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

<div><p>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 <i>via</i> 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.</p></div

Dysregulation of GJIC through signaling pathways other than MEK1/2 or PC-PLC.

<p>The following compounds inhibited GJIC neither through MEK1/2 nor PC-PLC: PFOSA (40 μM, 20 min), BzOOH (200 μM, 15 min), AA (70–100 μM, 15 min), Lau (150 μM, 10 min), BGA (30 μμM, 15 min), PCP (50 μM, 10 min) and Alachlor (185 μM, 25 min). The cells were treated with inhibitors of PC-PLC (D609, 50 μM, 20 min) or MEK1/2 (U0126, 20 μM, 30 min), or resveratrol (100 μM, 15 min) before addition of GJIC-dysregulator. At least three independent experiments were averaged ± SD. An ANOVA was conducted for each GJIC-dysregulator followed by a Dunnett’s post-hoc test to determine significance (at P<0.05 as indicated by an *) from cells treated with only the GJIC-dysregulator. The F-values for PFOSA, BzOOH, AA, Lau, BGA, PCP and alachlor were 1.0 (P = 0.426), 0.6 (P = 0.628), 0.7 (P = 0.565), 0.6 (P = 0.617), 2.1 (P = 0.131), 1.9 (P = 0.162) and 58.6 (P<0.001), respectively.</p

Dysregulation of GJIC through both MEK1/2 and PC-PLC.

<p>The following compounds inhibited GJIC through both MEK1/2 and PC-PLC: PFOA (80 μM, 10 min), NAC+BzOOH (cells were treated with 1 mM NAC for 15 min prior the addition of 200 μM BzOOH for 15 min), and R59022 (30–50 μM, 10 min). The cells were treated with inhibitors of PC-PLC (D609, 50 μM, 20 min) or MEK1/2 (U0126, 20 μM, 30 min), or resveratrol (100 μM, 15 min) before addition of GJIC-dysregulator. At least three independent experiments were averaged ± SD. An ANOVA was conducted for each GJIC-dysregulator followed by a Dunnett’s post-hoc test to determine significance (at P<0.05 as indicated by an *) from cells treated with only the GJIC-dysregulator. The F-values for PFOA and R59022 were 27.0 (P<0.001), 28.2 (P<0.001) and 20.9 (P<0.001), respectively.</p

Dysregulation of GJIC through MEK1/2.

<p>The following compounds inhibited GJIC through MEK1/2: TPA (10 nM, 30 min), EGF (5 ng/ml, 30 min), TRAP-6 (50 μM, 30 min) and lindane (60 μM, 25 min). The cells were treated with inhibitors of MEK1/2 (U0126, 20 μM, 30 min) or PC-PLC (D609, 50 μM, 20 min), or resveratrol (100 μM, 15 min) before addition of GJIC-dysregulator. At least three independent experiments were averaged ± SD. An ANOVA was conducted for each GJIC-dysregulator followed by a Dunnett’s post-hoc test to determine significance (at P<0.05 as indicated by an *) from cells treated with only the GJIC-dysregulator. The F-values for TPA, EGF, TRAP-6 and lindane were 156.563 (P<0.001), 750.742 (P<0.001), 135.648 (P<0.001) and 36.717 (P<0.001), respectively.</p

Dysregulation of GJIC through PC-PLC.

<p>The following compounds inhibited GJIC through PC-PLC: (<b>a)</b> Through the following PAHs: Flu (100 μM, 10 min), 1-MeFlu (70 μM, 10 min), Phe (70 μM, 10 min), 1-MeA (70 μM, 10 min), 9,10-DiMeA (100 μM, 10 min), Fla (70 μM, 10 min), Pyr (70 μM, 10 min) and 1-MePyr (70 μM, 10 min); (<b>b)</b> Other toxicants: PFDA (50 μM, 20 min), DiCuOOH (50 μM, 15 min), PCB 153 (50 μM, 30 min), and DDT (30 μM, 20 min). The cells were treated with inhibitors of PC-PLC (D609, 50 μM, 20 min) or MEK1/2 (U0126, 20 μM, 30 min), or resveratrol (100 μM, 15 min) before addition of GJIC-dysregulator. At least three independent experiments were averaged ± SD. An ANOVA was conducted for each GJIC-dysregulator followed by a Dunnett’s post-hoc test to determine significance (at P<0.05 as indicated by an *) from cells treated with only the GJIC-dysregulator. The F-values for Flu, 1-MeFlu, Phe, 1-MeA, 9,10-DiMeA, Fla, Pyr and 1-MeP were 71.8 (P<0.001), 75.6 (P<0.001), 57.7 (P<0.001), 737.3 (P<0.001), 74.2 (P<0.001), 58.4 (P<0.001), 67.4 (P<0.001) and 50.5 (P<0.001), respectively. The F-values for PFDA, DiCuOOH, PCB 153, and DDT were 13.1 (P = 0.002), 51.2 (P<0.001), 38.3 (P<0.001) and 87.5 (P<0.001), respectively.</p

Component score plot from principal component analysis showing distribution of different GJIC-dysregulators based on their effects on GJIC and alteration of these effects by D609, U0126 or resveratrol.

<p>Symbols represent different groups of GJIC-dysregulators: (<b>triangles</b> = PC-PLC-dependent compound, (<b>circles</b> = MEK1/2-dependent compounds), (<b>diamonds</b> = both PC-PLC- and MEK1/2-dependent compounds), (<b>squares</b> = neither PC-PLC- nor MEK1/2-dependent compounds).</p

Summary of toxicant-dependent regulatory pathways of GJIC.

<p>Each of the four pathways are designated as <b>A.</b> Mek-dependent and resveratrol sensitive, <b>B.</b> Phosphatidylcholine-specific phospholipase C (PC-PLC) and resveratrol sensitive, <b>C.</b> Mek and PC-PLC independent and resveratrol sensitive, and <b>D.</b> Mek and PC-PLC independent and resveratrol insensitive.</p