Hydroquinone impairs trophoblast migration and invasion via AHR-twist-IFITM1 axis

Abstract

Introduction: Embryo implantation is a tightly regulated process, critical for a successful pregnancy. After attachment of the blastocyst to the surface epithelium of the endometrium trophoblast migrate from the trophectoderm and invade into the stromal component of endometrium. Alterations on either process will lead to implantation failure or miscarriage. Volatile organic compounds (VOCs) such as benzene induce pregnancy complications, including preterm birth and miscarriages. The mechanism of this effect is unknown. The objective of this study was to elucidate the impact of benzene metabolite, Hydroquinone, on trophoblast function. We tested the hypothesis that Hydroquinone activates the Aryl hydrocarbon receptor (AhR) pathway modulating trophoblast migration and invasion.

Methods: First-trimester trophoblast cells (Sw.71) were treated with hydroquinone (6 and 25 μM). Trophoblast migration and invasion was evaluated using a 3D invasion/migration model. Gene expression was quantified by q-PCR and Western blot analysis.

Results: Hydroquinone impairs trophoblast migration and invasion. This loss is associated with the activation of the AhR pathway which reduced the expression of Twist1and IFITM1. IFITM1 overexpression can rescue impaired trophoblast migration.

Discussion: Our study highlights that hydroquinone treatment induces the activation of the AhR pathway in trophoblast cells, which impairs trophoblast invasion and migration. We postulate that activation of the AhR pathway in trophoblast suppress Twist1 and a subsequent IFITM1. Thus, the AhR-Twist1-IFITM1 axis represent a critical pathway involved in the regulation of trophoblast migration and it is sensitive to benzene exposure. These findings provide crucial insights into the molecular mechanisms underlying pregnancy complications induced by air pollution.

Keywords: AhR; Benzene; IFITM1; Implantation; Trophoblast; Twist1.

Figure 1:. Hydroquinone Adversely Affects Trophoblast Migration

(A) Trophoblast migration of Sw.71 is reduced at both 6μM and 25μM HQ from 24h-72h. (B) Quantification of the diameter of migration at specified time points. Each point represents 12 spheroids. Representative images of 6 independent experiments. Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by one-way ANOVA. (C) Model of the computer quantification approach for trophoblast migration.

Figure 2:. Hydroquinone Impairs Trophoblast Invasion.

(A) Early invasion presents significantly fewer protruding cells from the main spheroids when Sw.71 spheroids are treated with 25μM HQ at both 24 and 48 hours. (B) Quantification of early invading cells at 24 and 48 hours. Representative images from 6 independent experiments. Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by unpaired t-test or one-way ANOVA.

Figure 3:. Long term effect of Hydroquinone treatment on Trophoblast Invasion:

(A) After treatment with 6μM and 25μM HQ for a period ranging from 4 to 10 days, the invasion of Sw.71 spheroids is markedly inhibited. (B) Quantification of trophoblast invasion. Each point represents 12 spheroids. Representative images from 6 independent experiments Results are represented as the mean ± SEM.*p<0.05

Figure 4:. Activation of Aryl Hydrocarbon Receptor Pathway by Hydroquinone

(A) Cyp1a1 mRNA was upregulated placenta samples from pregnant mice exposed to 5ppm benzene for 24hours a day from E0.5 to E12.5. ***p<0.01, (B) Upon treatment with 6μM and 25μM HQ for 24 hours, a significant increase in the mRNA of Cyp1a1, the gene indicative of AhR pathway activation, is observed in monolayer Sw.71. ****p<0.0001 by unpaired t-test or one-way ANOVA (C) AhR Expression in trophoblast cells. Sw.71 trophoblast cells showed positive fluorescence (green) when stained with a specific anti-AhR antibody. Strong signal is detected in the nuclei of the cells as shown by co-localization with positive DAPI signal. Representative images from 4 independent experiments in triplicates. (D) 24-hour treatment of monolayer Sw.71 with 6μM and 25μM HQ results in nuclear translocation of the AHR protein. (E) Quantification of nuclear and cytoplasmic protein fractions Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by unpaired t-test or one-way ANOVA.

Figure 5:. Twist1 Knockout Sw.71 Spheroids Exhibit Impaired Migration and Invasion Similar to Hydroquinone-Treated Sw.71 Spheroids

(A) Untreated Twist1 KO Sw.71 spheroids display similar migration impairment as that observed in 25μM and 6μM HQ-treated WT Sw.71 spheroids. Each point represents 12 spheroids. (B) Quantification of trophoblast invasion. Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by one-way ANOV.

Figure 6:. Hydroquinone Suppresses Twist1 Expression

(A and B) Twist1 mRNA was reduced in a time dependent manner following exposure to 25μM HQ. (C and D) Twist1 protein was significantly reduced from HQ treatment (25μM) after 24h and 48h (E) Blockade of the AhR pathway using SR1 rescues the reduction in Twist1 mRNA observed after HQ treatment (6μM and 25μM). Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by unpaired t-test one-way ANOVA.

Figure 7:. Association of Twist1 Knockout with Cell Movement Biological Processes

(A) Chord plot comparison of shared genes within the top 20 enriched biological processes between Twist1 KO Sw.71 monolayer and WT Sw.71 monolayer. (B) Network analysis of the gene ontology term “Negative Regulation of Cell Migration”

Figure 8:. Hydroquinone and Twist1 KO Associated with Reduced Ifitm1 Expression

(A) Placentas samples obtained from pregnant mice exposed to continuous 5ppm benzene showed significantly decreased levels of Ifitm1 mRNA expression. (B) ifitm1 mRNA expression in Sw.71 trophoblast decreases in a time-dependent manner following treatment with 25μM HQ. (C) 24-hour treatment of WT Sw.71 monolayer with 25μM HQ showed decreased IFITM1 protein expression. IFITM1 protein expression is not detectable inTwist1 KO Sw.71 trophoblast cells. (D) IFITM1 mRNA expression levels are significantly reduced in Twist1 KO Sw.71 monolayer compared to WT Sw.71 cells. (E) IFITM1 mRNA expression in Sw.71 trophoblast cells following treatment with 6 and 25μM HQ. Protein levels of IFITM1 are reduced in both the WT Sw.71 monolayer treated with 6μM and 25μM HQ for 24h and in the Twist1 KO Sw.71 monolayer. (E-F) IFITM1 mRNA expression levels as significant lower in Twist1 KO Sw.71 cells and not affected by HQ treatment. (G) The AhR receptor antagonist SR1(1μM) rescues HQ induced inhibition of Ifitm1 expression in trophoblast cells. 24-hour treatment with 6μM and 25μM HQ in the WT Sw.71 monolayer group. (H) Model of Twsit1 regulation of IFITM1. Twist1 regulates basal expression levels of IFITM1 through its interaction with IRF-9 [84] Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by unpaired t-test one-way ANOVA.

Figure 9:. Overexpression of IFITM1 Rescues Impaired Migration in Twist1 KO Sw.71 Spheroids

(A) Transfection of Twist1 KO Sw.71 spheroids with an IFITM1 overexpression plasmid for 48h rescues the observed migration impairment in this group. Representative images of 5 independent experiments in triplicates. (B) Quantification of the diameter of migration after Ifitm1 overexpression. Each point represents 12 spheroids. (C) Confirmation of IFITM1 overexpression in WT and Twist1 KO Sw.71. OE (Overexpression) by Western blot. Results are represented as the mean ± SEM.*p<0.05, **p<0.01, ****p<0.0001 by one-way ANOVA.

Figure 10:. Overall Proposed Mechanism:

Benzene, through its metabolite HQ, activates the AhR pathway, which inhibits Twist1 expression and its downstream migration regulator factor IFITM1 leading to reduce trophoblast migration capacity.