Modulation of gap junctional intercellular communication in HaCaT cells by xenobiotics

Abstract

Gap junctions are clusters of transmembrane proteins which enable direct communication between adjacent cells. Gap junctions allow intercellular diffusion of small molecules like metabolites (such as glucose), second messengers (such as cAMP, ATP) and ions (such as Mg2+, K+). That process, called gap junctional intercellular communication (GJIC), allows coordination of many essential functions in multicellular organisms, such as directed cell differentiation, cell growth, cellular and tissue homeostasis. Moreover, dysfunctional GJIC has been linked to a variety of human diseases.The final aim of this project is to elucidate whether estrogenic acting chemicals play a role in GJIC. As a first step, two industrial chemicals, bisphenol A (BPA) and 4‐nonylphenol (4‐NP), were selected and assessed as modulators of GJIC in the human epidermal keratinocyte line HaCaT.To study GJIC of HaCaT cells, the scrape loading/dye transfer (SL/DT) assay was applied to HaCaT cultures grown at standard conditions with serial dilution treatments of either BPA or 4‐NP versus ‐a vehicle treatment (< 1% ethanol) for 24 hours. The SL/DT assay entails cutting a cell monolayer with a scalpel to allow for the incorporation of a fluorescent dye (Lucifer Yellow) into the ‘scraped’ cells. The dye transfer via opened gap junctions into adjacent cells is then investigated by fluorescence microscopy and image analysis. The number of Lucifer Yellow positive cells is a quantitative descriptor of GJIC activity in treated cells versus untreated cells. In addition to the results provided by the SL/DT functional assay, quantitative PCR was performed in parallel to assess the mRNA levels for GJIC protein connexins (Cx26, Cx36, and Cx43).The results of the SL/DT assays showed that both BPA and 4‐NP reduce GJIC in the human epidermal keratinocyte line in a dose range of 10 down to 0.1 μM. Currently qPCR experiments are being conducted to elucidate whether this is due to closed gap junctions or to modulation in expression of connexin encoding genes.With this research, these properties alongside qPCR may be relevant to answer the question of whether the observed modulation of GJIC in skin cells is due to an estrogenic activity. Furthermore, a better understanding of GJIC modulation may clarify the role of GJIC in disease and make it a future target for pharmacological, and particularly, dermatopharmacological approaches.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.