TCDD induces the hypoxia-inducible factor (HIF)-1α regulatory pathway in human trophoblastic JAR cells

Abstract

The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α) stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS) and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or N-acetylcysteine (a ROS scavenger). The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ), PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

Figure 1

Dose- and time-dependent effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on hypoxia-inducible factor-1 alpha (HIF-1α) stabilization in human trophoblastic cells. Western blots of a representative experiment were shown. (A) Human trophoblastic cells were treated with various doses of TCDD (0.2, 0.6, 2, 6, and 10 nM) for 1 h and immunodetected with an HIF-1α specific antibody; (B) Cells were incubated with 2 nM TCDD for various time periods (0.5–8 h); (C) Cells were pretreated with α-naphthoflavone (α-NF) (Aryl hydrocarbon receptor (AhR) antagonist) for 24 h. TCDD-induced HIF-1α stabilization was attenuated by pretreatment of the cells with α-NF (1, 5, and 10 μM). The mean densitometry data from independent experiments were normalized to the results obtained for cells in the absence of TCDD (as control group). DMSO (0.1% v/v) alone was used as the solvent control. The plots are presented as the mean ± standard deviation (SD) (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the control group. ## p < 0.01 compared with the 2 nM TCDD-treated group.

Figure 2

TCDD-mediated generation of reactive oxygen species (ROS) and nitric oxide (NO) in trophoblastic cells. (A,C) Cells were treated with the various doses of TCDD (0.2, 0.6, 2, 6, and 10 nM) with or without α-NF pretreatment (the AhR antagonist); (B,D) Cells were treated with 2 nM TCDD for different time periods; (A,B) ROS were measured through 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (H₂DCFDA) staining followed by flow cytometry; (C,D) NO production was detected using the Griess Reagent, as described in Methods; (E) The expression level of inducible NOS (iNOS) protein in the TCDD-treated cells with or without α-NF pretreatment was detected by western blotting and quantified by densitometry; (F) Pretreatment with α-NF reduced TCDD-induced ROS generation, NO production, and iNOS expression. The plots are presented as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the control group. ## p < 0.01 compared with the 2 nM TCDD-treated group.

Figure 3

ROS signaling and phosphoinositide-3-kinase (PI3K) pathway are involved in TCDD-induced HIF-1α stabilization and Akt phosphorylation. (A) Cells were pre-treated with wortmannin (PI3K inhibitor) or PD98059 (2'-amino-3'-methoxyflavone, the mitogen-activated protein kinase kinase (MEK) inhibitor) for 30 min and then incubated with 2 nM TCDD for 1 h; (B) Cells were pre-treated with N-acetylcysteine (NAC, ROS scavenger) or N^ω-nitro-l-arginine methyl ester hydrochloride (NAME, NO scavenger) for 30 min and then incubated with 2 nM TCDD for 1 h. The protein levels of HIF-1α, phospho-Akt (p-Akt), and total Akt (T-Akt) were immunodetected by western blotting; (C–F) The mean densitometry data from independent experiments were normalized to the results obtained for cells in the absence of TCDD (as control group). The plots are presented as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the control group. # p < 0.05; ## p < 0.01 compared with the 2 nM TCDD-treated group.

Figure 4

TCDD induced gene expression during cell invasion and vascularization. (A) Cells were treated with various concentrations of TCDD (0, 0.2, 0.6, 2, and 6 nM) for 24 h; (B) Cells were treated with 2 nM TCDD for different periods of time (0, 2, 4, 6, 12, and 24 h). The invading trophoblastic cells were collected and counted using a Matrigel invasion assay. Enhanced cell invasion occurred in a dose- and time-dependent manner; (C,D) Increased mRNA expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were revealed by a semi-quantitative RT-PCR analysis; (D) The increased active forms of MMP-9 and MMP-2 were detected in the cells treated with TCDD for 24 h by gelatin zymography; (E) Electrophoretogram of the RT-PCR products of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) amplified from TCDD-treated trophoblast cells; (F) Increased VEGF and PlGF mRNA levels were revealed by real-time quantitative PCR. The plots are the mean ± S.D. (n = 3). * p < 0.05 compared with each control group; ** p < 0.01 compared with each control group.

Figure 5

Peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator-1α (PGC-1α) are targets of TCDD. (A) The expression profiles of HIF-1α, uncoupling protein 2 (UCP2), PPARγ, PGC-1α, and mitochondrial transcription factor (TFAM) were assessed by western blotting; (B) The protein levels were quantified by densitometry. Reduced expression of PPARγ, PGC1α, TFAM, and UCP2 was found in the TCDD-treated trophoblastic cells. The plots are the mean ± S.D. (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 compared with each control group.

Figure 6

Schematic diagram of the TCDD induced HIF-1α molecular signaling. TCDD induced HIF-1α stabilization via the AhR or ROS-dependent activation of the PI3K/Akt pathway. A significant increase in invasion, MMP9 activity, and VEGF and PlGF gene expression was found in the TCDD-treated cells. Furthermore, down regulation of metabolic regulators PPARγ and PGC-1α was involved in the TCDD-induced insults. TFAM and UCP2, downstream targets of PGC1α, were concurrently suppressed and may lead to mitochondrial dysfunction. Taken together, our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity.