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, 151 (1), 23-34

Nicotine Directly Induces Endoplasmic Reticulum Stress Response in Rat Placental Trophoblast Giant Cells

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Nicotine Directly Induces Endoplasmic Reticulum Stress Response in Rat Placental Trophoblast Giant Cells

Michael K Wong et al. Toxicol Sci.

Abstract

Nicotine exposure during pregnancy leads to placental insufficiency impairing both fetal and neonatal development. Previous studies from our laboratory have demonstrated that in rats, nicotine augmented endoplasmic reticulum (ER) stress in association with placental insufficiency; however, the underlying mechanisms remain elusive. Therefore, we sought to investigate the possible direct effect of nicotine on ER stress in Rcho-1 rat placental trophoblast giant (TG) cells during differentiation. Protein and/or mRNA expression of markers involved in ER stress (eg, phosphorylated PERK, eIF2α, CHOP, and BiP/GRP78) and TG cell differentiation and function (eg, Pl-1, placental growth factor [Pgf], Hsd11b1, and Hsd11b2) were quantified via Western blot or real-time polymerase chain reaction. Nicotine treatment led to dose-dependent increases in the phosphorylation of PERK[Thr981] and eIF2α[Ser51], whereas pretreatment with a nicotinic acetylcholine receptor (nAChR) antagonist (mecamylamine hydrochloride) blocked the induction of PERK phosphorylation, verifying the direct involvement of nicotine and nAChR binding. We next investigated select target genes known to play essential roles in placental TG cell differentiation and function (Pl-1, Pgf, Hsd11b1, and Hsd11b2), and found that nicotine significantly augmented the mRNA levels of Hsd11b1 in a dose-dependent manner. Furthermore, using tauroursodeoxycholic acid, a safe bile acid known to improve protein chaperoning and folding, we were able to prevent nicotine-induced increases in both PERK phosphorylation and Hsd11b1 mRNA levels, revealing a potential novel therapeutic approach to reverse the deleterious effects of nicotine exposure in pregnancy. Collectively, these results implicate that nicotine, acting through its receptor, can directly augment ER stress and impair placental function.

Keywords: Rcho-1; endoplasmic reticulum stress; nicotine; placenta; tauroursodeoxycholic acid; trophoblast giant cell..

Figures

FIG. 1.
FIG. 1.
Different methods used to detect the presence of differentiated Rcho-1 TG cells. A, Phase contrast microscopic images (10×). White triangles identify several representative differentiated TG cells. B, Steady-state mRNA levels of Pl-1 as measured through RT-PCR. RT-PCR: real time-polymerase chain reaction
FIG. 2.
FIG. 2.
The effect of nicotine exposure (0.1–100 μM) on phosphorylation of PERK after 6 and 24 h in Rcho-1 TG cells. A, Specific targeted protein bands as detected by respective antibodies via Western blot. B, Protein levels of the ratio of P-PERK[Thr981]: PERK at 6 h and C, 24 h of nicotine exposure. All arbitrary values were expressed as means normalized to Amido Black ± SEM. All experiments were performed in quadruplicates (n = 4). Significant differences between treatment groups as determined by 1-way ANOVA (1WA) indicated by ** (P < .01) or *** (P < .001). Different letters represent means that are significantly different from one another according to Tukey’s posttest (P < .05). Nonsignificant differences (P > .05) indicated by n.s.
FIG. 3.
FIG. 3.
The effect of nicotine exposure (0.1–100 μM) on downstream targets of the PERK pathway after 6 and 24 h in Rcho-1 TG cells. A, Specific targeted protein bands as detected by respective antibodies via Western blot. B, Protein levels of the ratio of P-eIF2α[Ser51]: eIF2α at 6 h and C, 24 h of nicotine exposure. D, Protein levels of CHOP at 6 h and (E) 24 h of nicotine exposure. All arbitrary values were expressed as means normalized to Amido Black ± SEM. All experiments were performed in quadruplicates (n = 4). Significant differences between treatment groups as determined by 1-way ANOVA (1WA) indicated by ** (P < .01) or *** (P < .001). Different letters represent means that are significantly different from one another according to Tukey’s posttest (P < .05). Nonsignificant differences (P > .05) indicated by n.s.
FIG. 4.
FIG. 4.
The effect of nicotine exposure (0.1–100 μM) on BiP and PDI after 6 and 24 h in Rcho-1 TG cells. A, Specific targeted protein bands as detected by respective antibodies via Western blot. B, Protein levels of BiP at 6 h and C, 24 h of nicotine exposure. D, Protein levels of PDI at 6 h and E, 24 h of nicotine exposure. All arbitrary values were expressed as means normalized to Amido Black ± SEM. All experiments were performed in quadruplicates (n = 4). Non-significant differences (P > .05) indicated by n.s.
FIG. 5.
FIG. 5.
Pretreatment with MH (10 μM) blocked nicotine-induced PERK phosphorylation after 6 h in Rcho-1 TG cells. A, Specific targeted protein bands as detected by respective antibodies via Western blot. B, Protein levels of the ratio of P-PERK[Thr981]: PERK at 6 h. All arbitrary values were expressed as means normalized to Amido Black ± SEM. All experiments were performed in quadruplicates (n = 4). Significant differences between treatment groups as determined by 1-way ANOVA (1WA) indicated by * ( P < .05). Different letters represent means that are significantly different from one another according to Tukey’s posttest (P < .05).
FIG. 6.
FIG. 6.
The effect of nicotine exposure (1–10 μM) on markers of placental TG cell differentiation and function after 24 h in Rcho-1 TG cells. mRNA levels of A, Pl-1, B, Pgf, C, Hsd11b1, and D, Hsd11b2. All arbitrary values were expressed as means normalized to the geometric mean of β-Actin and Gapdh ± SEM. All experiments were performed in quadruplicates (n = 4). All experiments were performed in quadruplicates (n = 4). Significant differences between treatment groups as determined by 1-way ANOVA (1WA) indicated by * (P < .05). Different letters represent means that are significantly different from one another according to Tukey’s posttest (P < .05). Non-significant differences (P > .05) indicated by n.s.
FIG. 7.
FIG. 7.
Pretreatment with TUDCA (TUD; 100 μM) prevented the effects of nicotine on PERK phosphorylation and Hsd11b1 expression after 6 h in Rcho-1 TG cells. A, Specific targeted protein bands as detected by respective antibodies via Western blot. B, Protein levels of the ratio of P-PERK[Thr981]: PERK at 6 h. All arbitrary values were expressed as means normalized to Amido Black ± SEM. All experiments were performed in quadruplicates (n = 4). Significant differences between treatment groups determined by 1-way ANOVA (1WA) indicated by * (P < .05) or *** (P < .001). Different letters represent means that are significantly different from one another according to Tukey’s posttest ( P < .05).

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