (p-value<0.05) in luciferase assays. Different transcription factor binding patterns were identified in silico. Moreover, differences in cytokine secretion were also found among different promoter haplotypes. Polymorphisms of PTGDR gene influence basal promoter activity and gene expression, as well as the cytokine secretory pattern. Furthermore, an association between these positions and response to corticoid treatment was observed.

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Section: Methodsmentioning

confidence: 99%

PTGDR gene expression and response to dexamethasone treatment in an in vitro model

et al. 2017

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miR-4456/CCL3/CCR5 Pathway in the Pathogenesis of Tight Junction Impairment in Chronic Obstructive Pulmonary Disease

Ding

et al. 2021

Front. Pharmacol.

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Background: Cigarette smoke exposure (CSE) is a major cause of chronic obstructive pulmonary disease (COPD). The smoke disrupts cell-cell adhesion by inducing epithelial barrier damage to the tight junction (TJ) proteins. Even though the inflammatory mechanism of chemokine (C-C motif) ligand 3 (CCL3) in COPD has gained increasing attention in the research community, however, the underlying signaling pathway, remains unknown.Objectives: To identify the relationship of CCL3 in the pathogenesis of tight junction impairment in COPD and the pathway through which CSE causes damage to TJ in COPD via CCL3, both in vivo and in vitro.Methods: We screened the inflammatory factors in the peripheral blood mononuclear cells (PBMCs) from healthy controls and patients at each GOLD 1-4 stage of chronic obstructive pulmonary disease. RT-PCR, western blot, and ELISA were used to detect the levels of CCL3, ZO-1, and occludin after Cigarette smoke exposure. Immunofluorescence was applied to examine the impairment of the TJs in 16-HBE and A549 cells. The reverse assay was used to detect the effect of a CCR5 antagonist (DAPTA) in COPD. In the CSE-induced COPD mouse model, H&E staining and lung function tests were used to evaluate the pathological and physical states in each group. Immunofluorescence was used to assess the impairment of TJs in each group. ELISA and RT-PCR were used to examine the mRNA or protein expression of CCL3 or miR-4456 in each group.Results: The in vivo and in vitro results showed that CCL3 expression was increased in COPD compared with healthy controls. CCL3 caused significant injury to TJs through its C-C chemokine receptor type 5 (CCR5), while miR-4456 could suppress the effect of CCL3 on TJs by binding to the 3′-UTR of CCL3.Conclusion: miR-4456/CCL3/CCR5 pathway may be a potential target pathway for the treatment of COPD.

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Cell Culture Techniques: Corticosteroid Treatment in A549 Human Lung Epithelial Cell

Cited by 2 publications

References 7 publications

PTGDR gene expression and response to dexamethasone treatment in an in vitro model

PTGDR gene expression and response to dexamethasone treatment in an in vitro model

miR-4456/CCL3/CCR5 Pathway in the Pathogenesis of Tight Junction Impairment in Chronic Obstructive Pulmonary Disease

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