Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells

Abstract

Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.

Fig. 1.

Upregulation of ACSL3 mRNA expression by synthetic LXR agonists in primary human trophoblasts and BeWo cells. A: Primary trophoblasts were incubated in triplicate with vehicle (white bars) or T0901317 (1 μM, black bars) for 24 h in three independent placenta isolations. B: BeWo cells were incubated with vehicle (white bars), 9-cis RA (1 μM, gray bars), T0901317 (1 μM, bars with small dots), both agonists (bars with big dots), GW3965 (1 μM, striped bars), or GW3965 and 9-cis RA together (black bars) for 48 h. ACSL3 and SREBP-1 expression were analyzed by qRT-PCR and normalized to TBP. Each experiment was performed in triplicate. Data shown represents the means of three independent experiments performed in triplicate (n = 3) ± SEM, relative to control. *P < 0.05 and **P < 0.01 relative to control. ACSL, long-chain acyl-CoA synthetase; BeWo, human placental choriocarcinoma; LXR, liver X receptor; qRT-PCR, quantitative reverse transcription-PCR; SREBP-1, sterol-regulatory element binding protein-1; TBP, TATA box binding protein.

Fig. 2.

Expression of ACSL3, 5, and 6 are increased with synthetic LXR agonist, but only ACSL3 is predominantly expressed in BeWo cells. A: BeWo cells were incubated with vehicle (white bars), 9-cis RA (1 μM, gray bars), GW3965 (1 μM, striped bars), or both agonists together (black bars) for 48 h. Total RNA was analyzed for gene expression of the five ACSL isoforms by qRT-PCR normalized to TBP. The results are presented as means of three independent experiments performed in triplicate (n = 3) ± SEM relative to control. *P < 0.05 and **P < 0.01 B: Basal expression of ACSL, FATP, and ACSBG family members in BeWo cells (white bars) and placenta (black bars) normalized to B2M. The results are represented as 2^{(-delta Ct)} for the means of three independent BeWo cell experiments performed in triplicate (n = 3) and for the means of six independent placenta isolations performed in triplicate (n = 6) ± SEM. Ct values are indicated for placental tissue. ACSBG, acyl-CoA synthetase bubblegum; ACSL, long-chain acyl-CoA synthetase; B2M, beta-2-microglobulin; BeWo, human placental choriocarcinoma; FATP, fatty acid transport protein; LXR, liver X receptor; qRT-PCR, quantitative reverse transcription-PCR; SREBP-1, sterol-regulatory element binding protein-1; TBP, TATA box binding protein.

Fig. 3.

ACSL3 is a direct LXR target gene. A: Alignment of the mouse and human LXRE elements in the ACSL3 promoters. B: BeWo cells were transiently transfected with ACSL3-(−2202/+200)-LUC reporter and cotransfected with pLR (internal control), pcDNA3-MCS, pcDNA3-hRXRα, and/or pcDNA3-hLXRα expression vectors as indicated. After transfection, cells were incubated for 24 h with medium containing vehicle (0.2% DMSO, white bars), 9-cisRA (1 μM, gray bars), GW3965 (1 μM, striped bars), or both agonists (black bars). The data is a representative experiment of two independent experiments performed in triplicate (n = 3) ± SEM. C: Transient transfection with ACSL3-(−2202/+200)-LUC, ACSL3-(−138/+200)-LUC, and ACSL3-(−2202/+200-LXRE-mut)-LUC reporters. The cells were cotransfected with pcDNA3-MCS (empty vector, white bars), pcDNA3-hRXRα (gray bars), or pcDNA3-hLXRα and pcDNA3-hRXRα (striped and black bars). After transfection, cells were incubated in medium containing vehicle (0.1% DMSO, white, gray, and striped bars) or GW3965 (1 μM, black bars). The data is a representative experiment of four independent experiments performed in triplicate (n = 3) ± SEM. D: EMSA of nuclear extracts (2 µg) isolated from COS-1 cells transfected with either pcDNA3-hRXRα or pcDNA3-hLXRα expression vectors individually form a strong specific complex with the ACSL3 LXRE only when these extracts are combined (lane 5). Identical results were found for in vitro translated RXRα and LXRα proteins (lane 8). The competition experiments were performed using unlabeled LXRE (lanes 6) and LXRE-mutated oligonucleotides (lanes 7) as competitors in 10-fold molar excess. The upper array indicates the super-shift with LXRα antibody binding to the LXRα-RXRα-LXRE complex (lane 9). ACSL, long-chain acyl-CoA synthetase; BeWo, human placental choriocarcinoma; EMSA, electrophoretic mobility shift assay; LXR, liver X receptor; LXRE, liver X receptor responsive element; RXR, retinoid X receptor.

Fig. 4.

LXR activation increases acyl-CoA synthetase activity in BeWo cells, and ACSL3 silencing prevents increased acyl-CoA synthetase activity. BeWo cells were either treated directly or reverse-transfected with ACSL3 siRNA or negative control siRNA and incubated for 48 h in medium supplemented with vehicle (0.2% DMSO), 9-cis RA (1 μM), and/or GW3965 (1 μM). A: In cell lysates harvested from BeWo cells prestimulated with vehicle, 9-cis RA (1 μM), and/or GW3965 (1 μM) for 48 h. Acyl-CoA synthetase activity was analyzed in BeWo cell lysates by incubating the lysates in the presence of 100 μM [¹⁴C]OA for 8 min at 37°C. Generated [¹⁴C]OA-CoA was extracted, and the radioactivity was determined in a scintillation counter. Each experiment was performed in triplicate. The results are the means of three independent experiments performed in triplicate (n = 3) ± SEM. *P < 0.05 and **P < 0.01 relative to control (one tailed t-test). B: Total RNA was extracted and analyzed using qRT-PCR. ACSL3 mRNA expression (normalized to TBP) was compared in cells transfected with ACSL3 siRNA1 (black bar) or negative control siRNA (white bar). The results are the means of two independent experiments performed in duplicates (n = 2) ± SEM. Reduction in expression (in %) relative to negative control siRNA is indicated. C: ACSL3 and β-actin protein expression in cells transfected with control siRNA or ACSL3 siRNA. Each lane contains cells pooled from three wells (n = 2). The experiment was repeated once with similar results. D: Acyl-CoA synthetase activity in cell lysates from cells transfected with ACSL3 siRNA (black bar) or a negative control siRNA (white bar). The results are the means of three independent experiments performed in triplicate (n = 3) ± SEM. Reduction in expression (in %) relative to negative control siRNA is indicated. E: OA uptake in cells transfected with ACSL3 siRNA (black bar) or a negative control siRNA (white bar). A representative experiment of four independent experiments performed in triplicate is shown (n = 3) ± SEM. OA uptake (in %) relative to negative control siRNA is indicated. ACSL, long-chain acyl-CoA synthetase; BeWo, human placental choriocarcinoma; LXR, liver X receptor; OA, oleic acid; qRT-PCR, quantitative reverse transcription-PCR; siRNA, small interfering RNA.

Fig. 5.

LXR activation increases uptake of OA, EPA, ARA, and DHA in BeWo cells. BeWo cells were prestimulated with vehicle, 9-cis RA (1 μM) and/or GW3965 (1 μM) for 48 h. The fatty acid uptake was measured 3 h after the addition of [¹⁴C] fatty acids (100 μM) in the presence of vehicle (white bars) or triacsin C (gray bars). The black bars represent total fatty acid uptake (white bars) minus the triacsin C resistant uptake (gray bars). The results are presented as calculated fatty acid uptake (in nmol) normalized to protein (in μg) per well. A: Uptake of [¹⁴C]OA. B: Uptake of [¹⁴C]EPA. C: Uptake of [¹⁴C]ARA. D: Uptake of [¹⁴C]DHA. The data are representative experiments of two to four independent experiments performed in triplicate (n = 3) ± SEM. *P < 0.05 and **P < 0.01 relative to control. ARA, arachidonic acid; BeWo, human placental choriocarcinoma; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; LXR, liver X receptor; OA, oleic acid.