Phosphatidylcholine Specific PLC-Induced Dysregulation of Gap Junctions, a Robust Cellular Response to Environmental Toxicants, and Prevention by Resveratrol in a Rat Liver Cell Model

Abstract

Dysregulation of gap junctional intercellular communication (GJIC) has been associated with different pathologies, including cancer; however, molecular mechanisms regulating GJIC are not fully understood. Mitogen Activated Protein Kinase (MAPK)-dependent mechanisms of GJIC-dysregulation have been well-established, however recent discoveries have implicated phosphatidylcholine-specific phospholipase C (PC-PLC) in the regulation of GJIC. What is not known is how prevalent these two signaling mechanisms are in toxicant/toxin-induced dysregulation of GJIC, and do toxicants/toxins work through either signaling mechanisms or both, or through alternative signaling mechanisms. Different chemical toxicants were used to assess whether they dysregulate GJIC via MEK or PC-PLC, or both Mek and PC-PLC, or through other signaling pathways, using a pluripotent rat liver epithelial oval-cell line, WB-F344. Epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, thrombin receptor activating peptide-6 and lindane regulated GJIC through a MEK1/2-dependent mechanism that was independent of PC-PLC; whereas PAHs, DDT, PCB 153, dicumylperoxide and perfluorodecanoic acid inhibited GJIC through PC-PLC independent of Mek. Dysregulation of GJIC by perfluorooctanoic acid and R59022 required both MEK1/2 and PC-PLC; while benzoylperoxide, arachidonic acid, 18β-glycyrrhetinic acid, perfluorooctane sulfonic acid, 1-monolaurin, pentachlorophenol and alachlor required neither MEK1/2 nor PC-PLC. Resveratrol prevented dysregulation of GJIC by toxicants that acted either through MEK1/2 or PC-PLC. Except for alachlor, resveratrol did not prevent dysregulation of GJIC by toxicants that worked through PC-PLC-independent and MEK1/2-independent pathways, which indicated at least two other, yet unidentified, pathways that are involved in the regulation of GJIC. In conclusion: the dysregulation of GJIC is a contributing factor to the cancer process; however the underlying mechanisms by which gap junction channels are closed by toxicants vary. Thus, accurate assessments of risk posed by toxic agents, and the role of dietary phytochemicals play in preventing or reversing the effects of these agents must take into account the specific mechanisms involved in the cancer process.

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells

“…GJIC dysregulation by VIN has not been previously reported. Effective concentrations of VIN for inhibition of GJIC (30-min EC 50 ¼ 126 mM) are comparable with the chemicals like alachlor, perfluoroheptanoic acid, benzoylperoxide, or lipidic compounds, which required higher concentrations (>100 mM) to rapidly induce >50% inhibitory effect (Sovadinova et al, 2015;Upham et al, 1998Upham et al, , 2007. Also, endogenous or synthetic estrogens and androgens were found to affect GJIC and connexins not only via classical genomic mechanism (Ren et al, 2013), but also via rapid actions of non-genomic signaling (Iwase et al, 2006;Lyng et al, 2000;Pluciennik et al, 1996).…”

“…Also, endogenous or synthetic estrogens and androgens were found to affect GJIC and connexins not only via classical genomic mechanism (Ren et al, 2013), but also via rapid actions of non-genomic signaling (Iwase et al, 2006;Lyng et al, 2000;Pluciennik et al, 1996). GJIC thus represents a cellular event possibly altered by hormones and EDCs via different mechanisms, which might be chemical-, as well as cell type-specific (Osgood et al, 2013;Sovadinova et al, 2015). The immediate cell responses to MXC and VIN observed in this study, such as inhibition of GJIC and phosphorylation of MAPKs and Cx43, were most likely mediated by rapid mechanisms independent of ER or AR genomic signaling.…”

“…Cells were routinely cultured in 75 cm 2 tissue culture flasks (Costar, Cambridge, Massachusetts) in humidified 5% CO 2 atmosphere at 37 C, and routinely passaged every other day. (Ogawa et al, 2004;Sovadinova et al, 2015) and efficiency of these pretreatments was verified by positive controls with model chemicals known to dysregulate GJIC by a mechanism dependent on a given pathway: EGF was used as a positive control for U0126 (MEK1/2 inhibitor), anisomycin for SB202190 (p38 inhibitor), 1-MeA for D609 (PC-PLC inhibitor) and resveratrol. Pretreatments with ER agonist (E2, HPTE), ER antagonists (ICI 182 780), AR agonist (testosterone) and AR antagonists (flutamide, VIN M2) were done using concentrations ranging from 1 to 250 mM and 30 or 120 min incubation time.…”

“…Many EDCs, such as bisphenol A, DDT, lindane or phthalates, were found to induce rapid dysregulation of GJIC, with their effects shown to be structure-dependent and mediated via mechanisms including activation of specific kinases or phospholipases (Sovadinova et al, 2015;Vinken et al, 2009b). These data indicate that dysregulation of GJIC could be the key cellular process altered by EDCs via mechanisms independent on genomic signaling, which might contribute to their disruptive effects on epigenetic regulations, developmental programming, tissue development, and homeostasis.…”

“…These effects, which are relevant for transcriptional and epigenetic control of gene expression and normal cell behavior (Trosko, 2011), were studied in a rat liver epithelial cell line WB-F344. This cell line is a well-established in vitro model for assessment of GJIC and mechanistic studies in responses to environmental contaminants (Sovadinova et al, 2015;Upham et al, 2007Upham et al, , 2008, which has been validated by in vivo studies (Sai et al, 2000;Upham et al, 2009) and matches the most extensively used rodent model system, F344 rats, in the National Toxicology Program. Both of these EDCs exhibited dose, time and mechanistic differences on GJIC, MAPKs, and phospholipase C, which were elicited by the parental compounds and occurred independently of ER-or AR-mediated genomic mechanisms.…”