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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2265
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4267-4275
Copyright © 2005 by The Endocrine Society

Peroxisome Proliferator-Activated Receptor-{gamma} and Retinoid X Receptor Signaling Regulate Fatty Acid Uptake by Primary Human Placental Trophoblasts

W. Timothy Schaiff, Ibrahim Bildirici, Monica Cheong, Peggy L. Chern, D. Michael Nelson and Yoel Sadovsky

Departments of Obstetrics and Gynecology, and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence to: Yoel Sadovsky, M.D., Washington University School of Medicine, Department of Obstetrics and Gynecology, Campus Box 8064, 4566 Scott Avenue, St. Louis, Missouri 63110. E-mail: ysadovsky{at}wustl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Transplacental transfer of fatty acids from the maternal to the fetal circulation is essential for fetal development. The nuclear receptor peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) regulates fatty acid transport and storage in adipocytes and other cell types.

Objective: This study tested the hypothesis that PPAR{gamma} and its heterodimeric nuclear receptor partner, retinoid X receptor (RXR), regulate fatty acid uptake by human trophoblasts.

Design: Prospective basic laboratory in vitro research was conducted using primary term human trophoblasts.

Setting: The study was performed in the perinatal biology laboratory of an academic medical center.

Patients or Other Participants: Study materials were obtained from healthy pregnant women at a gestational age of 37–41 wk.

Interventions: There were no interventions.

Main Outcome Measures: Fat uptake and accumulation in human placental trophoblasts were measured.

Results: We initially demonstrated that activation of PPAR{gamma} and/or RXR with selective agonists increased the accumulation of neutral lipids in trophoblasts as well as uptake of free fatty acids. Furthermore, activation of PPAR{gamma} and RXR enhanced the expression of the fat droplet-associated protein adipophilin along with fatty acid transport protein (FATP)4, whereas expression of FATP2 was decreased by activation of RXR. Finally, we found that inhibition of p38 MAPK, which diminishes the activity of PPAR{gamma} in trophoblasts, inhibited fatty acid uptake and blocked the PPAR{gamma}- and RXR-dependent increases in adipophilin and FATP4 expression, yet stimulated the expression of FATP1, FATP2, and FATP3.

Conclusions: These data support a role for PPAR{gamma} and RXR in regulation of fatty acid transport and storage in human placental trophoblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSPORT OF FATTY acids from the maternal circulation across the placenta into the fetal circulation is essential for proper fetal growth and development. Insufficient in utero growth is associated with adulthood diseases, such as diabetes, hypertension, and cardiovascular disease (1, 2). Fetal demand for fatty acids is increased during the rapid fetal growth that occurs primarily during the latter stages of pregnancy (3). This period is also associated with acquisition of very-long-chain fatty acids, such as docosahexaenoic acid and arachidonic acid, which are essential for rapid brain growth and ocular development (reviewed in Refs. 3 and 4). Although the process of fatty acid uptake is paramount for fetal development, the mechanisms underlying this process are largely unknown.

The nuclear receptor peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) is essential for placental development and, consequently, fetal viability (5, 6). PPAR{gamma}-null murine embryos die at embryonic d 10.5 as a result of maldevelopment of the placental labyrinth. Interestingly, this defect is associated with absent lipid droplets in trophoblasts. The heterodimeric nuclear receptor partner of PPAR{gamma}, retinoid X receptor-{alpha} (RXR{alpha}), is also required for placental development. Ablation of the RXR{alpha} gene in mice results in embryonic death, with embryos exhibiting placental defects similar to those in PPAR{gamma}-null embryos, including improper labyrinth development and greatly reduced trophoblastic lipid droplets (7, 8). We previously demonstrated that PPAR{gamma} enhances the differentiation of cultured primary term human trophoblasts (9). This process is accompanied by an increase in accumulation of neutral lipids, particularly in syncytiotrophoblasts (10). Interestingly, the p38 MAPK, which is also essential for development of the murine placental labyrinth (11, 12), regulates the transcriptional activity of PPAR{gamma} (13, 14, 15). Inhibition of p38 MAPK activity leads to inhibition of PPAR{gamma}-induced cellular differentiation of trophoblasts, preadipocytes, and macrophages, and activation of p38 MAPK potentiates PPAR{gamma} activity (13, 14).

Based on this information, we hypothesized that PPAR{gamma} and its heterodimeric nuclear receptor partner RXR regulate fatty acid uptake by trophoblasts. We also surmised that p38 MAPK modulates this process. To test this hypothesis we measured the influence of PPAR{gamma}, RXR, or both on fatty acid accumulation and transport into primary human trophoblasts. We identified fatty acid-associated proteins that are targets for regulation by PPAR{gamma} and RXR, and using selective inhibitors we defined the role of p38 MAPK in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

The study was approved by the human studies committee of Washington University School of Medicine. All cell culture media and supplements were purchased from the Washington University Tissue Culture Support Center, except where specified. Primary human cytotrophoblasts were prepared from human placentas, derived from uncomplicated term (37–41 wk) pregnancies. Trophoblasts were isolated using the trypsin-DNase-Dispase/Percoll method as described by Kliman et al. (16) with previously published modifications (17). Cultures were plated at a density of 300,000 cells/cm2 and maintained in Earl’s medium 199 (M199) containing 10% fetal bovine serum (Hyclone, Logan, UT), 20 mM HEPES (Sigma Chemical Co., St. Louis, MO) (pH 7.4), 0.5 mM L-glutamine, penicillin (10 U/ml), streptomycin (10 µg/ml), and fungizone (0.25 µg/ml). The medium was changed every 24 h. Where indicated, cultures were supplemented with the PPAR{gamma}-specific agonist GW1929 (18) (2 µM; Calbiochem, La Jolla, CA), the RXR-specific agonist LG268 (19) (0.1 µM; a generous gift from Ligand Pharmaceuticals, San Diego, CA), the p38 MAPK inhibitors SB203580 or SB202190, or the noninhibitory control compound SB202474 (20, 21) (all at 30 µM; Calbiochem), dissolved in dimethylsulfoxide (Sigma). Dimethylsulfoxide was added to control cultures. The concentrations of GW1929 and LG268 used were the lowest concentration to induce maximal trophoblast differentiation as measured by human chorionic gonadotropin secretion (15) (data not shown). The concentration of p38 MAPK inhibitors used was as previously described (15).

Oil Red O staining

Primary trophoblasts were cultured for 72 h in the presence or absence of agonists for PPAR{gamma} and/or RXR and then stained with Oil Red O to visualize neutral lipid droplets and counterstained with hematoxylin by the Washington University Molecular Biology and Pharmacology core histology laboratory. Digital micrographs were obtained using a MagnaFire model S99802 digital imaging system (Optronics, Goleta, CA). Lipid droplets were quantified from digital images using Analysis digital imaging software (Soft Imaging Corp., Lakewood, CA). Nuclei were manually counted from the same images. Statistics were performed based on data obtained from eight to 10 random fields. The average number of lipid droplets was normalized to the number of nuclei.

Fatty acid transport assay

Transport of fatty acid into cultured human trophoblasts was measured with radiolabeled oleic acid using a modification of a previously described assay (22). Tritiated oleic acid uptake medium consisted of a 1:1 ratio of fatty acid-free BSA (Sigma) and unlabeled oleic acid (Nu Chek Prep, Elysian, MN) at 80 µM each, spiked with [3H]oleic acid (PerkinElmer Life Sciences, Boston, MA) at 10 million dpm/ml. The transport medium was prepared by adding the unlabeled and tritiated oleic acid dropwise with stirring to a solution of fatty acid-free BSA in Hank’s balanced salt solution at 40 C. The solution was stirred until cleared, then allowed to cool to room temperature.

Trophoblasts were cultured in six-well plates for 72 h in the presence or absence of PPAR{gamma} and/or RXR agonists with or without p38 MAPK inhibitors as described above. Triplicate wells were used for all conditions. At the end of the culture period, the cells were incubated 1 h at 37 C in serum-free DMEM. After incubation, the culture plates were allowed to equilibrate to room temperature for 5–10 min. After rinsing the wells twice with 2 ml of room temperature Hank’s balanced salt solution, 800 µl of transport medium was added to each well and incubated at room temperature for 3 min. The uptake was stopped by the addition of 3.5 ml/well ice-cold stop solution [0.1% fatty acid-free BSA, 500 µM phloretin (Sigma) in PBS]. The wells were rinsed twice with ice-cold stop solution, the cells lysed with 500 µl of 0.2% SDS, and the lysates added to 10 ml ScintiVerse II scintillation fluid (Fisher Scientific, Fair Lawn, NJ). The wells were rinsed with an additional 500 µl of 0.2% SDS and pooled into their respective scintillation vials. Samples were counted on a Packard 2500TR liquid scintillation analyzer (Packard, Downers Grove, IL). Nonspecific association of labeled fatty acid with trophoblasts was determined using duplicate cultures assayed in the presence of 500 µM phloretin. Because phloretin blocks receptor-mediated fatty acid transport (23, 24, 25) it was included in the preincubation, prewash, and fatty acid transport buffers in parallel cultures to determine how much of the observed radiolabeled fatty acid detected in the trophoblast cells was a result of passive uptake. By subtracting the counts in the presence of phloretin from those in the absence of phloretin, receptor-mediated fatty acid uptake was determined. Total [3H]oleic acid dpm in the absence of phloretin ranged from 20,000–50,000 dpm. The [3H]oleic acid dpm in the presence of phloretin was 30–50% of the counts in the absence of phloretin.

RT-PCR

RNA was purified from cultures of human primary trophoblasts using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. Cells were harvested at 48 h for expression screening because cultures at this time contain a roughly even mixture of cytotrophoblasts and syncytiotrophoblasts (9). For quantitative analysis, 72-h cultures were used to allow maximal trophoblast differentiation. Purified RNA was treated for 1 h at 37 C with DNase I (DNA-free; Ambion, Austin, TX) to remove contaminating DNA. cDNA was prepared from 1 µg RNA using the TaqMan Gold RT-PCR kit with the supplied random hexamer primers (Applied Biosystems, Branchburg, NJ). PCR was performed on reverse transcripts using primer pairs listed in Table 1Go. All sequences were checked for specificity using BLAST analysis (26). Quantitative real-time PCR was performed in duplicate using 3-µl samples of cDNA and 25 µl SYBR Green PCR Master Mix (Applied Biosystems) in a total reaction volume of 50 µl that contained 300 nM each of forward and reverse primers. Reactions were run and analyzed using an Applied Biosystems Geneamp 5700 sequence detection system. Dissociation curves were run on all reactions to ensure amplification of a single product with the appropriate melting temperature (data not shown). Samples were normalized to parallel reactions using primers specific for 18S RNA (Table 1Go). The fold increase relative to control cultures was determined by the 2–{Delta}{Delta}Ct method (27). All PCR primer pairs are listed in Table 1Go. All sequences were checked for specificity using BLAST analysis (26).


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  		TABLE 1. Primer sequences used for expression analysis and real-time quantitative PCR of human genes

 
Statistics

Data are expressed as mean ± SD. All comparisons were made by either paired t test or ANOVA with post hoc Bonferroni correction, where appropriate, using the Primer of Biostatistics software package (McGraw-Hill, Inc., New York, NY). Significance was determined at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of PPAR{gamma} and/or RXR enhances the uptake and accumulation of fatty acids in primary human trophoblasts

We initially tested whether activation of PPAR{gamma} or its heterodimeric nuclear receptor partner RXR influences the accumulation of fatty acid in cultured term primary human trophoblasts. As shown in Fig. 1Go, we found that activation of PPAR{gamma} and/or RXR caused an increased number of neutral lipid droplets within cultured trophoblasts. Not surprisingly, activation by both ligands led to greater activation than either ligand alone. Notably, activation of RXR consistently induced a greater accumulation of lipid droplets than did activation of PPAR{gamma}.



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  		FIG. 1. Activation of PPAR{gamma} and RXR induces accumulation of neutral lipids in primary human trophoblasts. Term primary human trophoblasts were cultured in the presence or absence of PPAR{gamma} agonist GW1929 (2 µM) and RXR agonist LG268 (0.1 µM), as described in Materials and Methods. A, Oil Red O staining of trophoblasts showing accumulation of neutral lipid droplets in cells incubated in the presence of PPAR{gamma} and RXR activation. Original image was captured at x400 magnification. Insets show staining magnified an additional x2. B, Oil Red O-stained lipid droplets were quantitated as described in Materials and Methods, based on data obtained from eight to 10 random fields of each experiment and expressed as mean number of droplets per nucleus. All paradigms were significantly different (P < 0.01, ANOVA). Data are representative of three independent experiments.

 
We next determined whether activation of PPAR{gamma} or RXR affected the transport of fatty acids into trophoblasts. We initially found that ligands for PPAR{gamma} and RXR up-regulated the uptake of radiolabeled oleic acid by primary trophoblasts cultured for 24, 48, and 72 h (Fig. 2AGo), a time span that supports differentiation of cytotrophoblasts into syncytium (9, 17). We next examined the distinct influence of RXR or PPAR{gamma} on fatty acid transport into trophoblasts. As shown in Fig. 2BGo, activation of RXR consistently induced a 50–60% increase in the uptake of oleic acid by trophoblasts. We also observed a significant increase, albeit to a slightly lesser degree, by PPAR{gamma} activation. We obtained similar results when we estimated cellular fat accumulation using the fluorescent long-chain fatty acid analog C1-BODIPY 500/510-C12 (Molecular Probes, Eugene, OR) in place of tritiated oleic acid (data not shown). Together, these data indicate that PPAR{gamma} and RXR enhance the uptake and accumulation of fatty acids in term human trophoblasts.



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  		FIG. 2. Activation of PPAR{gamma} and RXR induce increased uptake of fatty acids by primary human trophoblasts. A, Primary trophoblasts were incubated in the presence of LG268 (0.1 µM) and GW1929 (2 µM) at the time points indicated and then assayed for fatty acid transport into the cells as described in Materials and Methods. Data are expressed as relative uptake compared with control cultures. *, P < 0.05 relative to control cultures (paired t test). B, Primary trophoblasts were incubated for 72 h in the presence or absence of LG268 (0.1 µM) and GW1929 (2 µM) and then assayed for fatty acid transport into the cells. *, P < 0.05 relative to control cultures; **, P < 0.05 relative to control or GW1929 (ANOVA). Data are representative of four independent experiments.

 
The influence of PPAR{gamma} and RXR activation on expression of adipophilin and fatty acid transport proteins (FATPs) in human trophoblasts

We previously reported the microarray-based analysis of a large placental transcriptome data set that was derived from trophoblasts cultured in the absence or presence of PPAR{gamma} ligands (28, 29). Using T-generator, a novel microarray analysis tool that was developed in our lab (29), we found that different ligands for PPAR{gamma}, including troglitazone, GW1929, and other PPAR{gamma} agonists, consistently enhance the expression of the lipid droplet-associated protein adipophilin in cultured human trophoblasts. Whereas the expression of adipophilin in primary human trophoblasts has been shown (30), the expression of FATP isoforms in these cells has not been previously investigated. To examine trophoblast expression of FATP isoforms, we used RNA isolated from trophoblasts cultured for 48 h. At that time point, the cultures contain a mixture of cytotrophoblasts and syncytiotrophoblast, ensuring detection of FATP transcripts that might be expressed exclusively in either cytotrophoblast or syncytiotrophoblast phenotypes. As shown in Fig. 3Go, we confirmed the expression of transcripts for adipophilin (positive control) and FATP1, -2, -3, -4, and -6. In contrast, FATP5 was not expressed in trophoblasts.



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  		FIG. 3. Expression of adipophilin and FATPs in human trophoblasts. Primary human trophoblasts were incubated for 48 h in the absence of agonists. RNA was extracted from the cells at the end of the culture period and assayed for expression of adipophilin and FATPs as described in Materials and Methods. All controls and product sizes matched our predictions for each transcript tested.

 
Because ligand activation of PPAR{gamma} and RXR increased the uptake and accumulation of fatty acids in cultured trophoblasts, we sought to determine whether activation of PPAR{gamma} and RXR influenced the expression of adipophilin and FATP in cultured primary human trophoblasts. In our microarray data set, we found that the expression of FATP4 was up-regulated by PPAR{gamma} activation. As shown in Fig. 4Go, activation of either RXR or PPAR{gamma} enhanced the expression of adipophilin as well as FATP4, albeit to a lesser extent. Activation of RXR induced greater expression of adipophilin than did activation of PPAR{gamma} (5- to 10-fold vs. 2- to 4-fold relative to control, respectively), whereas simultaneous activation of RXR and PPAR{gamma} induced the highest increase in adipophilin expression (10- to 20-fold). The expression of FATP1 was enhanced by 1.5- to 3-fold only when both RXR and PPAR{gamma} were activated. Activation of RXR or PPAR{gamma}, either separately or simultaneously, had no effect on the expression of FATP3 or FATP6. In contrast, activation of RXR or concomitant activation of PPAR{gamma}/RXR consistently reduced expression of FATP2 to 40% of control. Taken together, these findings buttress our uptake studies and suggest that adipophilin and FATP4 likely play a role in placental fat accumulation.



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  		FIG. 4. Activation of RXR and PPAR{gamma} influences expression of adipophilin and FATPs in human trophoblasts. Primary human trophoblasts were incubated for 72 h in the presence or absence of LG268 (0.1 µM) and GW1929 (2 µM). At the end of the culture period, RNA was extracted from the cells and assayed for expression of adipophilin and FATPs by quantitative real-time PCR. Data are expressed as relative expression compared with control cultures for each respective gene. *, P < 0.05 relative to control cultures; **, P < 0.05 relative to all paradigms (ANOVA). Data are representative of four separate experiments.

 
p38 MAPK regulates fatty acid uptake and transporter expression in trophoblasts

Previous experiments in our laboratory and others have shown that active p38 MAPK is required for PPAR{gamma} activity and expression (13, 14, 15). We therefore hypothesized that inhibition of p38 MAPK might diminish fatty acid uptake by trophoblasts. To test this hypothesis we incubated trophoblasts in the presence or absence of p38 MAPK inhibitor SB202190 or the noninhibitory analog SB202474 (20, 21). As shown in Fig. 5Go, inhibition of p38 MAPK blocked PPAR{gamma} and RXR agonist-induced increases in fatty acid transport into trophoblasts, whereas the inactive analog had no affect on agonist-induced fatty acid transport. Interestingly, inhibition of p38 MAPK caused a small but significant decrease in fatty acid transport in the presence of RXR agonist but not in the presence of PPAR{gamma} agonist. This decrease was less pronounced when both PPAR{gamma} and RXR were activated. These data demonstrate that p38 MAPK is required for PPAR{gamma}- and RXR-dependent up-regulation of fatty acid transport.



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  		FIG. 5. Inhibition of p38 MAPK inhibits fatty acid uptake by human trophoblasts. Primary human trophoblasts were incubated for 72 h in the presence or absence of LG268 (0.1 µM) and GW1929 (2 µM) either with or without the p38 MAPK inhibitor SB202190 (30 µM) or the noninhibitory analog SB202474 (30 µM) and then assayed for fatty acid transport into the cells as described in Materials and Methods. Data are expressed as uptake relative to cultures without agonists for each p38 MAPK inhibitor experiment. Baseline uptake in the presence of SB202190 or SB202474 averaged 81 and 76% of control cultures, respectively. *, P < 0.05 relative to control cultures; **, P < 0.05 relative to all paradigms (ANOVA). Data are representative of three separate experiments.

 
To assess whether or not inhibition of p38 MAPK alters the expression of adipophilin or FATP, we cultured primary human trophoblasts in the absence or presence of RXR and/or PPAR{gamma} agonists either with or without p38 MAPK inhibitors and analyzed transcript expression using quantitative real-time PCR. For these assays we used the selective p38 MAPK inhibitors SB202190 and SB203580 as well as the noninhibitory analog SB202474. As shown in Fig. 6Go, A and E, incubation of primary human trophoblasts with the p38 MAPK inhibitors SB203580 or SB202190 blocked the increased expression of adipophilin and FATP4 induced by ligand-activated RXR and/or PPAR{gamma}. Notably, inhibition of p38 MAPK had little or no effect on baseline expression of these transcripts. As expected, the inactive analog did not affect transporter expression. Surprisingly, we found that p38 MAPK inhibition resulted in a profound increase in FATP2 expression (Fig. 6CGo), with a more modest enhancement of FATP1 and FATP3 expression (Fig. 6Go, B and D, respectively). Inhibition of p38 MAPK in primary trophoblasts induced a minimal increase in baseline FATP6 expression (1.5-fold). Taken together, these data indicate that stimulation of fatty acid uptake by PPAR{gamma} or RXR requires p38 MAPK activity. Moreover, inhibition of p38 MAPK exhibits a robust and selective influence on the expression of adipophilin and FATPs in human trophoblasts, with down-regulation of adipophilin and FATP4 and up-regulation of FATP1, FATP2, and FATP3.



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  		FIG. 6. Inhibition of p38 MAPK modulates the expression of adipophilin and FATPs. Primary human trophoblasts were incubated for 72 h in the presence or absence of LG268 (0.1 µM), GW1929 (2 µM), SB202190 (30 µM), SB203580 (30 µM), or SB202474 (30 µM) as indicated. RNA was extracted at the end of the culture period and assayed for adipophilin and FATP expression by quantitative real-time PCR. P < 0.05 for all cultures incubated in the presence of SB202190 or SB203580 relative to cultures without inhibitor, except for FATP6 in which only cultures incubated without RXR or PPAR{gamma} agonists exhibited a significant change (P < 0.05, paired t test). The differences between cultures in the presence or absence of SB202474 were not significant. Data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proper transfer of fatty acids from the maternal circulation across the placenta to the fetal circulation is essential for proper fetal development, particularly during the latter stage of gestation (reviewed in Refs. 4 and 31). Here we showed that RXR and PPAR{gamma} regulate fatty acid transport and accumulation in primary human trophoblasts. Notwithstanding a non-transporter-mediated uptake of 30–50%, the extent of increase in fatty acid transport observed in our study is similar to previously published results regarding fatty acid transport in response to PPAR{gamma} or RXR activation in other cell types (32, 33, 34).

We demonstrated the expression of proteins that are known to play a role in fatty acid uptake. Whereas the expression of adipophilin (30) and FATP1, FATP4, and FATP6 (35, 36, 37) in the whole placenta has been previously reported, we expanded these studies to include additional FATPs. Importantly, we found that the stimulation of fatty acid uptake and accumulation in trophoblasts by ligand-activated PPAR{gamma} and/or RXR is associated with enhanced expression of adipophilin and FATP4. These data are consistent with previous work from our laboratory in which we found that adipophilin protein expression is regulated by PPAR{gamma} and RXR (30) and also consistent with the known up-regulation of adipose differentiation-related protein, the murine ortholog of adipophilin, by PPAR{gamma} agonists in murine preadipocytes (38). The effect of PPAR{gamma} and RXR on other FATP family members in trophoblasts was modest, exhibiting an increase in FATP1 expression only when both PPAR{gamma} and RXR were activated. Although activation of PPAR{gamma} or RXR enhances FATP1 expression in other systems (33, 34, 39) and a PPAR{gamma} response element has been identified in the FATP1 promoter (39), the regulation of FATP1 expression by PPAR{gamma} and RXR is tissue specific, which could account for the difference in FATP1 regulation in trophoblasts vs. nontrophoblastic systems. In contrast, the observed decrease in FATP2 expression by activated RXR sets FATP2 apart from the other FATPs. Suppression of gene expression (40) and transcription (41) by ligand-activated RXR has been described in other systems, even though the mechanism of activated-RXR-mediated suppression has not been determined.

Although the increased fatty acid transport we observe when PPAR{gamma} and/or RXR are activated is consistent with that observed elsewhere under similar conditions (32, 33, 34), it is noteworthy that relative to the fold increase in the expression of adipophilin and FATP4, the fold increase in fatty acid transport is smaller. Whereas up-regulation of adipophilin and FATP4 is likely to play a significant role in regulation of fatty acid transport, additional mechanisms also affect net uptake and accumulation of fatty acids. For example, efflux of fatty acid out of the trophoblasts might have reduced the amount of accumulated fat. Additionally, the availability of intracellular proteins required for fatty acid movement within the cell, such as fatty acid-binding proteins (42, 43), might limit cellular fatty acid transport. Additional experiments are required to examine these and other mechanisms that might modulate fat transport in trophoblasts.

Earlier work from our laboratory (15) and others (13, 14) demonstrated that p38 MAPK modulates PPAR{gamma} activity. Here we expanded these findings and demonstrated that p38 MAPK modulates the influence of PPAR{gamma} and RXR on fatty acid uptake and expression of lipid droplet-related proteins. Inhibition of p38 MAPK activity blocked the increase in expression of adipophilin and FATP4 induced by activation of PPAR{gamma} or RXR but without affecting baseline expression of these proteins in trophoblasts. This effect of p38 MAPK in trophoblasts may be mediated by direct phosphorylation of PPAR{gamma} or RXR, as shown for other MAPKs (ERK and Jun N-terminal kinase) (44, 45, 46, 47, 48), and altered protein stability (15). Alternatively, p38 MAPK could be acting indirectly via phosphorylation of other proteins that modulate the transcriptional activity of PPAR{gamma} or RXR (49, 50, 51, 52). Additional experiments are required to distinguish among these possibilities.

The effect of p38 MAPK inhibition on the expression of FATPs other than FATP4 was unexpected. Unlike adipophilin or FATP4, inhibition of p38 MAPK markedly enhanced the expression of FATP2, as well as the expression of FATP1, FATP3, and FATP6, albeit to a lesser extent. Similar to our finding, inhibition of p38 MAPK with SB203580 in IL-1ß-stimulated primary human airway myocytes led to increased secretion of granulocyte-macrophage colony-stimulating factor (53). Together, our results establish that p38 MAPK activity is essential for enhanced expression of proteins that are stimulated by PPAR{gamma} and RXR, namely adipophilin and FATP4, and enhances fatty acid uptake. In contrast, when PPAR{gamma} and/or RXR exhibit minimal (FATP1) or no up-regulation (FATP3 and FATP6), or even RXR-mediated down-regulation (FATP2), inhibition of p38 MAPK enhances transcript expression. Our data suggest that FATP1, -2, -3, and -6 are unlikely to play a significant role in fatty acid transport into trophoblasts. In fact, our data are consistent with the possibility that FATP2 plays a role in fatty acid efflux from trophoblasts, which may be inhibitable by RXR and p38 MAPK activity.

Although highlighting the role of PPAR{gamma} and RXR in fat uptake by trophoblasts, our work did not address the role of adipophilin and FATP4 in enhanced fat accumulation in trophoblasts. Nevertheless, our data are consistent with the finding that adipose differentiation-related protein, the murine ortholog of adipophilin, facilitates selective uptake of long-chain fatty acids when transfected into COS-7 cells (54). FATP4 is the only FATP expressed in the small intestine, localized on the apical brush-border membrane of intestinal enterocytes, and is thought to be responsible for uptake and transport of dietary fatty acids (55). FATP4 is also localized to the apical brush-border membrane of yolk sac trophoblasts in E8 murine embryos (56). Together, these observations along with our data suggest that FATP4 might be responsible for uptake and transplacental transport of fatty acids from the maternal circulation. The phenotype of FATP4-deficient mice is inconsistent, with embryonic lethality by E9.5 reported by some researchers (56) yet neonatal lethality reported by others (57, 58). The reason for this discrepancy and the cause of death are unknown. Additional experiments are necessary to establish the role of FATP4 in fetal nutrition.

Although we focused on RXR as a hetero-partner of PPAR{gamma} in regulation of fatty acid transport in trophoblasts, we are cognizant of the fact that RXR is capable of forming homodimers as well as heterodimers with diverse nuclear receptors (reviewed in Ref. 59). Indeed, the reduction in FATP2 expression is observed with RXR activation, not PPAR{gamma}. Additionally, in some instances, such as fatty acid accumulation, RXR activation had a more profound affect than activation of PPAR{gamma}. These data suggest that RXR, possibly in conjunction with other nuclear receptors, might predominate in regulating aspects of fatty acid transport in trophoblasts. Additional studies using selective knockdown of PPAR{gamma}, RXR, or other nuclear receptors, as well as the FATPs and adipophilin, are necessary to determine the relative importance of each protein in fatty acid transport in trophoblasts. Finally, our studies did not address the expression of additional proteins that may modulate fat uptake and accumulation in trophoblasts, including fatty acid-binding proteins, perilipin, and TIP47. Information on additional proteins that regulate fat accumulation in trophoblasts may provide insights into strategies to correct insufficient transplacental fatty acid transport and thus support fetal growth and development.


   Acknowledgments
 
We thank Steve Smith, Elena Sadovsky, Tamar Solomon, and Lori Rideout for their assistance during these studies. We also thank Mark Leibowitz and Andres Negro-Vilar from Ligand Pharmaceuticals for generously providing LG268.


   Footnotes
 
This work was supported by National Institutes of Health Grants Building Interdisciplinary Research Careers in Women’s Health (a part of K12 HD-01459; to W.T.S.), R01-HD29190 (to D.M.N.), and R01-ES11597 and R01-HD45675 (both to Y.S.).

Results from this work were presented in part at the 2003 International Federation of Placenta Associations Meeting in Mainz, Germany.

First Published Online April 12, 2005

Abbreviations: E10.5, Embryonic d 10.5; FATP, fatty acid transport protein; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; RXR, retinoid X receptor.

Received November 24, 2004.

Accepted April 4, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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