Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m6A

Abstract

Background: Several epidemiological investigations demonstrated that maternal arsenic (As) exposure elevated risk of fetal growth restriction (FGR), but the mechanism remains unclear.

Objectives: This study aimed to investigate the effects of gestational As exposure on placental and fetal development and its underlying mechanism.

Methods: Dams were exposed to 0.15, 1.5, and $15\mathrm{mg}/L$ ${\text{NaAsO}}_2$ throughout pregnancy via drinking water. Sizes of fetuses and placentas, placental histopathology, and glycogen content were measured. Placental RNA sequencing was conducted. Human trophoblasts were exposed to ${\text{NaAsO}}_2$ ($2\mu M$) to establish an in vitro model of As exposure. The mRNA stability and protein level of genes identified through RNA sequencing were measured. $N^6\text{-Methyladenosine}$ ($m^{6}A$) modification was detected by methylated RNA immunoprecipitation-quantitative real-time polymerase chain reason (qPCR). The binding ability of insulin-like growth factor 2 binding protein 2 to the gene of interest was detected by RNA-binding protein immunoprecipitation-qPCR. Intracellular S-adenosylmethionine (SAM) and methyltransferase activity were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and colorimetry, respectively. In vitro ${\mathrm{As}}^{+3}$ methyltransferase (As3MT) knockdown or SAM supplementation and in vivo folic acid (FA) supplementation were used to evaluate the protective effect. A case-control study verified the findings.

Results: Sizes of fetuses (exposed to 1.5 and $15\mathrm{mg}/L$ ${\text{NaAsO}}_2$) and placentas (exposed to $15\mathrm{mg}/L$ ${\text{NaAsO}}_2$) were lower in As-exposed mice. More ${\text{glycogen}}^{+}$ trophoblasts accumulated and the expression of markers of interstitial invasion was lower in the $15\mathrm{mg}/L$ ${\text{NaAsO}}_2\text{-exposed}$ mouse group in comparison with control. Placental RNA sequencing identified cysteine-rich angiogenic inducer 61 (Cyr61) as a candidate gene of interest. Mechanistically, mice and cells exposed to As had lower protein expression of CYR61, and this was attributed to a lower incidence of Cyr61 $m^{6}A$. Furthermore, cells exposed to As had lower methyltransferase activity, suggesting that this could be the mechanism by which Cyr61 $m^{6}A$ was affected. Depletion of intracellular SAM, a cofactor for $m^{6}A$ methyltransferase catalytic domain, partially contributed to As-induced methyltransferase activity reduction. Either As3MT knockdown or SAM supplementation attenuated As-induced Cyr61 $m^{6}A$ down-regulation. In mice, FA supplementation rescued As-induced defective trophoblastic invasion and FGR. In humans, a negative correlation between maternal urinary As and plasma CYR61 was observed in infants who were small for gestational age.

Discussion: Using in vitro and in vivo models, we found that intracellular SAM depletion-mediated Cyr61 $m^{6}A$ down-regulation partially contributed to As-induced defective trophoblastic invasion and FGR. https://doi.org/10.1289/EHP12207.

Figure 1.

Effects of As exposure on interstitial migration and invasion in mouse placentas and human placental trophoblasts. (A and B) All pregnant mice except controls were exposed to $15\mathrm{mg}/\mathrm{L}$ ${\text{NaAsO}}_2$ by drinking water throughout pregnancy. All pregnant mice were sacrificed on GD18. Placental E-cadherin, vimentin, N-cadherin, MMP2, and MMP9 were measured by immunoblotting. (A) E-cadherin, Vimentin and N-cadherin ($n=3$ mice/group, repeated three times); (B) MMP2 and MMP9 ($n=3$ mice/group, repeated three times). (C–G) HTR-8/SVneo cells were exposed to ${\text{NaAsO}}_2$ ($2\mathrm{\mu }\mathrm{M}$). (C) Cell migration was detected by wound-healing assay ($n=3$ samples/group, repeated 2 times). (D) Cell migration was detected by transwell migration assay ($n=3$ samples/group, repeated 2 times). Representative photomicrographs are shown. Scale bars, $200\mathrm{\mu }\mathrm{m}$. (E) Cell invasion was detected by transwell invasion assay ($n=3$ samples/group, repeated 2 times). Representative photomicrographs are show. Scale bars, $200\mathrm{\mu }\mathrm{m}$. (F and G) Occuldin, vimentin, N-cadherin, MMP2, and MMP9 proteins in HTR8/SVneo cells were measured by immunoblotting ($n=3$ samples/group, repeated three times). All data are expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. Unpaired two-tailed Student’s t-test was used for A–G. *$p<0.05$, **$p<0.01$. Note: As, arsenic; As-H, $15\mathrm{mg}/\mathrm{L}$ ${\text{NaAsO}}_2$; CTRL, control; GD, gestational day; MMP2 and MMP9, matrix metalloproteinases (MMP)2 and MMP9; SEM, standard error of the mean; $\mathrm{\mu }\mathrm{M}$, $\mathrm{\mu }\text{mol}/\mathrm{L}$.

Figure 2.

RNA-seq on placentas from mice exposed to ${\text{NaAsO}}_2$. (A–G) All pregnant mice except controls were exposed to ${\text{NaAsO}}_2$ ($15\mathrm{mg}/\mathrm{L}$) by drinking water throughout pregnancy. All pregnant mice were sacrificed on GD 18. (A) Volcano plot of differentially expressed genes in mouse placentas. A total of 743 genes (red triangle) were up-regulated, and 1,889 genes (green dot) were down-regulated by at least 1.5-fold with a significance level ($p<0.05$). (B) KEGG pathway analysis. (C) Top 100 genes were screened based on the p-value ranking of all differential genes and then we screened 40 candidate genes among them, with the average FPKM $>10$ of all samples as the filter condition. RNA-seq of mouse placentae from three dams were analyzed per group. (D) Validation of trophoblasts development-related differentially expressed genes by real-time RT-PCR ($n=6$ mice/group). (E) Fold differences in differentially expressed genes between RNA-seq and real-time RT-PCR. (F) Placental CYR61 was detected by immunoblotting ($n=3$ mice/group, repeated three times). (G) Representative pictures of CYR61-positive cells in labyrinth zone and junctional zone. Scale bars, $20\mathrm{\mu }\mathrm{m}$ ($n=6$ mice/group). (H–J) HTR-8/SVneo cells were exposed to ${\text{NaAsO}}_2$ ($2\mathrm{\mu }\mathrm{M}$). (H) Cyr61 mRNA was measured by real-time RT-PCR ($n=6$ samples/group). (I) CYR61 protein was detected by immunoblotting ($n=3$ samples/group, repeated three times). (J) CYR61-positive cells were stained with by immunofluorescence ($n=3$ samples/group). All data are expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. RNA-seq data are reported in an Excel sheet and uploaded to the National Center for Biotechnology Information (GSE222092). Unpaired two-tailed Student’s t-test was used for D–F, H, I. *$p<0.05$, **$p<0.01$. Note: As, arsenic; As-H, $15\mathrm{mg}/\mathrm{L}$ NaAsO₂; Ceacam 3/13, carcinoembryonic antigen-related cell adhesion molecule 3/13; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FPKM, fragments per kilobase per million; Not DEG, not differential gene expression; RNA-seq, RNA sequencing; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; Sparcl1, SPARC-like protein 1; $\text{TGF-}\mathrm{\beta }2$, transforming growth factor $\mathrm{\beta }2$; $\mathrm{\mu }\mathrm{M}$, $\mathrm{\mu }\text{mol}/\mathrm{L}$.

Figure 3.

The role of CYR61 in induced inhibition of migration and invasion in As-exposed human placental trophoblasts. HTR-8/SVneo cells were exposed to ${\text{NaAsO}}_2$ ($2\mathrm{\mu }\mathrm{M}$). (A–D) HTR-8/SVneo cells were transfected with CYR61 overexpression plasmid and then exposed to NaAsO₂ ($2\mathrm{\mu }\mathrm{M}$). (A) The efficiency of CYR61 overexpression was determined by real-time RT-PCR ($n=6$ samples/group). (B) Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ($n=2$ samples/group, repeated three times), and $\mathrm{\beta }\text{-actin}$ was used as a loading control. (C) Cell migration was determined by transwell migration assay ($n=3$ samples/group, repeated two times). (D) Cell invasion was measured by transwell invasion assay ($n=3$ samples/group, repeated 2 times). (E–H) HTR-8/SVneo cells were transfected with CYR61 knockdown plasmid and then exposed to NaAsO₂ ($2\mathrm{\mu }\mathrm{M}$). (E) The efficiency of CYR61 knockdown was determined by real-time RT-PCR ($n=6$ samples/group). (F) Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ($n=2$ samples/group, repeated three times); $\mathrm{\beta }\text{-actin}$ was used as a loading control. (G) Cell migration was determined by transwell migration assay ($n=3$ samples/group, repeated two times). (H) Cell invasion was measured by transwell invasion assay ($n=3$ samples/group, repeated 2 times). Scale bars, $200\mathrm{\mu }\mathrm{m}$. All data were expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. Unpaired two-tailed Student’s t-test was used for A and E. When ANOVA showed $p<0.05$, the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for B–D and F–H. *$p<0.05$, **$p<0.01$. Note: ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; CYR61 OE, CYR61 overexpression; shCYR61, CYR61 shRNA; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; $\mathrm{\mu }\mathrm{M}$, $\mathrm{\mu }\text{mol}/\mathrm{L}$.

Figure 4.

Effect of ${\mathrm{m}}^6\mathrm{A}$ modification on Cyr61 mRNA stability and CYR61 protein in As-exposed human placental trophoblasts. HTR-8/SVneo cells were exposed to ${\text{NaAsO}}_2$ ($2\mathrm{\mu }\mathrm{M}$). (A) HTR8/SVneo cells were treated with actinomycin D ($5\mathrm{\mu }\mathrm{g}/\mathrm{mL}$), followed by detection of Cyr61 mRNA at indicated times ($n=3$ samples/group, repeated two times). (B) The ${\mathrm{m}}^6\mathrm{A}$ content was measured by EpiQuik ${\mathrm{m}}^6\mathrm{A}$ methylation quantification kit ($n=5$ samples/group, repeated two times). (C) Schematic representation of the position of ${\mathrm{m}}^6\mathrm{A}$ motifs with CYR61 transcript. (D) The ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ Cyr61 levels were measured by MeRIP-qPCR ($n=3$ samples/group). (E) Schematic representation of the interaction between Cyr61 mRNA and IGF2BP1/2/3 proteins. (F) The interaction between Cyr61 mRNA and IGF2BP2 proteins was detected by RIP-qPCR ($n=3$ samples/group). (G) The expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases and demethylases was measured by real-time RT-PCR ($n=6$ samples/group). (H) The ${\mathrm{m}}^6\mathrm{A}$ methylase activity was measured by Epigenase ${\mathrm{m}}^6\mathrm{A}$ methylase activity/inhibition assay kit ($n=6$ samples/group). (I) A schematic model of the reversible ${\mathrm{m}}^6\mathrm{A}$ methylation. SAM is not only a methyl donor for ${\mathrm{m}}^6\mathrm{A}$ methylation but also a cofactor for catalytic domain of Mettl3. (J) Intracellular SAM content was measured by LC-MS/MS ($n=6$ samples/group). All data were expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. Unpaired two-tailed Student’s t-test was used for A, B, D, F, G, H, and J. *$p<0.05$, **$p<0.01$. Note: ActD, actinomycin D; Alkbh5, a-ketoglutarate-dependent dioxygenase alkB homolog 5; ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FTO, fat-mass obesity-associated protein; IGF2BP1/2/3, insulin-like growth factor 2 mRNA-binding protein one-half/3; LC-MS/MS, liquid chromatography–tandem mass spectrometry; ${\mathrm{m}}^6\mathrm{A}$, ${\mathrm{N}}^6\text{-methyladenosine}$; MeRIP-qPCR, methylated RNA immunoprecipitation–qPCR; Mettl3/14, methyltransferase-like 3/14; Rbm15/15b, RNA-binding motif protein 15/15b; RIP-qPCR, RNA-binding protein immunoprecipitation–qPCR; RT-PCR, reverse transcription polymerase chain reaction; SAH, s-adenosyl-l-homocysteine; SAM, s-adenosylmethionine; SEM, standard error of the mean; $\mathrm{\mu }\mathrm{M}$, $\mathrm{\mu }\text{mol}/\mathrm{L}$; WTAP, wilms tumor 1-associating protein.

Figure 5.

Effect of As3MT knockdown on Cyr61 ${\mathrm{m}}^6\mathrm{A}$ modification in As-exposed human placental trophoblasts. HTR8/SVneo cells were exposed to $2\mathrm{\mu }\mathrm{M}$ ${\text{NaAsO}}_2$ in presence or absence of As3MT-shRNA (A) The efficiency of As3MT knockdown was determined by real-time RT-PCR ($n=6$ samples/group). (B) Intracellular SAM content was measured by LC-MS/MS ($n=6$ samples/group). (C) The ${\mathrm{m}}^6\mathrm{A}$ methylase activity was measured by Epigenase ${\mathrm{m}}^6\mathrm{A}$ methylase activity/inhibition assay kit ($n=6$ samples/group). (D and E) HTR8/SVneo cells were treated with actinomycin D ($5\mathrm{\mu }\mathrm{g}/\mathrm{mL}$), followed by detection of Cyr61 mRNA at indicated times ($n=3$ samples/group, repeated two times). (F) CYR61, Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ($n=3$ samples/group). (G) Cell migration was determined by transwell migration assay ($n=3$ samples/group, repeated two times). (H) Cell invasion was measured by transwell invasion assay ($n=3$ samples/group, repeated two times). Scale bars, $200\mathrm{\mu }\mathrm{m}$. All data were expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. When ANOVA showed $p<0.05$, the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–H. Asterisks indicate comparison with the control group. Hashtags indicate comparison with the As group. *p and ^#$p<0.05$, **p and ^##$p<0.01$. Note: ActD, actinomycin D; ANOVA, analysis of variance; As, arsenic; As3MT, arsenite methyltransferase; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MMP2, matrix metalloproteinases 2; RT-PCR, reverse transcription polymerase chain reaction; SAM, s-adenosylmethionine; shAs3MT 1#, As3MT shRNA 1#; shAs3MT 2#, As3MT shRNA 2#; SEM, standard error of the mean; $\mathrm{\mu }\mathrm{M}$, $\mathrm{\mu }\text{mol}/\mathrm{L}$.

Figure 6.

Effect of SAM supplementation on induced inhibition of Cyr61 ${\mathrm{m}}^6\mathrm{A}$ modification in As-exposed human placental trophoblasts. HTR8/SVneo cells were incubated with ${\text{NaAsO}}_2$ ($2\mathrm{\mu }\mathrm{M}$) in presence or absence of SAM ($10\mathrm{\mu }\mathrm{M}$). (A) Intracellular SAM content was measured by LC-MS/MS ($n=6$ samples/group). (B) The ${\mathrm{m}}^6\mathrm{A}$ methylase activity was measured by Epigenase ${\mathrm{m}}^6\mathrm{A}$ methylase activity/inhibition assay kit ($n=6$ samples/group). (C) HTR8/SVneo cells were treated with actinomycin D ($5\mathrm{\mu }\mathrm{g}/\mathrm{mL}$), followed by detection of Cyr61 mRNA at indicated times ($n=3$ samples/group, repeated two times). (D and E) CYR61, Vimentin, N-cadherin, and MMP2 proteins were determined by immunoblotting ($n=3$ samples/group, repeated three times). (F) Cell migration was determined by transwell migration assay ($n=3$ samples/group, repeated two times). (G) Cell invasion was measured by transwell invasion assay ($n=6$ samples/group, repeated two times). Scale bars, $200\mathrm{\mu }\mathrm{m}$. All data were expressed as $\text{mean}\pm \text{SEM}.$ The numeric data are shown in Table S8. When ANOVA showed $p<0.05$, the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–G. Asterisks indicate comparison with the control group. Hashtags indicate comparison with the As group. *p and ^#$p<0.05$, **p and ^##$p<0.01$. Note: ANOVA, analysis of variance; As, arsenic; ActD, actinomycin D; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; MMP2, matrix metalloproteinases 2; RT-PCR, reverse transcription polymerase chain reaction; SAM, s-adenosylmethionine; SEM, standard error of the mean; $\mathrm{\mu }\mathrm{M}$, $\mathrm{\mu }\text{mol}/\mathrm{L}$.

Figure 7.

Effect of FA supplementation on placental development and fetal growth restriction in As-exposed mice. Pregnant mice were divided into four groups: CTRL, FA, As, and $\mathrm{As}+\text{FA}$ groups. In the As and $\mathrm{As}+\text{FA}$ groups, pregnant mice drank ultrapure water containing ${\text{NaAsO}}_2$ ($15\mathrm{mg}/\mathrm{L}$) throughout pregnancy. In FA and $\mathrm{As}+\text{FA}$ groups, pregnant mice were administered FA ($150\mathrm{\mu }\mathrm{g}/\mathrm{kg}$) by gavage daily throughout pregnancy. All pregnant mice were sacrificed on GD18. (A) Fetal weight ($n=8$ dams/group, 12–15 fetuses per dam). (B) Crown–rump length ($n=8$ dams/group, 12–15 fetuses per dam). (C) Placental weight ($n=8$ dams/group, 12–15 fetuses per dam). (D) Placental diameter ($n=8$ dams/group, 12–15 fetuses per dam). (E) Placental cross-section was stained with H&E. Scale bars, $500\mathrm{\mu }\mathrm{m}$ (left); $200\mathrm{\mu }\mathrm{m}$ (right) ($n=6$ mice/group). (F) The percentage of labyrinth zone area in the entire placenta area ($n=6$ mice/group). (G) The ratio of cross-sectional thickness of labyrinth zone to junctional zone ($n=6$ mice/group). (H) Placental SAM content was determined by LC-MS/MS ($n=6$ mice/group). (I) The ${\mathrm{m}}^6\mathrm{A}$ methylase activity was measured by Epigenase ${\mathrm{m}}^6\mathrm{A}$ methylase activity/inhibition assay kit ($n=6$ mice/group). (J) Placental CYR61 and MMP2 were measured by immunoblotting ($n=3$ mice/group, repeated two times). All data were expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. When ANOVA showed $p<0.05$, the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–D and F–J. *$p<0.05$, **$p<0.01$. Note: ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FA, folic acid; H&E, hematoxylin and eosin; JZ, junctional zone; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LZ, labyrinth zone; MMP2, matrix metalloproteinases 2; SAM, s-adenosylmethionine; SEM, standard error of the mean.

Figure 8.

Based on a case–control study: association among maternal urinary As concentration, plasma CYR61 content, and fetal growth restriction. (A) Maternal urinary As concentration in AGA and SGA infants was determined by hydride generation-atomic fluorescence spectrometry ($n=45$ human sample/group). (B) Maternal plasma CYR61 content in AGA and SGA infants was measured by ELISA ($n=45$ human sample/group). (C) The correlation between maternal urinary As concentration and plasma CYR61 content in AGA infants ($n=45$ human sample). (D) The correlation between maternal urinary As concentration and plasma CYR61 content in SGA infants ($n=45$ human sample). (E) Placental CYR61-positive cells in SGA infants and AGA infants were stained by immunohistochemistry ($n=18$ human sample/group). Representative histology images were showed. Blue arrows indicate CYR61-positive cells. Scale bars, $20\mathrm{\mu }\mathrm{m}$. (F) Placental vimentin, MMP2, and MMP9 were detected by immunoblotting ($n=18$ human sample/group). The data in A and B are expressed as $\text{mean}\pm \text{SD}$. The data in A–D are represented by dots indicating individual values. The data in E and F are expressed as $\text{mean}\pm \text{SEM}$. The numeric data are shown in Table S8. Two-tailed Student’s t-test was used for A, B, E, and F. The Spearman correlation was used for C and D. *$p<0.05$, **$p<0.01$. Note: AGA, appropriate for gestational age; As, arsenic; CYR61, cysteine-rich angiogenic inducer 61; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; SEM, standard error of the mean; SGA, small for gestational age.