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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0808
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 5 3045-3053
Copyright © 2005 by The Endocrine Society

PL74, a Novel Member of the Transforming Growth Factor-ß Superfamily, Is Overexpressed in Preeclampsia and Causes Apoptosis in Trophoblast Cells

Hongshi Li, Jamal Dakour, Larry J. Guilbert, Bonnie Winkler-Lowen, Fiona Lyall and Donald W. Morrish

Departments of Medicine (H.L., J.D., D.W.M.) and Medical Microbiology and Immunology (L.J.G., B.W.-L.), University of Alberta; Edmonton, Alberta, Canada T6G 2S2; and Institute of Medical Genetics (F.L.), University of Glasgow, Glasgow G3 8SJ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Donald W. Morrish, 362 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail: dmorrish{at}gpu.srv.ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PL74, a novel member of the TGFß superfamily that has highest expression in placenta, is a multifunctional peptide that can induce differentiation, inhibit inflammatory stimulation of TNF{alpha}, and execute apoptosis after p53 overexpression and cytotoxic injury. To study its expression and function in placenta and preeclampsia, we first determined mRNA expression in nine normal and 10 preeclamptic placentas. PL74 mRNA was overexpressed by 57.3% in preeclampsia. Transfection of PL74 into term cytotrophoblasts resulted in increased apoptosis by terminal uridine deoxynucleotidyl nick end labeling labeling (control, 2.8 ± 0.5%; PL74, 19.1 ± 0.2%; P < 0.005). Addition of PL74 protein to HTR8/SVneo extravillous cytotrophoblast cells showed a dose-response (0–100 ng/ml) inhibition of [3H]thymidine uptake and increase in apoptosis shown by terminal uridine deoxynucleotidyl nick end labeling and histone-associated DNA fragment ELISA (control, 0.11 ± 0.01 absorbance units; PL74, 0.21 ± 0.01; P < 0.01). PL74 did not alter cytotrophoblast invasion using a Matrigel in vitro invasion assay. Cytokine regulation of PL74 mRNA expression in term cytotrophoblasts showed that epidermal growth factor and IFN{gamma} increased PL74 expression, but TGFß and TNF{alpha} had no effect. Transfection of antisense PL74 into term cytotrophoblast cells resulted in an inhibition of spontaneous differentiation at 2 and 24 h of culture (control vector, 30.8 ± 3.1% and 26.4 ± 1.2%; antisense PL74, 17.6 ± 1.8%and 12.6 ± 1.4% syncytial units, at 2 and 24 h respectively; P < 0.01). We conclude that PL74 is overexpressed in preeclampsia and may thus promote apoptosis of cytotrophoblasts at the expense of differentiation. PL74 secretion is induced by IFN{gamma} and may play a role in abnormal placental responses in preeclampsia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TGFß SUPERFAMILY is a large group of cytokines that have activity regulating proliferation, differentiation, extracellular matrix formation, immune defense, and organogenesis (1). In 1996, we identified PL74, a new member of the TGFß superfamily, from a subtractive library of in vitro differentiating human trophoblasts (2). This peptide has been separately identified by several groups and called macrophage inhibitory cytokine 1 (MIC-1) (3), growth differentiation factor 15 (4, 5), prostate-derived factor (6), nonsteroidal antiinflammatory drug-activated gene 1 (7), HT-1080 (8), placental bone morphogenetic protein (9, 10), and placental TGFß (11, 12). PL74 is divergent from other members of the TGFß superfamily but has highest similarity to bone morphogenetic proteins 5–8 (31–35% homology) and the dpp- and Vgl-related group (growth differentiation factors 1, 7, and 9; 31% homology) and lesser similarity to TGFß1–3 (21–25%). It is widely expressed with greatest expression in human tissues in the placenta (8, 9, 13). PL74 has a unique secretory mechanism in which the propeptide is not required for correct folding and secretion but acts as a quality control determinant of protein folding (14, 15). Multiple functions for PL74 include inhibition of hematopoietic precursor proliferation (9); as a downstream executor of apoptosis as induced by nonsteroidal antiinflammatory drugs, p53 overexpression, radiation, or chemotherapeutic drug exposure (11, 12, 15, 16); as an inhibitor of lipopolysaccharide-induced production of TNF{alpha} by macrophages (3); and as an inducer of cartilage formation and early bone formation in rats (6). Additional studies of MIC-1 found elevated levels in women who subsequently had cardiovascular events, linking possible activated macrophage overproduction to atherosclerotic events (17). Regulatory factors described for PL74 secretion include androgen stimulation in prostate (6) and stimulation of production in monocytes of IL-1ß, TNF{alpha}, IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TGFß1 (3). There is thus an apparent link between PL74 (MIC-1) and immune function.

During pregnancy, maternal plasma levels of PL74 (named MIC-1 in these studies) increase 15-fold (18). Both maternal serum and immunostaining assessment of MIC-1 quantity in preeclamptic tissues has been performed but not found to be altered compared with normal placentas (19). These data thus suggest any potential effects of PL74 on trophoblast function may be on a paracrine basis. Although PL74 has high expression in placenta, its function in this tissue is unknown. Because of its diverse functional capabilities, we wished to further study possible paracrine function and regulation of PL74 in human trophoblast cells and to determine whether there were altered expression in preeclampsia.


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

Placenta collections and the study protocol were approved by the University of Alberta Research Ethics Board. Placental samples were collected within 0.5 h after delivery and kept on ice in culture medium until freezing at –70 C. Samples for extraction were randomly chosen from the interior of cotyledons in areas without obvious infarcts or other pathology. Placental bed biopsies were obtained under direct visualization, after approval by the Joint Ethics Committee of Newcastle and North Tyneside, and snap frozen in liquid nitrogen as previously described (20). Preeclampsia was defined according to the Report of the National High Blood Pressure Education Program Working Group criteria as hypertension starting during pregnancy after 20 wk duration with blood pressure more than 140 systolic and more than 90 diastolic, and proteinuria of at least 1 (21).

PL74 identification and protein purification

The sequence was identified from a subtractive cDNA library (2) (GenBank U51731). A placental 5' stretch cDNA library (BD Biosciences, Clontech, Palo Alto, CA) was screened to obtain an insert containing the entire gene, which was cloned into pBluescript. Complete nucleotide sequencing was performed by primer walking using the dideoxy method using an Applied Biosystems (Foster City, CA) automated sequencer. Regions of disagreement with published sequences were resequenced three times for confirmation. Polyclonal antibodies to a unique sequence of PL74 as previously described (6) were generated in rabbits by ResGen Invitrogen Corp. (Huntsville, AL). Full-term placental trophoblast cultures were prepared as described (22) and the culture medium harvested for extraction of PL74 protein. Protein from pooled medium was precipitated with 100 mM zinc sulfate, centrifuged, lyophilized, and resuspended in PBS (pH 7.4). The IgG fraction of the antibody was purified by protein-A Sepharose column chromatography (Pharmacia Biotech/Amersham, Uppsala, Sweden) and then coupled by cyanogen bromide to an activated Sepharose column (Pharmacia). Resuspended protein was added to the column, and immunopurified PL74 protein eluted using 100 mM glycine (pH 2.7). Protein was run on Western blots to determine purity. Protein content was assayed by Bio-Rad (Hercules, CA) protein assay.

Northern blot hybridization

cDNA probes of the entire coding region were prepared using Wizard Minipreps purification system (Promega, Madison, WI), run on 1% agarose gels, purified with Geneclean II, and labeled with [32P]dCTP using the random DNA labeling system (Invitrogen, Carlsbad, CA). Total mRNA from either term normal (n = 10) or preeclamptic placentas (n = 10) or cultured term cytotrophoblast or extravillous cytotrophoblast HTR8/SVneo cells (see below) were prepared using RNeasy kits (QIAGEN Inc., Valencia, CA), run on 1.2% agarose-formaldehyde denaturing gels, and transferred to Nytran membrane (Schleicher and Schuell, Keene, NH). Hybridization was performed at 42 C for 20 h using 5x piperazine-N,N'-bis(20-ethanol sulfonic acid), 50% formamide, 5x Denhardt’s solution, 1% SDS, 0.1% sodium pyrophosphate, and 1% salmon sperm DNA. The blots were washed with 1x standard saline citrate (SSC)/0.4% SDS for 15 min at room temperature, 0.5x SSC/0.4% SDS for 15 min at room temperature, and 0.1% SSC/0.4% SDS for 45 min at 65 C. Equality of lane loading was determined by reprobing the membrane with an 18 S rRNA probe (American Type Culture Collection, Manassas, VA).

Western blots

Tissue was extracted in lysis buffer containing 0.5% Nonidet P-40, 14 µM leupeptin (Roche Molecular Biochemicals, Laval, Canada), 10 µM pepstatin A (Sigma Chemical Co., St. Louis, MO), 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM benzamidine (Sigma), and 0.01 mg/ml trypsin inhibitor (Sigma) in PBS and incubated at 4 C for 1 h. Lysates were centrifuged at 13,000 rpm at 4 C for 5 min, and supernatants were collected for determination of protein content (Bio-Rad). SDS-PAGE was carried out by standard methods using 10% premade gels from Bio-Rad. Aliquots of sample lysates containing equivalent amounts of protein were added to 0.25 vol of 4-fold concentrated reducing sample buffer (0.25 M Tris/HCl, pH 6.8; 8% wt/vol SDS; 40% wt/vol glycerol; 2.84 M ß-mercaptoethanol; and 5% wt/vol bromphenol blue) and boiled for 10 min before loading onto gels. After electrophoresis, proteins were transferred electrophoretically onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h using 7% milk protein and 2% albumin, and the 1:1000 primary antibody to PL74 in 2% milk powder was added overnight at 4 C. Membranes were washed three times with PBS/Tween 20 and then incubated with 1:10,000 secondary antibody, washed four times, and placed in developer solution (ECL, Amersham Biosciences, Piscataway, NJ) for 1 min. Membranes were then transferred to cassettes for autoradiography.

Cell culture

Term human cytotrophoblast preparations were prepared as previously described using trypsin-DNase I digestion, which produces a cell preparation over 95% pure for cytotrophoblast with fewer than five vimentin-positive cells per105 cells (22, 23). Cells were plated at 6–8 x 106 cells per dish in 100-mm petri dishes (Corning) for mRNA studies and cultured in 10% FBS/DMEM/penicillin/streptomycin as described (22). Cells were attached for 2 h, and then the medium was changed to serum-free DMEM for cytokine addition studies. These cells rapidly differentiate spontaneously over 18–24 h toward a syncytial phenotype including up-regulation of most syncytial gene products and formation of some morphological syncytia but have a villous cytotrophoblast phenotype in early (<3 h) culture (24). Cytokine regulation in cytotrophoblast was determined by exposing the cells for 3 h to 10 ng/ml epidermal growth factor (EGF), 10 ng/ml TGFß1, 10 ng/ml TNF{alpha}, or 100 U/ml IFN{gamma}, and the cells were extracted for PL74 mRNA analysis. Three separate experiments were performed for EGF and IFN{gamma} or control, and one for TNF{alpha} and TGFß1. Expression of PL74 mRNA in cells cultured for 18 h when they had differentiated toward syncytium was also performed. In experiments testing antisense PL74 transfection, the term cytotrophoblast cells were further purified using combined HLA class I and II and CD9 immunopurification and cultured in 10% FBS/IMDM as previously described (25) to allow quantitation of syncytial formation by desmoplakin immunostaining (26, 27). Purified cells were frozen after preparation and thawed and replated for experiments. Transfections were performed either immediately after replating (~6–8 h after first trypsinization) or at 24 h in culture. Unpurified and immunopurified cells behave essentially identically in culture, except immunopurified cells differentiate more slowly, permitting better assessment of changes in differentiation rates (23, 24, 28, 29). A first-trimester extravillous cytotrophoblast cell line (HTR8/SVneo) (30, 31, 32) was kindly obtained from Dr. C. Graham, Queen’s University, Kingston, Canada, and cultured in 10% FBS/DMEM.

Transfections, apoptosis quantitations, [3H]thymidine labeling, and desmoplakin quantitation of syncytial formation

The PL74 coding sequence was cloned into pcDNA.3 (Invitrogen) in the 5' to 3' direction for sense transfectants, and in the 3' to 5' direction for antisense transfectants. Transient transfections were performed using Lipofectin (GIBCO-BRL, Gaithersburg, MD). Six micrograms of purified plasmid DNA and 15 µl Lipofectin were added to cells and processed according to the manufacturer’s instructions. Transfections were analyzed 72 h after transfection. Control transfectants contained empty vector alone. Transfection efficiency was determined by cotransfecting ß-galactosidase and measuring the expressed galactosidase activity in a colorimetric assay (Stratagene, La Jolla, CA). Sense transfections were performed using human term cytotrophoblast cells without further immunopurification. Apoptosis in these transfected cells was determined using terminal uridine deoxynucleotidyl nick end labeling (TUNEL) (Cell Death Detection Kit; Roche) on cells fixed with 1:1 cold methanol:acetone on the plates and photographed. Three separate experiments with three to four replicate cultures were performed on cells immediately after plating and attachment and on two experiments 24 h after plating. Between 460 and 1046 cells per microscope field were counted for each replicate using at least four separate microscope fields. For studies of antisense transfection effect on cytotrophoblast differentiation, cryopreserved CD9/HLA class I/II-purified cells were used. Desmoplakin staining was performed as previously described (27). Formation of syncytia was quantitated by counting nuclei with desmoplakin-stained cell outlines (26, 27). The ratio of multinucleated cell groups (at least two nuclei) to total number of nuclei was calculated as a measure of syncytial unit formation and expressed as a percentage. Two separate transfection experiments were performed immediately after thawing and reconstitution of the cells, and two experiments were done 24 h after replating. Eight randomly chosen microscope fields were counted for each experiment to determine percent syncytial unit formation, and 50–132 nuclei per field were counted in each experiment.

The effects of PL74 protein on apoptosis and [3H]thymidine uptake was measured in HTR8/SVneo cells. Doses of 0, 10, and 100 ng/ml protein were added to quadruplicate cultures for 48 h, and percent apoptotic cells (100–160 cells per plate counted) was measured using TUNEL. In separate experiments, to confirm the induction of apoptosis, HTR8/SVneo cells were exposed to 10 ng/ml PL74 protein for 24 h and cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes), specific for apoptosis, were measured (Cell Death Detection ELISA; Roche). For [3H]thymidine labeling experiments, doses of 0, 1, 3, and 7 ng/ml PL74 protein were added to triplicate wells for each dose for 48 h. The cells were then exposed to 5 µCi/ml [3H]thymidine (Amersham) for 4 h. Cells were washed with PBS and trypsinized using 0.25% trypsin/10 mM EDTA for 5 min, centrifuged, and counted in a liquid scintillation counter.

In vitro cytotrophoblast invasion assay

The effects of PL74 protein on HTR8/SVneo cell invasion were determined using an in vitro Matrigel (Becton Dickinson-Collaborative Biomedical Products, Franklin Lakes, NJ) invasion assay as previously described (33). In brief, transwells (Corning-Costar, Acton, MA) were coated with growth-factor-free Matrigel (1:15 dilution) using serum-free medium and allowed to dry. One hundred microliters of serum-free DMEM were added to each gel, and the Matrigel was allowed to rehydrate for 1 h. Then, 50,000 HTR8/SVneo cells were added in 100 µl DMEM in the presence or absence of 6 ng/ml PL74 to the transwell, and 750 µl medium was added to each well under the insert and the transwell replaced. Cells were incubated for 24 h under 20% oxygen. Medium was removed and the transwell rinsed three times with PBS, and the inside of the transwell was wiped with a cotton swab. Five hundred microliters of cold methanol were added to the outer well, and the insert was allowed to fix for 1 min and was then rinsed three times with PBS. Five hundred microliters of 1% toluidine blue/1% sodium borate solution were added to the transwells and stained overnight. The membrane was then cut out and mounted on a slide and sealed. Cells invading through to the bottom of the membrane were counted. The experiment was performed in duplicate.

Immunostaining of the placental implantation site

Placental bed biopsies of normal and preeclamptic pregnancies were obtained as previously described (20). Slides of frozen tissue sections stored at –70 C were allowed to slowly come to room temperature and fixed in acetone for 5 min and then 100% ethanol for 5 min and then washed in distilled water twice for 5 min each. Slides were blocked with 0.01% horse serum in PBS. Primary antibody (1:500) with or without blocker was added to slides for 24 h at 4 C and washed twice with PBS. Biotinylated second antibody was added for 30 min at room temperature. After washing, endogenous peroxidase was blocked with 1% H2O2 in methanol for 15 min at room temperature and then washed in PBS. ABC complex (Vectastain ABC Universal Elite Kit; Vector Laboratories, Burlingame, CA) was added for 30 min at room temperature and then washed, and diaminobenzidine (Sigma) was added for 5 min. The slides were dehydrated through a series of alcohols from 30–100%, treated with Histoclear for 1 min, and then mounted with DePeX.

Statistical analysis

Comparison of means was by ANOVA or t test. Results are expressed as the mean ± SEM.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical parameters in normal and preeclamptic women

Table 1Go lists the clinical features of the nine normal and 10 preeclamptic women from whom placentas were obtained.


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  		TABLE 1. Characteristics of controls and patients with preeclampsia

 
PL74 mRNA expression in normal and preeclamptic placentas and cultured cells

PL74 mRNA and protein expression in nine normal and 10 preeclamptic placentas are shown in Fig. 1Go. After normalization to 18 S rRNA, densitometry showed that the PL74/18 S rRNA ratio was 0.75 ± 0.03 (mean ± SEM) in normal placentas and 1.18 ± 0.05 for preeclamptic placentas, a 57% increase (P < 0.01, ANOVA). PL74 protein expression by Western blot was qualitatively increased in preeclamptic placentas. PL74 mRNA expression in HTR8/SVneo extravillous cytotrophoblast cells and in term human cytotrophoblast and cells undergoing syncytialization after 18 h are shown in Fig. 2Go. HTR8/SVneo cells have a low but present expression of PL74. Newly isolated term cytotrophoblasts during early culture (Fig. 2Go, time zero) are phenotypically villous cytotrophoblasts and do not express PL74 but increase expression rapidly as they differentiate into syncytium. EGF promotes differentiation (2, 24) and hence increases PL74 expression in very early culture (see Fig. 5BGo), but by 18 h expression has become maximal and EGF cannot further increase expression (representative experiment shown in Fig. 2Go; repeated three times).



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  		FIG. 1. PL74 mRNA (top) and protein expression (bottom) in term normal and preeclamptic placentas and 18 S rRNA (middle) for nine normal and 10 preeclamptic placentas.

 


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  		FIG. 2. Expression of PL74 mRNA in term cytotrophoblast, in term syncytialized trophoblast at 18 h of culture, and in HTR8/SVneo extravillous cytotrophoblast cells.

 


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  		FIG. 5. A, Cytokine regulation of PL74 mRNA expression in term cytotrophoblast at 3 h of culture. B, Densitometric normalization of EGF, IFN{gamma}, and control PL74 mRNA expression; mean ± SEM of three separate experiments.

 
Effects on term cytotrophoblast apoptosis and differentiation

Transient transfection of sense PL74 into term human cytotrophoblast cells immediately after preparation and plating (~6 h) significantly increased apoptosis from 2.8 ± 0.5% (control) to 19.1 ± 0.2% (PL74 transfected) by TUNEL (three separate placental experiments; P < 0.05, ANOVA). Transfection of sense PL74 performed 24 h after plating of cells in two separate experiments showed a similar induction of apoptosis by TUNEL (empty vector control, 3.5 ± 0.5%; sense PL74, 16.2 ± 2.2%; P < 0.05, ANOVA). Transient transfection of an antisense PL74 construct into term cytotrophoblast cells immediately after replating and reconstitution of cells (2 h of culture, ~8 h after first trypsinization) decreased formation of syncytial units from 30.8 ± 3.1% of nuclei (empty vector control) to 17.6 ± 1.8% (antisense PL74 transfected) (two separate transfections; P < 0.01, ANOVA). When the antisense transfection was delayed until 24 h after replating of cells to allow further spontaneous differentiation to occur, antisense PL74 also induced a significant reduction in formation of syncytial units (control, 26.4 ± 3.4%; antisense PL74, 12.6 ± 1.2%; two separate experiments; P < 0.01, t test). A representative experiment is shown in Fig. 3Go. Transfection efficiency as determined by cotransfection with galactosidase was 15–20%.



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  		FIG. 3. Transfection of term normal cytotrophoblast cells with either empty vector control immediately after replating cells (A) or antisense PL74 (B) and transfection performed 24 h after replating cells (C, control; D, antisense PL74). Arrows mark mononuclear cytotrophoblast cells (C) or syncytial units comprising at least two nuclei (S).

 
Effects on HTR8/SVneo extravillous cytotrophoblast proliferation and apoptosis

There was a significant dose-related reduction in [3H]thymidine labeling, with a 45% decrease with 7 ng/ml peptide (Table 2Go) (P < 0.01, ANOVA). There was a dose-related significant increase in apoptosis at each dose over a dose range of 0–100 ng/ml PL74 protein (Table 3Go) (P < 0.01, ANOVA). A representative micrograph of TUNEL of the three doses of PL74 demonstrating an increasing percentage of TUNEL-positive cells is shown in Fig. 4Go. Apoptosis was also quantitated by measuring histone-associated DNA fragments by ELISA. These results showed that exposure to 10 ng/ml PL74 protein for 24 h significantly increased DNA fragment formation (control, 0.11 ± 0.01 absorbance units; PL74, 0.21 ± 0.01 absorbance units; P < 0.01, ANOVA).


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  		TABLE 2. Dose-response reduction in [3H]thymidine uptake in HTR8/SVneo cells by PL74 protein

 

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  		TABLE 3. Dose-response increase in apoptosis induced by PL74 protein

 


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  		FIG. 4. TUNEL of HTR8/SVneo cells treated with 0 (A), 10 (B), or 100 (C) ng/ml PL74 protein. Arrows mark apoptotic TUNEL-positive cells.

 
Cytokine regulation of PL74 expression in term cytotrophoblasts

EGF and IFN{gamma} significantly increased PL74 expression in early-culture (3-h) term cytotrophoblast cells (Fig. 5AGo) (three separate placental experiments; P < 0.05, ANOVA), but TNF{alpha} and TGFß did not significantly affect expression. Densitometric normalization is shown in Fig 5BGo.

Effects on HTR8/SVneo invasion

PL74 protein at 6 ng/ml did not affect in vitro invasion (control, 1980 ± 13 cells invading; PL74, 1982 ± 9 cells invading).

Expression of PL74 protein in placental bed biopsies

Immunostaining of normal placental bed biopsies is shown in Fig. 6Go. Positive staining was observed in multinucleated giant cells (Fig. 6Go, A and C), in mononuclear invading extravillous cytotrophoblast cells (Fig. 6BGo), and in endovascular trophoblast (Fig. 6CGo). Myometrium was also positive for PL74 staining (Fig. 6CGo). No significant differences in staining intensity were observed between normal and preeclamptic placentas.



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  		FIG. 6. Placental bed biopsies of normal (A, B, C, and F) and preeclamptic (D and E) placentas. A, Deciduas with spiral artery; B, placental bed with spiral artery; C, myometrium; D, placental bed with spiral artery; E, parallel section to D stained with cytokeratin (arrows indicate trophoblast cells); F, negative control. MG, multinucleate trophoblast giant cell; evC, mononuclear extravillous cytotrophoblast; ET, endovascular trophoblast; M, myometrial cell; SA, transformed spiral artery.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our experiments have demonstrated that PL74 is overexpressed in preeclamptic placenta and can induce dose-dependent apoptosis as well as cytotrophoblast differentiation into syncytium. IFN{gamma} and EGF both induce PL74 in cytotrophoblasts.

In normal trophoblast biology, multiple factors are known that regulate differentiation of cytotrophoblast, a process in which mononuclear cytotrophoblast cells fuse to form the syncytium (34). The inhibition of differentiation by PL74 antisense transfection indicates a normal function of PL74 is to enhance cytotrophoblast differentiation into syncytium. This effect would continue throughout pregnancy because PL74 (MIC-1) increases with gestation in parallel with the increase in placental size and is primarily a syncytial product with only sporadic expression in other placental subtypes (19). Our data also indicate that PL74 promotes differentiation both in phenotypic cytotrophoblast as well as in partly differentiated cytotrophoblast, because the effect was noted both in early (2 h) and late (24 h) cells in culture. Several other syncytial products also either promote (hCG, syncytin, corticosteroids, and estradiol) or inhibit (TGFß1 and prostaglandins) differentiation (34, 35, 36, 37, 38). Thus, the syncytium can act as a paracrine regulator of cytotrophoblast differentiation and hence its own formation. This regulatory control system exists in addition to other differentiation regulation by decidual and macrophage products (CSF-1, GM-CSF), and cytotrophoblast (leukemia inhibitory factor) (39).

Apoptosis and fusion (the defining event of cytotrophoblast differentiation) appear to share the same initial steps, including the initiation stages of apoptosis (expression of initiator caspases) and externalization of phosphatidylserine on the membrane (37). It is of interest that Huppertz et al. (40, 41, 42) have proposed that cytotrophoblast fusion to form syncytium (differentiation) is linked with activation of apoptosis to regulate trophoblast turnover and shown that blocking caspase 8 activation inhibits morphological differentiation. The current data indicate PL74 can also induce apoptosis in cytotrophoblast both at early stages of culture when a cytotrophoblast phenotype is predominant and later in culture when a syncytial phenotype predominates, and is therefore consistent with this hypothesis. These dual actions of PL74 operating over the same time course suggest PL74 can act as a switch between apoptosis and differentiation. In our experiments, there was an increasing effect of apoptosis from 0–100 ng/ml; it is known that mean pregnancy serum levels of PL74 (MIC-1) range from 11–38 ng/ml over gestation (19) so that the effects of PL74 we observed are within the physiological range. In other cell systems, PL74 also has been shown to have either prodifferentiative or proapoptotic capabilities (6, 11, 12, 43). Our current studies are thus consistent with a dual functional capability for PL74.

In preeclampsia, there is impaired extravillous cytotrophoblast invasion into the endometrium with concomitant defective remodeling of spiral arteries, abnormalities in a large number of circulating substances, and increased apoptosis of invasive extravillous cytotrophoblasts (44, 45, 46, 47). The current data relate particularly to the regulation of apoptosis in normal and preeclamptic placentas. The results show that PL74 is overexpressed in preeclamptic placentas and causes a dose-dependent inhibition of proliferation and increase in apoptosis in the extravillous cytotrophoblast cell line HTR8/SVneo as well as increased apoptosis in term villous cytotrophoblasts (Figs. 2Go and 4Go and Tables 1–3GoGoGo). Several studies demonstrate an increase in apoptosis in both extravillous invading cytotrophoblasts and in villous trophoblast in preeclampsia (45, 48, 49, 50). Our data thus suggest that overexpression of PL74 may be one of the pathophysiological factors occurring in preeclampsia that induce the observed increase in apoptosis. However, the cause of the overexpression of PL74 in preeclampsia is unknown. In our studies, extravillous cytotrophoblast invasiveness itself was not specifically impaired as assessed by an in vitro invasion assay. In contrast to our results, Marjono et al. (19) found that neither MIC-1 (PL74) expression as assessed by immunostaining nor maternal serum levels were altered in preeclampsia. The differing results may be a result of technical differences, in that immunostaining is a less quantifiable measurement than mRNA expression. The lack of difference in maternal serum concentrations between normal and preeclamptic women (19) also suggests that the differences in MIC-1 (PL74) expression are small and that PL74 actions are predominantly paracrine. We have observed similar events in our studies of adrenomedullin expression in normal and preeclampsia, where maternal serum levels are similar in normal and preeclampsia yet placental production is clearly reduced (29).

In trophoblasts, we found that TNF{alpha} and TGFß1 do not alter expression, but IFN{gamma} and EGF strongly induce its expression. The EGF effect is most likely because of EGF’s effect to enhance syncytialization of the cytotrophoblast in vitro and hence an increase in PL74-synthesizing cells (24). However, IFN{gamma} does not alter differentiation and so is likely a direct stimulatory effect on gene expression. In contrast, in the prostate, androgens induce prostate-derived factor (PL74) (6), but in monocytes, IL-1ß, TNF{alpha}, IL-2, GM-CSF, and TGFß1 all induce secretion of MIC-1 (PL74), whereas IFN{gamma} has no effect (3). Thus, regulation of PL74 appears to be cell-type specific.

Our data are consistent with the hypothesis of Bootcov et al. (3) that PL74 (MIC-1) may limit macrophage proinflammatory responses. This would consist of a feedback loop between IFN{gamma} acting on the trophoblast, which responds by releasing PL74 (MIC-1) that in turn inhibits macrophage IFN{gamma} production. Furthermore, IFN{alpha} and -ß inhibit expression of EGF receptors in first-trimester invasive trophoblast cells and are antiproliferative to these cells (51, 52) and so may inhibit invasiveness. However, we did not observe an inhibition of trophoblast invasiveness by PL74; therefore, IFN{gamma} would not be acting through PL74 to achieve such an effect. IFN{gamma} in combination with TNF{alpha} induces apoptosis of trophoblast, although IFN{gamma} alone produces little if any apoptosis (27). In our experiments, this lack of IFN{gamma} effect on trophoblast, plus the finding that TNF{alpha} did not induce PL74, suggest IFN{gamma}/TNF{alpha} combinations do not act through PL74 to induce apoptosis. Additionally, EGF also induces PL74, and EGF protects the trophoblast against TNF{alpha}/IFN{gamma}-induced apoptosis (27).

The stimulation of PL74 by IFN{gamma} is of physiological importance because multiple actions of IFN{gamma} in the placenta have been demonstrated. In the mouse, IFN{gamma} from uterine natural killer cells is required for triggering of decidual spiral artery modification (53), induction of trophoblast differentiation from mixed ectoplacental cone cells (54), and stimulation of trophoblast nitric oxide (both inducible and endothelial nitric oxide synthase) during the implantation process (55). It has been further demonstrated in humans that IFN{gamma} induces placental indoleamine 2,3-dioxygenase, which in turn suppresses monocyte proliferation and is thought to be a major mechanism by which the placenta suppresses maternal immune responses against the placenta (56). It is unknown whether any of these actions are mediated through PL74, but additional experiments to investigate these possibilities are warranted.

Our studies have shown that PL74 is mainly produced by syncytium. However, PL74-positive immunostaining can be demonstrated in trophoblast cell columns and sporadic stromal cells in the first trimester, with a shift to predominantly syncytial staining and sporadically positive stromal cells, macrophages, cytotrophoblast, and endothelial cells at term (19). To further investigate sources of PL74, we immunostained placental bed biopsies from normal and preeclamptic pregnancies (Fig. 6Go). These data show strong PL74 staining in multinucleated giant cells, mononuclear extravillous cytotrophoblasts in the decidua, and endovascular trophoblast cells. In addition, myometrium stained positive. No significant differences were noted in staining intensity between normal and preeclamptic placentas. Because immunostaining is qualitative, it was not able to differentiate the relatively small degree of overexpression of PL74 detectable by Northern and Western blotting. These results indicate that, besides being a major syncytial product, trophoblast cells invading the myometrium and the myometrium itself may produce PL74 or be a target of PL74 action (because immunostaining does not definitively differentiate between synthesis or binding to receptors of PL74). The data thus suggest an additional site of regulatory control of both trophoblast and myometrial growth and apoptosis.

In conclusion, the data are most consistent with PL74 being a counter-regulatory factor to macrophage proinflammatory IFN{gamma} secretion. PL74 can also cause a dose-dependent inhibition of cell growth and apoptosis of extravillous invasive cytotrophoblast cells as well as promote differentiation of term cytotrophoblast cells. The overexpression of PL74 in preeclampsia may thus contribute to villous and invasive cytotrophoblast death and poor uterine invasion in this disease.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research.

First Published Online February 10, 2005

Abbreviations: EGF, Epidermal growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MIC, macrophage inhibitory cytokine; SSC, standard saline citrate; TUNEL, terminal uridine deoxynucleotidyl nick end labeling.

Received May 3, 2004.

Accepted January 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. A. Keelan and M. D. Mitchell
Cytokines, Hypoxia, and Preeclampsia
Reproductive Sciences, September 1, 2005; 12(6): 385 - 387.
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