Alpha-1 Antitrypsin-Induced Endoplasmic Reticulum Stress Promotes Invasion by Extravillous Trophoblasts

Abstract

Alpha-1 antitrypsin (A1AT) is a glycoprotein that has been shown to protect tissues from proteolytic damage under various inflammatory conditions. Several studies show that A1AT may be associated with pre-eclampsia. However, the role of A1AT expression in placental physiology is not fully understood. In the present study, we aim to characterize the expression and function of placental A1AT. A1AT knockdown is found to reduce the expression of the serine protease HTRA1 in a trophoblast cell line. In addition, A1AT overexpression (A1AT-OE) increases the expression of HTRA1, IL6, CXCL8, and several markers of endoplasmic reticulum (ER) stress. Treatment with tunicamycin or thapsigargin, which induces ER stress, increases HTRA1 expression. Furthermore, immunohistochemistry reveals that HTRA1 is expressed in trophoblasts and the endometrial decidual cells of human placentas. An invasion assay shows that A1AT and HTRA1 stimulate cell invasion, but treatment with the ER stress inhibitors reduces the expression of HTRA1 and ER stress markers and prevents cell invasion in A1AT-OE trophoblasts. These results suggest that endogenous A1AT regulates inflammatory cytokine expression and HTRA1-induced trophoblast invasion via the induction of ER stress. It is concluded that an imbalance in the functional link between A1AT and ER stress at the maternal-fetal interface might cause abnormal placental development.

Keywords: alpha-1 antitrypsin; cell invasion; endoplasmic reticulum stress; extravillous trophoblast; high-temperature requirement A serine peptidase 1.

Figure 1

Alpha-1 antitrypsin (A1AT) regulates the expression of HTRA1 in an extravillous trophoblast cell line. (a) RNA was extracted from human extravillous trophoblast HTR8/SVneo cells that had been transfected with non-targeting control siRNA (Ctrl, 50 nM) or A1AT siRNA (A1AT knockdown (A1AT-KD), 50 nM) 24 h previously. The effect on A1AT, HTRAs, IL6, and CXCL8 mRNA expression was determined by qPCR (n = 3 per treatment). GAPDH mRNA was used as the reference gene. The values are the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01. (b) mRNA expression of A1AT, HTRAs, IL6, and CXCL8 in HTR8/SVneo cells that had been transfected with an empty vector (Ctrl) or an A1AT expression vector (A1AT overexpression (A1AT-OE)). GAPDH mRNA was used as the reference gene. The values are the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01. (c) Lysates prepared from HTR8/SVneo cells treated as above were subject to immunoblotting. GAPDH served as the loading control. The values are the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01. (d) A1AT-KD and A1AT-OE HTR8/SVneo cells were incubated in Matrigel-coated transwells for 48 h. Representative images of the cells that passed through the transmembranes are shown in the panels on the left. The numbers of cells that passed through the transmembranes were counted and are presented as the means ± SEMs of the three independent experiments. * p < 0.05, ** p < 0.01. (e) A1AT-KD or A1AT-OE HTR8/SVneo cells were cultured for 48 h and the numbers of cells were determined using a WST-8 assay. ** p < 0.01. (f) The expression of MMP2 and MMP9 in A1AT-OE HTR8/SVneo cells was measured. GAPDH was used as the reference gene. The values are the means ± SEMs from three independent experiments.

Figure 2

HTRA1 regulates invasion by extravillous trophoblast cells. (a) Tissue sections of human placenta were stained with anti-HTRA1, HLA-G (positive control for extravillous trophoblast), or normal rabbit IgG (negative control). (b,c) Extravillous trophoblast HTR8/SVneo cells were transfected with two different HTRA1 siRNAs (HTRA1-KD1 and -KD2, each 50 nM) and incubated for 24 h. (b) The mRNA expression of HTRAs and A1AT was determined (n = 3). GAPDH was used as the reference gene. The values are the means ± SEMs. ** p < 0.01. (c) Lysates prepared from HTR8/SVneo cells that had been transfected with HTRA1-KD1 or -KD2 were subject to immunoblotting. GAPDH served as a loading control. (d) HTR8/SVneo cells that had been transfected with HTRA1-KD1 or -KD2 were incubated in Matrigel-coated transwells for 48 h. Representative images of the cells that passed through the transmembranes are shown in panels on the left. The numbers of cells that passed through the transmembranes were counted and are presented as the means ± SEMs of three independent experiments. ** p < 0.01. (e) HTR8/SVneo cells that had been transfected with HTRA1-KD1 or -KD2 were cultured for 48 h, and then the numbers of cells were determined using a WST-8 assay. ** p < 0.01. (f) The mRNA expression of MMP2 and MMP9 in HTR8/SVneo cells that had been transfected with HTRA1-KD1 or -KD2 was measured. GAPDH was used as the reference gene. The values are the means ± SEMs of the three independent experiments.

Figure 3

A1AT-induced HTRA1 expression and cell invasion are regulated by unfolded protein response (UPR) signaling. (a) The mRNA expression of endoplasmic reticulum (ER) stress markers in HTR8/SVneo cells that had been transfected with A1AT-OE was measured. GAPDH was used as the reference gene. The values are the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01. (b) The protein levels of ER stress markers were measured by immunoblotting lysates from A1AT-OE HTR8/SVneo cells. GAPDH served as a loading control. (c) HTR8/SVneo cells were treated with tunicamycin (5 μM) or thapsigargin (100 nM) for 48 h, and then the mRNA expression of A1AT, HTRA1, and ER stress markers was measured using qPCR. GAPDH was used as the reference gene. The values are the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01. (d) A1AT-OE HTR8/SVneo cells were treated with GSK2606414 (GSK, 250 nM), Kira6 (1 μM), or AEBSF (300 μM) for 24 h, and then the mRNA expression of HTRAs was measured. GAPDH was used as the reference gene. The values are the means ± SEMs of three independent experiments. ** p < 0.01. (e) A1AT-OE HTR8/SVneo cells were treated with GSK2606414 (GSK, 250 nM), Kira6 (1 μM), or AEBSF (300 μM) in Matrigel-coated transwells for 24 h. Representative images of the cells that passed through the transmembranes are shown in the panels on the left. The numbers of cells that passed through the transmembranes were counted and are presented as the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 4

Diagram illustrating the proposed mechanism linking A1AT and invasion by extravillous trophoblast cells. Abnormal A1AT expression induces ER stress, which leads to the upregulation of HTRA1 and results in invasion by trophoblast cells. ER stress increases A1AT expression. In addition, A1AT stimulates IL6 and CXCL8 expression.