help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1199
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/4/2344    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Meadows, J. W.
Right arrow Articles by Myatt, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Meadows, J. W.
Right arrow Articles by Myatt, L.
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2344-2350
Copyright © 2005 by The Endocrine Society

Expression and Localization of Adipophilin and Perilipin in Human Fetal Membranes: Association with Lipid Bodies and Enzymes Involved in Prostaglandin Synthesis

Juliana W. Meadows, Brad Pitzer, Diane E. Brockman and Leslie Myatt

Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Dr. Juliana Meadows, Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0526. E-mail: cvq7{at}cdc.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lipid storage droplets (LSDs) are subcellular storage depots for triglycerides (TGs) and cholesterol esters surrounded by specific populations of proteins that are necessary for their formation. We have previously described the appearance of LSDs in human fetal membranes with advancing gestation and labor. Perilipin and adipophilin are functional/structural proteins located on the surfaces of intracellular LSDs. Adipophilin and perilipin were both immunolocalized to the amnion epithelium and amnion fibroblasts in human fetal membranes. Adipophilin was also localized to the choriodecidual layer, whereas perilipin was localized to the chorion trophoblasts. Although immunohistochemical data show an apparent increase in adipophilin, but not perilipin, expression in fetal membranes with advancing gestation and labor, Western analysis of tissue homogenate supernatant revealed no significant changes in adipophilin and perilipin expression. However, Western analysis of the floating lipid-rich layer from the tissue homogenate revealed an abundance of adipophilin and perilipin as well as other enzymes (cytosolic phospholipase A2, prostaglandin endoperoxide, and microsomal-associated prostaglandin E synthase-1) involved in prostaglandin synthesis. The association of these enzymatically active proteins with LSDs suggests that LSDs may be foci for signaling via the arachidonic acid cascade in fetal membranes. The structural and functional roles of adipophilin and perilipin in gestation and labor remain to be determined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOST MAMMALIAN CELLS package neutral lipids in intracellular droplets as storage depots for triglycerides (TGs) and cholesterol esters (CE). These lipid droplets are surrounded by a phospholipid monolayer and are coated by specific populations of proteins necessary for their formation and function (1). Many animals use these small lipid storage droplets (LSDs) as an energy source or in membrane biogenesis. Several proteins have been identified in abundance on the surfaces of these LSDs, including adipose differentiation-related protein (ADRP or adipophilin), perilipin, and caveolin (2, 3, 4). ADRP is found primarily on smaller droplets, whereas perilipins are present on large droplets (5). The roles of ADRP and perilipin in lipid droplet assembly are as yet unclear.

Adipophilin is the human homolog of murine ADRP, a 52-kDa protein that was cloned from a mouse adipocyte cDNA library (6, 7). It is related to a family of lipid-associated proteins that include perilipin and the mannose-6-phosphate receptor-targeting protein, TIP47 (8, 9). Adipophilin is highly expressed on the surface of lipid droplets in a wide variety of cells and tissues that store or synthesize lipids (7), including hepatocytes, adipocytes, mammary epithelial cells, steroidogenic cells, and muscle cells (10, 11). It has been proposed that adipophilin functions in LSD formation (12), fatty acid uptake (12), and milk lipid secretion (13, 14). Buechler et al. (15) demonstrated that increasing free fatty acids in the presence of nuclear peroxisome proliferator-activated receptor {gamma} ligands, such as 15d-prostaglandin (15d-PGJ2) and ciglitazone, induced the up-regulation of adipophilin mRNA and protein expression in blood monocytes (16), indicating that peroxisome proliferator-activated receptor {gamma} may mediate the induction of adipophilin expression.

Perilipins are found exclusively on lipid droplet surfaces (1, 2, 17) and were first identified in adipocytes as proteins that are polyphosphorylated by protein kinase A upon lipolytic stimulation (18). Perilipin is a 62-kDa phosphoprotein that coats lipid droplets and segregates with the lipid droplets floated from cell homogenates (19, 20). It is a single copy gene that gives rise to three protein isoforms by mRNA splicing. Perilipin A is the most abundant isoform in both adipose and steroidogenic cells, whereas the B and C isoforms occur primarily in adipose and steroidogenic cells, respectively (20, 21, 22, 23). Perilipin appears to be restricted to adipocytes and steroidogenic cells, which are specialized for lipid storage (2). Lipolysis is catalyzed by hormone-sensitive lipase (HSL). Phosphorylation of HSL by PKA activates the lipolytic reaction. It has been suggested that perilipin blocks access of HSL to the lipid droplet surface, but upon phosphorylation serves as a docking platform for HSL, allowing HSL to access and hydrolyze lipid. The nonphosphorylated perilipin exerts a protective effect and suppress lipolysis (1, 18, 24).

Lipid bodies are typically sparse in normal cells, but increase in number and size in cells associated with inflammation. Weller and Dvorak (26) reported that lipid droplet formation is a coordinated cellular response and is mediated by protein kinase C activation, which ultimately leads to the mobilization and deposition of lipids and proteins in discrete intracellular domains. Ultrastructural immunogold localization has detected PGH synthase on lipid bodies of various cells, including human mast cells, neutrophils, eosinophils, monocytes, and murine fibroblast (27). Recent evidence has also shown colocalization of cytosolic phospholipase A2 (cPLA2) and its activating protein kinases, including extracellular signal-regulated kinase 1/2 and p85 and p38 MAPKs, on lipid bodies in monocytic U937 cells (28). Together, these data suggest that lipid bodies may be structurally distinct intracellular sites active in extracellular ligand-induced arachidonate release and eicosanoid formation.

PG production by intrauterine tissues at parturition has a pivotal role in the initiation and maintenance of labor (29, 30, 31) Conversion of the unstable PG endoperoxide (PGH2) to PGE2 is catalyzed by PGE synthase (PGES). We and others have previously shown the presence of both PGES enzyme isoforms in human fetal membranes (32, 33). In particular, microsomal-associated PGES-1 (mPGES-1) presented with a punctate immunostaining pattern similar to that of the lipid stain Sudan Black B, suggesting a possible association with lipid droplets in the fetal membranes (32). The presence of enzymes involved in PG synthetic pathway on lipid droplets suggests the potential involvement of lipid droplets in PG synthesis during labor.

In this study we investigated the presence of adipophilin and perilipin in human fetal membranes at preterm and term, with and without labor (PTL and PTNL, respectively). We also investigated the presence of cPLA2, PGHS-2, mPGES, and cPGES in the floating fat cake of total tissue homogenates and their association with adipophilin and perilipin.


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

Rabbit antihuman perilipin polyclonal and mouse antihuman adipophilin antibody were purchased from Research Diagnostics, Inc. (Flanders, NJ). Rabbit antihuman PGES polyclonal antibody was purchased from Cayman Chemical (Ann Arbor, MI), and mouse monoclonal anti-p23 was obtained from Affinity Bioreagents, Inc. (Golden, CO). Mouse anti-cPLA2 monoclonal and rabbit anti-PGHS-2 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Oxford Biomedical Research (Oxford, MI). Protease inhibitors, including pepstatin, leupeptin, 4-(2-aminoethyl) benzenesulfonyl fluoride, N{alpha}-p-tosyl-L-lysine-chloromethyl ketone, and sodium orthovanadate, were purchased from Calbiochem (San Diego, CA). Prestained SeeBlue Plus 2 protein marker and Tris-glycine gels were obtained from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Vectastain Elite avidin-biotin-peroxidase complex (ABC) and aminoethylcarbazole were purchased from Vector Laboratories, Inc. (Burlingame, CA), and hematoxylin was obtained from Biomeda Corp. (Foster City, CA).

Tissue collection and preparation

Tissues were collected according to the guidelines set forth in a protocol that is in compliance with the institutional review board of University of Cincinnati (Cincinnati, OH). Human fetal membranes (n = 5 patients/group) were collected immediately after delivery at term following labor (TL; 39.5 ± 0.7 wk), at term with no labor (TNL; 39.7 ± 0.5 wk), PTL (34.6 ± 1.8 wk), or PTNL (30.2 ± 2.7 wk). Term was defined as 37 completed weeks or more, and labor was defined as regular contractions, 2–3 min apart, accompanied by cervical change. TNL patients received elective cesarean sections, and PTNL patients received elective cesarean sections due to nonlabor-related complications. For immunohistochemistry, a 3- to 6-cm strip of reflected membranes was cut, rolled, flash-frozen in liquid nitrogen, and stored at –80 C. Cryosections (7 µm) of membrane rolls were cut just before immunostaining.

For SDS-PAGE analysis, separated amnion and choriodecidua were flash-frozen at collection in liquid nitrogen and stored at –80 C. The amnion and choriodecidua samples (n = 3 patients/group) were homogenized at 4 C in homogenizing buffer (250 mM sucrose and 50 mM HEPES, pH 7.4) with a hand-held Tissue Tearor (speed, 18) in the presence of protease inhibitors [0.7 µg/ml pepstatin, 10 µg/ml leupeptin, 200 µM 4-(2-aminoethyl) benzenesulfonyl fluoride, 100 µM N{alpha}-p-tosyl-L-lysine-chloromethyl ketone, and 200 µM sodium orthovanadate] and centrifuged at 1000 x g for 15 min at 4 C. The supernatant as well as the floating lipid-rich layer were collected for SDS-PAGE. The floating lipid-rich layer was removed with blunt forceps and solubilized in 2x sample buffer.

Immunohistochemistry

Cryosections of membrane rolls were immunostained as we described previously (34). Briefly, serial frozen sections were allowed to air-dry, hydrated, and immunostained using the Vectastain Elite ABC method with the mouse kit for monoclonal antibody against adipophilin and the rabbit antibody kit for polyclonal antibody against perilipin. The negative immunological control on serial sections included the absence of preimmune rabbit serum or an isotype-matched mouse myeloma Ig.

After air-drying, all slides were blocked using the respective animal serum corresponding to the secondary antibody for 30 min at room temperature. Excess blocking solution was removed, and respective primary antibody was added and incubated for 1 h at 37 C. The slide was rinsed for three 5-min cycles in PBS. Incubation with secondary antibody was at 37 C for 60 min. All slides were then rinsed three times for 5 min each time in PBS. The ABC complex was incubated with all slides for 30 min at room temperature, followed by three 5-min rinse cycles in PBS. Aminoethylcarbazole was used as peroxidase substrate and allowed to develop for 5–10 min. The slides were then rinsed with filtered water (Millipore Corp., Bedford, MA), counterstained with hematoxylin for 3 min, then mounted in PBS/glycerol (1:9).

Tissue sections were also stained for the presence of lipid droplets using the Sudan Black B method (35). Briefly, cryosections were air-dried and hydrated in PBS for 5 min. The sections were incubated in Sudan Black B for 10 min, followed by a quick rinse in 70% ethanol and water. The sections were counterstained with Mayer’s Carmalum for 30 min, rinsed, and mounted in PBS/glycerol (1:9).

SDS-PAGE and Western blotting

The post 1000 x g supernatant of amnion and choriodecidua homogenate was diluted in 2x sample buffer containing 0.25 M Tris (pH 6.8), 20% glycerol, 2% sodium dodecyl sulfate, 5% ß-mercaptoethanol, and 0.02% bromophenol blue and heated at 100 C for 5 min. The floating lipid-rich layer was solubilized as follows: 0.1 g wet weight lipid was added to 600 µl (1:6 ratio) of 2x sample buffer, mixed, and heated at 100 C for 5 min. Protein samples (10 µg/lane) and solubilized lipid-rich layer (20 µl/lane) were loaded, separated using 12% Tris/glycine gels (Invitrogen Life Technologies, Inc.), and run at 40 mA/gel. Invitrogen’s prestained SeeBlue Plus 2 protein marker was loaded as standard. The gels were then electroblotted onto nitrocellulose membranes (Osmonics, Inc., Minnetonka, MN). The blots were blocked for 1 h in Tris-buffered saline (TBS) consisting of 100 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 0.1% (vol/vol) Tween 20, and 5% (wt/vol) nonfat dried milk at room temperature with agitation. The blots were then incubated with a 1:1000 dilution of antiperilipin, anti-mPGES1 (0.5 µg/ml), anti-cPGES, anti-cPLA2, or anti-PGHS-2 for 1 h at 37 C. One hundred and fifty microliters of the unpurified, ready to use antiadipophilin antibody were used for immunoblotting. The blots were washed three times in TBS containing 0.1% (vol/vol) Tween 20 and incubated for 1 h at room temperature with horseradish peroxidase-conjugated donkey antirabbit (1:2000) for perilipin, mPGES-1, and PGHS-2 in TBS with 5% nonfat dry milk. Donkey antimouse (1:2000) was used for adipophilin, cPGES, and cPLA2. The washing steps were repeated, and the ECL (Amersham Biosciences, Arlington Heights, IL) chemiluminescence detection system was used to identify the presence of bands. The resulting band intensities were quantitated using an Alpha Imager 5.0 scanning densitometer (Alpha Innotech, San Leandro, CA). For comparison purposes, the intensity of protein bands from the lipid-rich layer was normalized to the original tissue wet weight. Statistical differences between groups were analyzed using Kruskal-Wallis ranked ANOVA as a nonparametric method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
None of the negative controls for immunostaining showed any staining with aminoethylcarbazole (data not shown). Adipophilin was immunolocalized to the amnion epithelium, amnion fibroblasts, and the fibrin present in the choriodecidual layer of the fetal membranes at preterm and term, with or without labor (Fig. 1Go). Adipophilin presented with a punctate staining pattern in both the amnion epithelium and amnion fibroblasts. Immunohistochemistry showed an apparent increase in adipophilin immunostaining with advancing gestation and with labor. Western analysis of adipophilin in the tissue homogenate supernatant revealed a distinct band at 52 kDa (Fig. 2Go); however, scanning densitometry revealed no significant difference in band intensity among the four gestational groups (PTNL, PTL, TNL, and TL; data not shown). Similarly, perilipin was immunolocalized to the amnion epithelium, amnion fibroblasts, and chorion trophoblasts of the fetal membranes of all four groups of tissue (Fig. 3Go). A punctate immunostaining pattern for perilipin was observed only in the amnion fibroblasts in all four tissue groups; both amnion epithelium and chorion trophoblast had a diffuse cytoplasmic staining. There was no observable difference in location or staining intensity for perilipin in any of the cell types between preterm or term tissues, with or without labor. Western blot analysis of amnion and choriodecidua homogenate supernatants for perilipin showed a consistent band at 62 kDa (Fig. 4Go). Scanning densitometry again revealed no significant differences in band intensities among the groups of tissues, as confirmed by Kruskal-Wallis ranked ANOVA (P > 0.05; data not shown).



View larger version (111K):
[in this window]
[in a new window]
  		FIG. 1. Immunostaining of adipophilin in human fetal membranes at preterm and term, with or without labor. A, PTNL; B, PTL; C, TNL; D, TL. Positive adipophilin immunostaining was observed in the amnion epithelium and amnion fibroblasts and in association with fibrin present in the choriodecidual layer in all gestational groups. Controls showed no immunostaining for adipophilin (data not shown).

 


View larger version (50K):
[in this window]
[in a new window]
  		FIG. 2. Western analysis of adipophilin in fetal membrane total tissue homogenates in the PTNL, PTL, TNL, and TL groups. A distinct adipophilin band at 52 kDa was observed, with no significant differences in band intensity among the four groups of tissues. A, Amnion; C, choriodecidua.

 


View larger version (167K):
[in this window]
[in a new window]
  		FIG. 3. Perilipin immunostaining in human fetal membranes at preterm and term, with or without labor. A, PTNL; B, PTL; C, TNL; D, TL. Perilipin was immunolocalized to the amnion epithelium, amnion fibroblasts, and chorion trophoblasts of all four gestational groups. No immunostaining was observed in controls (data not shown).

 


View larger version (27K):
[in this window]
[in a new window]
  		FIG. 4. Western blot analysis of perilipin in fetal membrane total homogenates at preterm and term, with or without labor. A 62-kDa perilipin band was observed with antiperilipin antibody, with no apparent differences among the four groups of tissues.

 
Western blot analysis of the solubilized lipid-rich layers revealed an abundance of adipophilin (Fig. 5AGo) as well as perilipin (Fig. 5BGo) from both amnion and choriodecidual tissues. In addition, enzymes associated with PGE2 synthesis, such as cPLA2 (Fig. 5CGo), PGHS-2 (Fig. 5DGo), and mPGES-1 (Fig. 5EGo), were detected in the Western immunoblot of the solubilized lipid-rich layer of both tissues. In the Western analysis of the lipid-rich layer, adipophilin (Fig. 5Go, A and F) was significantly more abundant (P < 0.05) in the PTL and TNL groups in the amnion and in the PTL group in the choriodecidua compared with the other gestational age groups. Perilipin concentrations (Fig. 5BGo), in contrast, showed no significant differences between amnion and choriodecidua or across gestational age and with or without labor. Cytoplasmic PLA2 (Fig. 5CGo) was weakly detected only in the PTL, TNL, and TL groups in the amnion tissue lipid-rich layer, whereas no cPLA2 was observed in choriodecidua. A band at 72 kDa was recognized by the anti-PGHS-2 antibody on Western blotting in the lipid-rich layer of both tissue types (Fig. 5DGo), with no difference in band intensity among the four groups. Two distinct bands at 39 and 73 kDa were observed on the blots probed with the antihuman mPGES-1 antiserum (Fig. 5EGo). However, the sizes of these bands did not correspond to native mPGES-1, which is 16 kDa. The 39-kDa band was not observed in the TL and TNL groups in the amnion. The intensity of the upper 73-kDa band was significantly less in TL in the amnion tissue lipid-rich layer compared with the other three groups of tissues (P < 0.05). There was no significant difference in the intensity of the 73-kDa band across the four groups in choriodecidua.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 5. Western blot analysis of adipophilin (A), perilipin (B), cPLA2 (C), PGHS-2 (D), and mPGES-1 (E) on the floating lipid-rich layer from total fetal membrane homogenate of amnion and choriodecidual tissues. Adipophilin, perilipin, cPLA2, PGHS-2, and mPGES-1 were all observed in the lipid-rich layer of the amnion and choriodecidua tissue homogenates, except for cPLA2 which seemed to be absent from choriodecidua. Lane 1, PTNL; lane 2, PTL; lane 3, TNL; lane 4, TL. A representative figure is shown. F, Quantitation of adipophilin expression in lipid-rich layer by scanning densitometry. *, Significantly greater than PTNL and TL (P < 0.05, by ANOVA); +, significantly greater than PTNL, TNL, and TL (P < 0.05, by ANOVA). Values are the mean ± SD (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PGs have a significant role in the initiation and maintenance of labor. It is well documented that during labor, PGE2 production is increased dramatically (29, 36, 37). This increase in PGE2 is associated with increased expression of cPLA2 and PGHS-2 (38, 39, 40, 41, 42, 43, 44, 45). PGE synthases are involved in the terminal step of PGE2 synthesis, i.e. conversion of the endoperoxide PGH2 to the primary PG, PGE2. Our previous studies have shown the presence of the distinct PGES isoforms, mPGES-1 and cPGES, in human fetal membranes (32, 33). In particular, mPGES-1 presented a punctate immunostaining pattern in the fetal membranes, which suggested a possible association with lipid droplets (32). Increased lipid concentrations were observed in fetal membranes throughout gestation (25); however, the role of lipid droplets in parturition and the regulation of their formation are unclear (46). Lipid staining with Sudan Black B in the fetal membranes showed an increase in lipid droplets from early to late gestation and with labor (32). It is possible that lipid droplets have a potential role in energy reserve, mobilization of arachidonic acid, and production of PGE2 at parturition (47). Several proteins have been found to be associated with lipid droplets; among these are adipophilin and perilipin (2, 3, 4).

Adipophilin is a prominent lipid droplet-associated protein that is found on many cell types. Association of adipophilin with cells that accumulate triacylglycerol-rich lipid droplets in the cytoplasm has led to the speculation that adipophilin has an integral role in lipid droplet formation. It has been reported that minute lipid droplets in preadipocytes are coated with adipophilin; after differentiation, the number of adipophilin-coated lipid droplets is increased. However, after the onset of perilipin expression, lipid droplets lose their adipophilin coating and acquire a coat of perilipin (1), suggesting a possible structural function for adipophilin. It is also possible that adipophilin and perilipin have a competitive or exclusive relationship at the lipid droplet surface.

Based on these observations, we studied the expression of adipophilin and perilipin in human fetal membranes. We demonstrated by immunohistochemistry in fetal membranes that adipophilin is present in amnion epithelium, and amnion fibroblasts and in association with fibrin in the choriodecidual layer. Adipophilin had the highest expression in the amnion epithelium and showed a punctate immunostaining pattern similar to that we previously reported for mPGES-1 (32). Immunohistochemistry revealed an apparent increase in adipophilin staining in the membranes with advancing gestation and labor. However, Western blot analysis of the total tissue homogenate showed no significant increase in adipophilin from PTNL to TL; therefore, we examined the lipid-rich layer. Perilipin was also immunolocalized to the amnion epithelium, amnion fibroblasts, and chorion trophoblasts of the fetal membranes, but mainly with a diffuse cytoplasmic pattern of immunostaining. Contrary to our expectations, immunohistochemistry revealed that perilipin is apparently not associated with lipid droplets, except in amnion fibroblasts. This could be due to a mutually exclusive interaction with adipophilin. Perilipin expression in adipocytes correlated with the disappearance of adipophilin, after which lipid droplets acquire a coating of perilipin (1).

Surprisingly, and in contrast to the immunohistochemical data, Western blot analysis of amnion and choriodecidua total tissue homogenate supernatant showed no differences in adipophilin or perilipin expression when comparing preterm and term tissues, with or without labor. However, analysis of the floating lipid-rich layer revealed an abundance of adipophilin and perilipin that was presumably either still associated with lipid droplets or had become trapped in the lipid-rich layer during tissue homogenization. Additional investigation of the floating lipid-rich layer revealed the presence of cPLA2, PGHS-2, and mPGES-1, enzymes involved in PG synthesis. However, we failed to detect an abundance of cytosolic isoforms of PGES, which we have previously shown in amnion and choriodecidua homogenates (32), by Western analysis of the floating lipid-rich layer, suggesting that we are not observing nonspecific trapping in the lipid layer.

It is well established that cPLA2 is present in fetal membranes and placental tissues and plays a key role in PGE2 synthesis coupled to PGHS-2 (49). We observed the presence of cPLA2 in Western blotting of amnion and choriodecidua tissue homogenates as well as in immunohistochemical staining of the human fetal membranes (our unpublished observations). The absence of cPLA2 in the Western blot of the lipid-rich layer of choriodecidua suggests that other PLA2 isoforms, presumably secretory PLA, mediate PG synthesis at labor in choriodecidua. Together with the weak expression of cPLA2 in the lipid-rich layer of amnion, this finding also argues against nonspecific trapping of enzymes in the lipid layer. With the onset of labor, the expression of PGHS-2 mRNA and protein is dramatically increased in fetal membranes (49, 50). It is therefore understandable that PGHS-2 is found in abundance in the amnion and choriodecidua lipid-rich layer. However, there were no significant differences among the four gestational groups. Because PGHS-2 is associated with several cellular fractions, it is possible that the increase in PGHS-2 expressed at labor is associated with some other cellular site in addition to lipid droplets. The terminal enzymes involved in PG synthesis are mPGES-1 and cPGES. The cytosolic isoforms of PGES were noticeably absent from the lipid-rich layer of both amnion and choriodecidua. However, we did detect two bands in the lipid-rich layer that were recognized by anti-mPGES-1 antibody at higher molecular masses than the native 16-kDa mPGES-1 we have previously observed in the tissue homogenates (32). These could be aggregates of mPGES-1 protein or association of mPGES-1 with other proteins within the lipid-rich layer. We previously observed a 180-kDa band immunoreactive with mPGES-1 antibody along with its native monomeric 16-kDa band in immunoblots of Wistar Institute Susan Hayflick (WISH) homogenates (51).

Structural (adipophilin) and functional (perilipin) proteins of lipid droplets are seen in the amnion epithelial, amnion fibroblasts, and chorion trophoblasts. Adipophilin increases with gestation and labor, suggesting an increase in lipid droplet formation, in agreement with observations of Sudan Black B lipid staining (48, 52). Perilipin, in contrast, appeared to be present throughout gestation without a change in concentration. The association of enzymes in the arachidonic acid cascade with the lipid-rich layer suggests that lipid droplets may be foci for PGE2 production via cPLA2, PGHS-2, and mPGES-1 at the time of labor. It is possible that this is a novel route for PG production in fetal membranes that has been previously unrecognized, because paraffin fixation of tissues will remove lipid droplets before immunohistochemistry, and the lipid-rich layer of cellular homogenates is usually discarded; therefore, any proteins associated with it would not be measured.


   Footnotes
 
First Published Online January 18, 2005

Abbreviations: ABC, Avidin-biotin-peroxidase complex; ADRP, adipose differentiation-related protein; CE, cholesterol ester; cPLA2, cytosolic phospholipase A2; HSL, hormone-sensitive lipase; LSD, lipid storage droplet; mPGES, microsomal-associated prostaglandin E synthase; PG, prostaglandin; PGES, prostaglandin E synthase; PGHS-2, PG endoperoxide; PTL, preterm with labor; PTNL, preterm no labor; TBS, Tris-buffered saline; TG, triglyceride; TL, at term with labor; TNL, at term without labor.

Received June 23, 2004.

Accepted January 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Londos C, Brasaemle DL, Schultz CJ, Segrest JP, Kimmel AR 1999 Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin Cell Dev Biol 10:51–58[CrossRef][Medline]
  2. Brown DA 2001 Lipid droplets: proteins floating on a pool of fat. Curr Biol 11:R446–R449
  3. van Meer G 2001 Caveolin, cholesterol, and lipid droplets? J Cell Biol 152:F29–F34
  4. Ostermeyer AG, Paci JM, Zeng Y, Lublin DM, Munro S, Brown DA 2001 Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets. J Cell Biol 152:1071–1078[Abstract/Free Full Text]
  5. Imamura M, Inoguchi T, Ikuyama S, Taniguchi S, Kobayashi K, Nakashima N, Nawata H 2002 ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts. Am J Physiol 283:E775–E783
  6. Jiang HP, Serrero G 1992 Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein. Proc Natl Acad Sci USA 89:7856–7860[Abstract/Free Full Text]
  7. Heid HW, Moll R, Schwetlick I, Rackwitz HR, Keenan TW 1998 Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res 294:309–321[CrossRef][Medline]
  8. Miura S, Gan JW, Brzostowski J, Parisi MJ, Schultz CJ, Londos C, Oliver B, Kimmel AR 2002 Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J Biol Chem 277:32253–32257[Abstract/Free Full Text]
  9. Lu X, Gruia-Gray J, Copeland NG, Gilbert DJ, Jenkins NA, Londos C, Kimmel AR 2001 The murine perilipin gene: the lipid droplet-associated perilipins derive from tissue-specific, mRNA splice variants and define a gene family of ancient origin. Mamm Genome 12:741–749[Medline]
  10. McManaman JL, Zabaronick W, Schaack J, Orlicky DJ 2003 Lipid droplet targeting domains of adipophilin. J Lipid Res 44:668–673[Abstract/Free Full Text]
  11. Brasaemle DL, Barber T, Wolins NE, Serrero G, Blanchette-Mackie EJ, Londos C 1997 Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 38:2249–2263[Abstract]
  12. Gao J, Serrero G 1999 Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake. J Biol Chem 274:16825–16830[Abstract/Free Full Text]
  13. McManaman JL, Palmer CA, Wright RM, Neville MC 2002 Functional regulation of xanthine oxidoreductase expression and localization in the mouse mammary gland: evidence of a role in lipid secretion. J Physiol 545:567–579[Abstract/Free Full Text]
  14. Mather IH, Keenan TW 1998 Origin and secretion of milk lipids. J Mammary Gland Biol Neoplasia 3:259–273[CrossRef][Medline]
  15. Buechler C, Ritter M, Duong CQ, Orso E, Kapinsky M, Schmitz G 2001 Adipophilin is a sensitive marker for lipid loading in human blood monocytes. Biochim Biophys Acta 1532:97–104[Medline]
  16. Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR{gamma}2, a lipid-activated transcription factor. Cell 79:1147–1156[CrossRef][Medline]
  17. Garcia A, Sekowski A, Subramanian V, Brasaemle DL 2003 The central domain is required to target and anchor perilipin A to lipid droplets. J Biol Chem 278:625–635[Abstract/Free Full Text]
  18. Londos C, Brasaemle DL, Gruia-Gray J, Servetnick DA, Schultz CJ, Levin DM, Kimmel AR 1995 Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells. Biochem Soc Trans 23:611–615[Medline]
  19. Egan JJ, Greenberg AS, Chang MK, Londos C 1990 Control of endogenous phosphorylation of the major cAMP-dependent protein kinase substrate in adipocytes by insulin and ß-adrenergic stimulation. J Biol Chem 265:18769–18775[Abstract/Free Full Text]
  20. Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ, Londos C 1991 Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J Biol Chem 266:11341–11346[Abstract/Free Full Text]
  21. Greenberg AS, Egan JJ, Wek SA, Moos Jr MC, Londos C, Kimmel AR 1993 Isolation of cDNAs for perilipins A and B: sequence and expression of lipid droplet-associated proteins of adipocytes. Proc Natl Acad Sci USA 90:12035–12039[Abstract/Free Full Text]
  22. Servetnick DA, Brasaemle DL, Gruia-Gray J, Kimmel AR, Wolff J, Londos C 1995 Perilipins are associated with cholesteryl ester droplets in steroidogenic adrenal cortical and Leydig cells. J Biol Chem 270:16970–16973[Abstract/Free Full Text]
  23. Blanchette-Mackie EJ, Dwyer NK, Barber T, Coxey RA, Takeda T, Rondinone CM, Theodorakis JL, Greenberg AS, Londos C 1995 Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes. J Lipid Res 36:1211–1226[Abstract]
  24. Londos C, Brasaemle DL, Schultz CJ, Adler-Wailes DC, Levin DM, Kimmel AR, Rondinone CM 1999 On the control of lipolysis in adipocytes. Ann NY Acad Sci 892:155–168[Abstract/Free Full Text]
  25. Bleasdale JE, Johnston JM 1984 Prostaglandins and human parturition; regulation of arachidonic acid metabolism. Rev Perinatal Med 5:151–191
  26. Weller PF, Dvorak AM 1994 Lipid bodies: intracellular sites for eicosanoid formation. J Allergy Clin Immunol 94:1151–1156[CrossRef][Medline]
  27. Dvorak AM, Morgan E, Schleimer RP, Ryeom SW, Lichtenstein LM, Weller PF 1992 Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils. J Histochem Cytochem 40:759–769[Abstract]
  28. Yu W, Bozza PT, Tzizik DM, Gray JP, Cassara J, Dvorak AM, Weller PF 1998 Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies. Am J Pathol 152:759–769[Abstract]
  29. Challis JR, Lye SJ, Gibb W 1997 Prostaglandins and parturition. Ann NY Acad Sci 828:254–267[Free Full Text]
  30. Gibb W 1998 The role of prostaglandins in human parturition. Ann Med 30:235–241[Medline]
  31. Weiss G 2000 Endocrinology of parturition. J Clin Endocrinol Metab 85:4421–4425[Free Full Text]
  32. Meadows JW, Eis AL, Brockman DE, Myatt L 2003 Expression and localization of prostaglandin E synthase isoforms in human fetal membranes in term and preterm labor. J Clin Endocrinol Metab 88:433–439[Abstract/Free Full Text]
  33. Alfaidy N, Sun M, Challis J, Gibb W 2003 Expression of membrane prostaglandin E synthase in human placenta and fetal membranes and effect of labor. Endocrine 20:219–226[CrossRef][Medline]
  34. Zarlingo TJ, Eis AL, Brockman DE, Kossenjans W, Myatt L 1997 Comparative localization of endothelial and inducible nitric oxide synthase isoforms in haemochorial and epitheliochorial placentae. Placenta 18:511–5120[CrossRef][Medline]
  35. Kiernan J 1990 Lipids. In: Kiernan JA, ed. Histological and histochemical methods: theory and practice. New York: Pergamon Press; 207
  36. Smith WL 1992 Prostanoid biosynthesis and mechanisms of action. Am J Physiol 263:F181–F191
  37. Olson DM, Skinner K, Challis JR 1983 Prostaglandin output in relation to parturition by cells dispersed from human intrauterine tissues. J Clin Endocrinol Metab 57:694–699[Abstract]
  38. Macchia L, Di Paola R, Guerrese MC, Chiechi LM, Tursi A, Caiaffa MF, Haeggstrom JZ 1997 Expression of prostaglandin endoperoxide H synthase 1 and 2 in human placenta at term. Biochem Biophys Res Commun 233:496–501[CrossRef][Medline]
  39. Mijovic JE, Zakar T, Nairn TK, Olson DM 1997 Prostaglandin-endoperoxide H synthase-2 expression and activity increases with term labor in human chorion. Am J Physiol 272:E832–E840
  40. Zuo J, Lei ZM, Rao CV, Pietrantoni M, Cook VD 1994 Differential cyclooxygenase-1 and -2 gene expression in human myometria from preterm and term deliveries. J Clin Endocrinol Metab 79:894–899[Abstract]
  41. Erkinheimo TL, Saukkonen K, Narko K, Jalkanen J, Ylikorkala O, Ristimaki A 2000 Expression of cyclooxygenase-2 and prostanoid receptors by human myometrium. J Clin Endocrinol Metab 85:3468–3475[Abstract/Free Full Text]
  42. Gibb W, Sun M 1996 Localization of prostaglandin H synthase type 2 protein and mRNA in term human fetal membranes and decidua. J Endocrinol 150:497–503[Abstract]
  43. Gijon MA, Leslie CC 1999 Regulation of arachidonic acid release and cytosolic phospholipase A2 activation. J Leukocyte Biol 65:330–336[Abstract]
  44. Leslie CC 1997 Properties and regulation of cytosolic phospholipase A2. J Biol Chem 272:16709–16712[Free Full Text]
  45. Skannal DG, Brockman DE, Eis AL, Xue S, Siddiqi TA, Myatt L 1997 Changes in activity of cytosolic phospholipase A2 in human amnion at parturition. Am J Obstet Gynecol 177:179–184[CrossRef][Medline]
  46. Sonek J, Gabbe SG, Iams JD, Kniss DA 1991 Morphologic changes in the human amnion epithelium that accompany labor as seen with scanning and transmission electron microscopy. Am J Obstet Gynecol 164:1174–1180[Medline]
  47. Okita JR, MacDonald PC, Johnston JM 1982 Mobilization of arachidonic acid from specific glycerophospholipids of human fetal membranes during early labor. J Biol Chem 257:14029–14034[Abstract/Free Full Text]
  48. Meadows JW, Pitzer B, Brockman D, Myatt L 2004 Adipophilin and signal transduction from lipid droplets in human fetal membranes. J Soc Gynecol Invest 11:77A
  49. Mijovic JE, Zakar T, Nairn TK, Olson DM 1999 Prostaglandin endoperoxide H synthase mRNA expression in the human amnion and decidua during pregnancy and in the amnion at preterm labour. Mol Hum Reprod 5:182–187[Abstract/Free Full Text]
  50. Slater D, Dennes W, Sawdy R, Allport V, Bennett P 1999 Expression of cyclo-oxygenase types-1 and -2 in human fetal membranes throughout pregnancy. J Mol Endocrinol 22:125–130[Abstract]
  51. Meadows JW, Brockman D, Pitzer B, Myatt L 2003 Effect of interleukin-1 and lipopolysaccharide on prostaglandin E synthase expression in amnion-derived WISH cells. J Soc Gynecol Invest 10:146A
  52. Meadows JW, Pitzer B, Brockman D, Myatt L 2004 Expression and localization of perilipin A in human amnion and chorion. J Soc Gynecol Invest 11:231A 77A



This article has been cited by other articles:


Home page
 FASEB J.Home page
H.-C. Wan, R. C. N. Melo, Z. Jin, A. M. Dvorak, and P. F. Weller
Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies
FASEB J, January 1, 2007; 21(1): 167 - 178.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/4/2344    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Meadows, J. W.
Right arrow Articles by Myatt, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Meadows, J. W.
Right arrow Articles by Myatt, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals