Expression, Localization, and Activity of the Aryl Hydrocarbon Receptor in the Human Placenta

Abstract

The human placenta is an organ between the blood of the mother and the fetus, which is essential for fetal development. It also plays a role as a selective barrier against environmental pollutants that may bypass epithelial barriers and reach the placenta, with implications for the outcome of pregnancy. The aryl hydrocarbon receptor (AhR) is one of the most important environmental-sensor transcription factors and mediates the metabolism of a wide variety of xenobiotics. Nevertheless, the identification of dietary and endogenous ligands of AhR suggest that it may also fulfil physiological functions with which pollutants may interfere. Placental AhR expression and activity is largely unknown. We established the cartography of AhR expression at transcript and protein levels, its cellular distribution, and its transcriptional activity toward the expression of its main target genes. We studied the profile of AhR expression and activity during different pregnancy periods, during trophoblasts differentiation in vitro, and in a trophoblast cell line. Using diverse methods, such as cell fractionation and immunofluorescence microscopy, we found a constitutive nuclear localization of AhR in every placental model, in the absence of any voluntarily-added exogenous activator. Our data suggest an intrinsic activation of AhR due to the presence of endogenous placental ligands.

Keywords: ARNT; AhR; CYP1A1; CYP1B1; benzo-a-pyrene; ontogeny; placenta; trophoblast.

Figure 1

Placental expression of aryl hydrocarbon receptor (AhR) and relevant biomarkers during pregnancy. Total mRNAs were extracted from chorionic villi of eight placentae at 8–9 weeks of amenorrhea (WA), at 12–14 WA and at 37–39 WA (term). (A) Levels of βhCG, hPL, PLGF, GH2, CYP11A1, and CYP19 were determined by RT-qPCR and normalized to the geometric mean of HPRT, RPL13, and 18S transcripts, and reported to CK7 (KRT7). The means (long horizontal lines) and standard deviations (short horizontal lines) of eight independent experiments are shown. ** compared to 8–9 WA p < 0.01; ^## compared to 12–14 WA p < 0.01; ^### compared to 12–14 WA p < 0.001; **** compared to 8–9 WA p < 0.0001; ^#### compared to 12–14 WA p < 0.0001. Ct is the raw value of the gene of interest without normalization to reference genes and to KRT7. (B) Levels of AhR, AhRR, and ARNT were determined by RT-qPCR and normalized to the geometric mean of HPRT, RPL13, and 18S transcripts, and reported to CK7 (KRT7). The means (long horizontal lines) and standard deviations (short horizontal lines) of eight independent experiments are shown. ** compared to 8–9 WA p < 0.01; ^## compared to 12–14 WA p < 0.01; *** compared to 8–9 WA p < 0.001; ^### compared to 12–14 WA p < 0.001; **** compared to 8–9 WA p < 0.0001; ^#### compared to 12–14 WA p < 0.0001. Ct is the raw value of interest gene without normalization to reference genes and to KRT7. (C) Total protein were analyzed by immunoblotting using AhR antibody alone or pre-incubated with its blocking peptide, AhR nuclear translocator (ARNT) antibody, and β-Actin, the last used as a loading control. The graph represents the total amount of AhR protein (2 bands) relative to β-Actin levels determined by quantification of immunoblot analysis using the Odyssey System Imager. The means (long horizontal lines) and standard deviations (short horizontal lines) of four distinct placentae per stage of pregnancy are shown (* p < 0.05). (D) Levels of CYP1A1, CYP1B1, and CYP1A2 were determined by RT-qPCR as above. The means (long horizontal lines) and standard deviations (short horizontal lines) of eight independent experiments are shown. Ct is the raw value of interest gene without normalization to reference genes and to KRT7. * compared to 8–9 WA p < 0.05; ^# compared to 12–14 WA p < 0.05; ** compared to 8–9 WA p < 0.01.

Figure 2

AhR localization in chorionic villi of placenta at different periods of pregnancy. (A) Chorionic villi from placentae for different periods of pregnancy (as indicated) were fixed in 4% PFA, included in agarose, and permeabilized with 0.5% Triton. Images are of confocal microscopy after immunostaining with anti-AhR antibody alone (HPA029723) (green, upper images) or anti-AhR pre-incubated with a blocking peptide (lowest image), of anti-cytokeratin 7 antibody (red, to underline the trophoblasts) and DAPI (blue, for nuclei). The overlay images are shown at right. Images are representative of four independent experiments. (B) Immunoblotting was carried out using anti-AhR antibody after cell fractionation of freshly harvested term chorionic villi to separate nuclear and cytosolic extracts. α-Tubulin and Lamin A/C are nuclear and cytosolic markers, respectively. N: Nucleus, C: Cytosol. The bar graph represents the total amount of AhR protein (two bands) normalized as following: Nuclear AhR level was normalized by Lamin A/C signal then nuclear contamination by cytosolic fraction (α-Tubulin/Lamin A/C ratio) was subtracted; cytosolic AhR level was normalized by α-Tubulin level then cytosolic contamination by nuclear fraction (Lamin A/C/ α-Tubulin ratio) was subtracted. Protein levels were determined by quantification of immunoblot analysis using the Odyssey System Imager. The means (long horizontal lines) and standard deviations (short horizontal lines) of three distinct villi are shown (*** p < 0.001).

Figure 2

AhR localization in chorionic villi of placenta at different periods of pregnancy. (A) Chorionic villi from placentae for different periods of pregnancy (as indicated) were fixed in 4% PFA, included in agarose, and permeabilized with 0.5% Triton. Images are of confocal microscopy after immunostaining with anti-AhR antibody alone (HPA029723) (green, upper images) or anti-AhR pre-incubated with a blocking peptide (lowest image), of anti-cytokeratin 7 antibody (red, to underline the trophoblasts) and DAPI (blue, for nuclei). The overlay images are shown at right. Images are representative of four independent experiments. (B) Immunoblotting was carried out using anti-AhR antibody after cell fractionation of freshly harvested term chorionic villi to separate nuclear and cytosolic extracts. α-Tubulin and Lamin A/C are nuclear and cytosolic markers, respectively. N: Nucleus, C: Cytosol. The bar graph represents the total amount of AhR protein (two bands) normalized as following: Nuclear AhR level was normalized by Lamin A/C signal then nuclear contamination by cytosolic fraction (α-Tubulin/Lamin A/C ratio) was subtracted; cytosolic AhR level was normalized by α-Tubulin level then cytosolic contamination by nuclear fraction (Lamin A/C/ α-Tubulin ratio) was subtracted. Protein levels were determined by quantification of immunoblot analysis using the Odyssey System Imager. The means (long horizontal lines) and standard deviations (short horizontal lines) of three distinct villi are shown (*** p < 0.001).

Figure 3

Indoleamine 2,3-dioxygenase 1 enzyme (IDO1) protein expression in placenta from different periods of pregnancy. Total protein extracts from placenta at different stages of pregnancy, as indicated, were analyzed by immunoblotting using anti-IDO1 and anti-TDO2 antibodies. β-Actin was used as a loading control. Quantification of IDO1 immunoblots relative to β-Actin was determined using the Odyssey System Imager and is shown in the bar scale graph. * compared to 8–9 WA, p < 0.05, n = 4 (lower panel).

Figure 4

AhR expression and immunolocalization during CT differentiation into ST. (A) Total mRNA was extracted from freshly isolated trophoblasts before culture (VCT₀) and after 72 h of culture when cells are differentiated into ST. The levels of AhR mRNA were determined by quantitative RT-qPCR. Data were normalized to HPRT, RPL0, SDHA, and 18S mRNA levels. The means (long horizontal lines) and standard deviations (short horizontal lines) of at least n = 10 independent experiments are presented. (B) Western blot with protein extracts from purified trophoblast before culture (VCT₀), after 24 h of culture (VCT), and after 72 h culture (ST). Membranes were immunoblotted with anti-AhR (WH0000196-M2 antibody) and β-Actin antibodies (loading control). Blot quantifications are shown in the bar graphs representing total AhR level (left panel) or upper 130-band (right panel) relative to β-Actin. * compared to VCT₀, p < 0.05; ** compared to VCT₀, p < 0.01. (C) Trophoblasts after 24 h of culture (VCT) and 72 h (ST) were fixed in 4% PFA and permeabilized with methanol. Images are of epifluorescence microscopy after immunostaining AhR (green), E-cadherin (membrane protein, in red), and nuclei (DAPI, blue). A control with non-specific IgG is shown in the lower panel and overlays in the right panel. (D) Freshly isolated cytotrophoblast before culture (VCT₀) and after 72 h of culture (ST) were subjected to RT-qPCR after mRNA extraction. Levels of CYP1A1, CYP1A2, and CYP1B1 are presented as the mean ± standard deviation of at least six independent experiments and normalized to HPRT, RPL0, SDHA, and 18S mRNA levels. ** compared to VCT₀, p < 0.05; **** compared to VCT₀, p < 0.0001.

Figure 5

AhR expression, localization, and activity in the BeWo cell line. (A) Total mRNA were extracted from BeWo cells at various cell densities: 24 h hours after seeding cells reach about 50% of confluence; 48 h after seeding, they reached about 70% confluence, and about 90% 72 h after seeding. Levels of AhR, AhRR, and ARNT were determined by RT-qPCR and normalized to the geometric mean of HPRT, RPL13, and Ubiquitin C transcripts. The means (long horizontal lines) and standard deviations (short horizontal lines) of four independent experiments are shown. ** compared to 50% confluence p < 0.01; ^## compared to 70% p < 0.001; **** compared to 50% confluence p < 0.0001. Ct is the raw value of gene without normalization to reference genes (B) Total proteins were analyzed by immunoblotting using AhR antibody alone or pre-incubated with its blocking peptide, using ARNT and β-Actin antibodies, used as a loading control (left panel). The graph represents the amount of total AhR protein relative to β-Actin level determined by quantification of the immunoblot using the Odyssey System Imager (right panel). The means (long horizontal lines) and standard deviations (short horizontal lines) of three independent experiments are shown. (C) BeWo cells were fixed in 4% PFA and permeabilized with 0.1% Triton. Images are of epifluorescence microscopy after staining of AhR alone (green, upper images), AhR pre-incubated with a blocking peptide (middle images), or IgG (lower images) and DAPI (blue, for nuclei). The overlay images are shown at right. Images are relative to five independent experiments. (D) Immunoblotting was carried out using anti-AhR antibody after cell fractionation of BeWo cells to separate the nuclear and cytosolic extracts. α-Tubulin and Lamin A/C are nuclear and cytosolic markers, respectively. N: Nucleus, C: Cytosol. (E) Levels of CYP1A1, CYP1B1, and CYP1A2 were determined and normalized to the geometric mean of HPRT, RPL13, and Ubiquitin C transcripts. The means (long horizontal lines) and standard deviations (short horizontal lines) of at least three independent experiments are shown. ** compared to 50% confluence p < 0.01; **** compared to 50% confluence p < 0.0001. Ct is the raw value of interest gene without normalization to reference genes.

Figure 5

AhR expression, localization, and activity in the BeWo cell line. (A) Total mRNA were extracted from BeWo cells at various cell densities: 24 h hours after seeding cells reach about 50% of confluence; 48 h after seeding, they reached about 70% confluence, and about 90% 72 h after seeding. Levels of AhR, AhRR, and ARNT were determined by RT-qPCR and normalized to the geometric mean of HPRT, RPL13, and Ubiquitin C transcripts. The means (long horizontal lines) and standard deviations (short horizontal lines) of four independent experiments are shown. ** compared to 50% confluence p < 0.01; ^## compared to 70% p < 0.001; **** compared to 50% confluence p < 0.0001. Ct is the raw value of gene without normalization to reference genes (B) Total proteins were analyzed by immunoblotting using AhR antibody alone or pre-incubated with its blocking peptide, using ARNT and β-Actin antibodies, used as a loading control (left panel). The graph represents the amount of total AhR protein relative to β-Actin level determined by quantification of the immunoblot using the Odyssey System Imager (right panel). The means (long horizontal lines) and standard deviations (short horizontal lines) of three independent experiments are shown. (C) BeWo cells were fixed in 4% PFA and permeabilized with 0.1% Triton. Images are of epifluorescence microscopy after staining of AhR alone (green, upper images), AhR pre-incubated with a blocking peptide (middle images), or IgG (lower images) and DAPI (blue, for nuclei). The overlay images are shown at right. Images are relative to five independent experiments. (D) Immunoblotting was carried out using anti-AhR antibody after cell fractionation of BeWo cells to separate the nuclear and cytosolic extracts. α-Tubulin and Lamin A/C are nuclear and cytosolic markers, respectively. N: Nucleus, C: Cytosol. (E) Levels of CYP1A1, CYP1B1, and CYP1A2 were determined and normalized to the geometric mean of HPRT, RPL13, and Ubiquitin C transcripts. The means (long horizontal lines) and standard deviations (short horizontal lines) of at least three independent experiments are shown. ** compared to 50% confluence p < 0.01; **** compared to 50% confluence p < 0.0001. Ct is the raw value of interest gene without normalization to reference genes.

Figure 6

AhR localization in BeWo after incubation with benzo-(a)-pyrene. BeWo cells (at 70% confluence) were either used as control (solvent DMSO) or incubated with 1 µM B(a)P for the indicated times, in the presence or absence of a pre-incubation (1h before B(a)P) with 3 µM CH223191, an inhibitor of AhR, as indicated. (A) BeWo cells were fixed in 4% PFA and permeabilized with methanol. Images are of epifluorescence microscopy (400× magnification) after staining AhR with WH0000196-M2 antibody (green) and nuclei (DAPI). Overlays are in the right panel. (B) Cells were fractionated into nuclear (N) and cytosolic (C) fractions and subjected to Western blot. Immunoblotting was carried out using AhR (WH0000196-M2) antibody, and fractions purity was assessed with α-Tubulin (cytosolic) and Lamin A/C (nuclear).

Figure 7

Impact of benzo-(a)-pyrene on AhR transcriptional activity. (A) Total RNA extracted from control BeWo cells (Co-DMSO) or incubated with 1 µM of B(a)P for 24 h, were analyzed by RT-qPCR using primers for AhR, ARNT, and AhRR. Results are normalized to three reference genes (HPRT, RPL13, and Ubiquitin C) and normalized to CK7 (KRT7) and are presented as fold change. The means (long horizontal lines) and standard deviations (short horizontal lines) of three different experiments are shown. (B) BeWo cells (control (Co-DMSO) or cells incubated with 1 µM B(a)P for 24 h, in the presence or absence of 3 µM CH223191, a specific AhR inhibitor (1 h before B(a)P)), were subjected to RT-qPCR after total RNA extraction. Levels of CYP1A1, CYP1B1, and CYP1A2 are presented as fold change, after normalization as above. * compared to Co-DMSO, ^# compared to B(a)P; *p < 0.05; ** or ^## p < 0.01; *** p < 0.001; **** or ^#### p < 0.0001 (n = 5). (C) Total protein extracts from chorionic villi (explants) of control term placenta (Co-DMSO) or incubated with 1 µM B(a)P for 24 and 48 h, respectively, were subjected to Western blotting. Immunoblotting was carried out with anti-AhR, anti-IDO1, and anti-Vinculin (as loading control). The bar graph represents the total amount of AhR protein (two bands) or IDO1 relative to Vinculin level determined by quantification of immunoblots using the Odyssey System Imager. The means (long horizontal lines) and standard deviations (short horizontal lines) of three different placentae at term are shown. * compared to DMSO, p < 0.05.

Figure 8

Summary of the results on AhR and relevant biomarkers’ changes during pregnancy. First trimester villi (on the left) evolve into term villi (on the right). Changes in placental biomarkers between the first trimester and term are found in the orange box, and changes in AhR, its partner ARNT, its repressor AhRR, phase I and II enzymes, and IDO1 between first trimester and term are found in the higher blue box. Changes in AhR and phase I enzyme during VCT differentiation into ST are found in the lower blue box. On the right side, changes in AhR, ARNT, AhRR, phase I enzymes, and IDO1 after incubation of BeWo cells or villi explants with 1 µM BaP are showed. Nuclear localization of AhR in the different cell types is visualized with a green bullet. Increase in a marker is represented by ↗, decrease by ↘, and no change by ≈.