Palmitic Acid Impedes Extravillous Trophoblast Activity by Increasing MRP1 Expression and Function

Abstract

Normal function of placental extravillous trophoblasts (EVTs), which are responsible for uteroplacental vascular remodeling, is critical for adequate delivery of oxygen and nutrients to the developing fetus and normal fetal programming. Proliferation and invasion of spiral arteries by EVTs depends upon adequate levels of folate. Multidrug resistance-associated protein 1 (MRP1), which is an efflux transporter, is known to remove folate from these cells. We hypothesized that palmitic acid increases MRP1-mediated folate removal from EVTs, thereby interfering with EVTs' role in early placental vascular remodeling. HTR-8/SVneo and Swan-71 cells, first trimester human EVTs, were grown in the absence or presence of 0.5 mM and 0.7 mM palmitic acid, respectively, for 72 h. Palmitic acid increased ABCC1 gene expression and MRP1 protein expression in both cell lines. The rate of folate efflux from the cells into the media increased with a decrease in migration and invasion functions in the cultured cells. Treatment with N-acetylcysteine (NAC) prevented the palmitic acid-mediated upregulation of MRP1 and restored invasion and migration in the EVTs. Finally, in an ABCC1 knockout subline of Swan-71 cells, there was a significant increase in invasion and migration functions. The novel finding in this study that palmitic acid increases MRP1-mediated folate efflux provides a missing link that helps to explain how maternal consumption of saturated fatty acids compromises the in utero environment.

Keywords: MRP1; extravillous trophoblast; in utero environment; placenta; saturated fatty acid.

Figure 1

Effect of palmitic and linoleic acids on cytotoxicity, MRP1 protein levels, and ABCC1 gene expression in placental cell lines. Cell viability vs. concentration curves of HTR-8/SVneo, JEG-3, BeWo, and Swan-71 cells upon treatment with palmitic (A) and linoleic acid (B), determined by efflux assay. Effect of 0.5 mM palmitic acid on MRP1 protein expression in HTR8/SVneo (C) and Swan-71 cells (D) determined by Western blotting. Grayscale ratios were calculated with the help of ImageJ (E,F). ABCC1 mRNA expression upon treatment with 0.5 mM palmitic acid in HTR8/SVneo (G) and Swan-71 cells (H) determined by RT-qPCR. Mean values were obtained from three independent experiments and error bars indicate SD. SP, sodium palmitate. * p < 0.05 compared to controls.

Figure 2

Effect of palmitic acid on [³H]-folate efflux and localization of MRP1 in HTR8/SVneo cells. (A) Effect of length of exposure to 0.5 mM palmitic acid (0, 6, 12, 24, 48, and 72 h) on [³H]-folate efflux in HTR8/SVneo cells. Cell lysates were measured for radioactivity at 0, 30, 60, and 120 min. Mean values were obtained from three independent experiments and error bars indicate SD. *p < 0.05 compared to controls. (B) MRP1 expression in HTR8/SVneo and Swan-71 cells after treatment with palmitic acid at 0 and 72 h determined by immunofluorescence. Nuclei were stained with DAPI and MRP1 was stained with Alexa Fluor 488. Images have been merged using Photoshop software.

Figure 3

Effect of palmitic acid on migration and invasion in HTR8/SVneo and Swan-71 cells. Migration (A) and invasion (C) in HTR8/SVneo cells after exposure to palmitic acid for 0 to 72 h and migration (E) and invasion (G) in Swan-71 cells after the same exposures. Images were captured from the underside of the transwell inserts. Quantification (B,D,F,H) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars indicate SD. * p < 0.05 compared to controls.

Figure 4

Effect of NAC on MRP1 expression and migration and invasion in palmitic acid-treated HTR-8/SVneo and Swan-71 cells. MRP1 expression in palmitic acid-treated HTR8/SVneo (A) and Swan-71cells (B) with or without NAC pretreatment determined by Western blotting. Grayscale ratios were calculated with the help of ImageJ (C,D). Migration (top row) and invasion (bottom row) of palmitic acid-treated HTR8/SVneo cells (E) and Swan-71 cells (F) with or without NAC pretreatment. Images were captured from the underside of the transwell inserts. Quantification (G–J) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars represent SD. * p < 0.05 compared to controls.

Figure 5

Effect of MK-571 on migration and invasion in palmitic acid-treated HTR-8/SVneo and Swan-71 cells. Migration (A) and invasion (C) in HTR-8/SVneo cells with or without palmitic acid exposure and MK-571 pretreatment and migration (E) and invasion (G) of Swan-71 cells after the same exposures. Images were captured from the underside of the transwell inserts. Quantification (B,D,F,H) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars represent SD. * p < 0.05 compared to controls.

Figure 6

Effect of ABCC1 knockout on migration and invasion in Swan-71 cells exposed to palmitic acid. Migration (A) and invasion (C) in Swan-71 ABCC1 knockout and wild-type cells with or without exposure to palmitic acid. Images were captured from the underside of the transwell inserts. Quantification (B,D) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars represent SD. * p < 0.05.