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The epidermal growth factor receptor (EGFR) signaling pathway has essential roles in maintaining homeostasis of various tissues by regulating cell proliferation and differentiation. Deregulation of the EGFR signaling pathway is associated with various chronic diseases including chronic obstructive pulmonary disease. Cigarette smoke (CS) is known to activate EGFR, which is linked to chronic obstructive pulmonary disease. The biological sequence from CS exposure to EGFR activation is initiated by oxidative stress caused by intracellular reactive oxygen species (ROS) and the depletion of glutathione, which led to EGFR ligand secretion and EGFR activation. We hypothesized that reducing exposure to CS constituents contributes to preventing CS‐inducible EGFR activation. Therefore, we examined the aerosol from heated tobacco products (HTPs) because the aerosol contains fewer chemical constituents at lower levels than CS. We exposed primary human bronchial epithelial cells from four donors to the extracts of CS from a 1R6F reference cigarette or HTP aerosol from three in‐market products, including our DT3.0a. The biological sequence from ROS to EGFR activation was assessed. CS induced all the tested endpoints although inter‐donor differences were observed, whereas HTPs elicited most of the biological events at higher concentrations; however, EGFR phosphorylation was not observed even at fivefold higher concentration than CS. Overall, our results indicate that HTPs are less effective than CS to elicit ROS‐induced EGFR activation. The reduced‐risk potential of HTPs on EGFR‐related diseases should be investigated further. In addition, testing with multiple donors is warranted when considering the individual differences in responses of primary cells to stimuli. We examined a biological sequence leading to EGFR activation by exposing bronchial epithelial cells from four different donors to cigarette smoke (CS) or aerosol from three in‐market heated tobacco products (HTPs). CS and HTP aerosols elicited oxidative stress and EGF ligand secretion, whereas higher concentrations of HTPs than CS were required for the same effects. EGFR activation was only observed after CS exposure. This is the first step in investigating the reduced‐risk potential of HTPs in EGFR signaling‐mediated adverse outcomes. [ABSTRACT FROM AUTHOR]

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Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2‐related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three‐dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments. Bronchial epithelium models in particular are useful for the evaluation of substances that directly contact the respiratory tract, such as CS. In the present study, we used 3D‐cultured human bronchial epithelial cells (HBECs) to assess activation of transcription factors and relevant gene expression in response to AqCSE, primarily focusing on NRF2 and nuclear factor‐kappa B (NF‐κB) pathways. The 3D‐cultured HBECs exposed to AqCSE showed expression of NRF2 and its nuclear translocation in addition to upregulation of genes related to oxidative stress. Our results suggest that the NRF2 pathway was the dominant pathway when 3D‐cultured HBECs were exposed to AqCSE at a low dose, supporting our previous findings that NRF2 was the most sensitive transcription factor in response to AqCSE. Expression and nuclear translocation of NF‐κB were not increased, although proinflammatory genes were upregulated. However, another inflammation‐related transcription factor, activation protein 1, was induced by AqCSE. Gene classification analysis suggested that induction of the inflammatory response by AqCSE was dependent on NRF2 and activation protein 1 rather than NF‐κB. We assessed activation of transcription factors and relevant signaling pathways when human bronchial epithelial cells in three‐dimensional culture were exposed to aqueous cigarette smoke extract (AqCSE). Our results suggest that the nuclear factor erythroid 2‐related factor 2 (NRF2) pathway is the dominant pathway at low‐dose exposure, supporting our previous findings that NRF2 is the most sensitive transcription factor to AqCSE. Moreover, the AqCSE‐induced inflammatory response is dependent on NRF2 and activation protein 1 rather than nuclear factor‐kappa B. [ABSTRACT FROM AUTHOR]

Background: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated.Methods: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) β1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis.Results: TGF-β1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-β1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model.Conclusion: Our findings indicate that TGF-β1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process. [ABSTRACT FROM AUTHOR]