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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-1472
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 4988-4994
Copyright © 2006 by The Endocrine Society

Induction of Surfactant Protein A Expression by Cortisol Facilitates Prostaglandin Synthesis in Human Chorionic Trophoblasts

Kang Sun, Diane Brockman, Begona Campos, Brad Pitzer and Leslie Myatt

Department of Obstetrics and Gynecology (K.S., D.B., B.C., B.P., L.M.), University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267; and School of Life Sciences (K.S.), Fudan University, Shanghai 200433, China

Address all correspondence and requests for reprints to: Dr. Kang Sun, Department of Obstetrics and Gynecology, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267. E-mail: sunkang2000{at}yahoo.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Surfactant protein A (SP-A) may be an important link between the maturation of fetal organs and the initiation of parturition. However, the local expression of SP-A and the effect of SP-A on prostaglandin synthesis in human fetal membranes have not been resolved.

Objective: Our objective was to examine SP-A expression and the effect of SP-A on prostaglandin synthesis in human fetal membranes.

Design: SP-A expression was examined with immunohistochemistry and PCR. The effect of SP-A on prostaglandin synthesis was investigated in cultured human chorionic trophoblasts.

Patients: Patients were normal-term pregnant women undergoing elective cesarean sections.

Results: Both SP-A protein and mRNA were present in amnion epithelial cells, fibroblasts, and chorionic trophoblasts. Cortisol (10–7 and 10–6 M, 24 h) induced SP-A expression in cultured chorionic trophoblasts, which could be blocked by the glucocorticoid receptor antagonist RU486. Treatment of chorionic trophoblasts with SP-A (10–100 µg/ml, 24 h) caused a dose-dependent increase of prostaglandin E2 release and an induction of cyclooxygenase type 2 but not cytosolic phospholipase A2 and microsomal prostaglandin E synthase expression.

Conclusions: SP-A can be synthesized locally in human fetal membranes, which can be induced by glucocorticoids. SP-A appeared to induce prostaglandin E2 synthesis in chorionic trophoblasts via induction of cyclooxygenase type 2 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING FETAL DEVELOPMENT, the type II alveolar cells of fetal lung eventually synthesize and release surfactant into pulmonary secretions (1), which is intermittently discharged into the amniotic fluid (2, 3). Surfactant is a complex mixture consisting of phospholipids, nonpolar lipids, and proteins (1, 4). One of the most abundant apoproteins specifically associated with pulmonary surfactant is surfactant protein A (SP-A) (4). SP-A concentration in the amniotic fluid increases dramatically in the third trimester of pregnancy, from less than 3 µg/ml at 30–31 wk to greater than 24 µg /ml at 40–41 wk (2, 3). An earlier study revealed the presence of both SP-A and other apoproteins such as SP-B and SP-D in the amniotic epithelium and chorio-decidual layers (5). Because high levels of surfactant proteins are present in the amniotic fluid at late gestation, adsorption or absorption of surfactant proteins onto or into the fetal membranes is likely to occur. Adsorption of SP-B onto the human amnion epithelium was indeed demonstrated by Newman et al. (6). Therefore, it would be of interest to examine whether SP-A mRNA can also be detected in the fetal membranes or whether the presence of SP-A in the fetal membranes is just simply a reflection of SP-A from amniotic fluid.

Animal studies have strongly indicated that signals from the maturing fetus trigger the onset of parturition (7). For example, in the sheep, glucocorticoids derived from the maturing fetal hypothalamus-pituitary-adrenal axis play a crucial role in triggering parturition (7, 8). Although the specific mechanisms initiating parturition may vary among different species, glucocorticoids have been proposed as the factor synchronizing fetal maturation with the triggering mechanisms of parturition in nearly all species studied (9). It is well known that glucocorticoids accelerate lung maturation by enhancing surfactant synthesis in the pulmonary alveolar cells (10, 11). Evidence has been obtained from early studies that the phospholipid content of surfactant provides a source of arachidonic acid that can be used by the amnion for prostaglandin synthesis (12, 13). Recently there is direct evidence pointing to SP-A as the key link between the maturing fetus and the initiation of parturition in the mouse (14). It has been very well recognized that increased prostaglandin (PGE2 and PGF2{alpha}) biosynthesis as a result of inflammation-like responses in intrauterine tissues is one of the key events leading to parturition in both term and preterm human labor because these compounds evoke uterine contractions as well as cervical softening and effacement (15, 16). Human fetal membranes are generally regarded as the major sources of prostaglandins at the end of pregnancy (15, 16). However, it is not clear whether SP-A affects prostaglandin synthesis in human fetal membranes.

To address the above issues, we examined whether the human fetal membranes express SP-A mRNA, thus enabling SP-A to be synthesized locally. We also studied whether glucocorticoids, the hormone inducing lung maturation, affect SP-A expression in the fetal membranes. Finally, we tested whether SP-A regulates the expression of prostaglandin-synthesizing enzymes and subsequent prostaglandin release in cultured human chorionic trophoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection and preparation

Human fetal membranes were collected according to the guidelines set forth in a protocol that is in compliance with the Institutional Review Board of University of Cincinnati. The reflected membranes were collected immediately after elective Cesarean section at term. For amnion epithelial cell, fibroblast, and chorionic trophoblast preparations, amnion was peeled off chorion and both of the layers were placed in ice-cold normal saline for subsequent preparation. For immunohistochemical staining, a 3- to 6-cm membrane strip was rolled and snap-frozen in liquid nitrogen, and cryosections of membrane rolls were cut.

Immunohistochemical staining of SP-A in human fetal membranes

Cryosections of human fetal membrane rolls were immunostained for SP-A using the Vectastain Elite ABC method with monoclonal antibody against human SP-A (Lab Vision Corp., Fremont, CA). All the slides were blocked using the respective animal serum corresponding to the secondary antibody. The negative immunological control included the absence of primary antibody and an isotype-matched mouse myeloma Ig. Aminoethylcarbazole was used as peroxidase substrate, and the slides were counterstained with hematoxylin.

Amnion epithelial cell, fibroblast, and chorionic trophoblast preparations

Amnion and chorion were washed in cold PBS (pH 7.5). For amnion epithelial cell preparation, amnion tissue was digested with 0.125% trypsin (Sigma Chemical Co., St. Louis, MO) and 0.02% DNase (Sigma) twice for 30 min at 37 C. The digestion media were collected, and the remaining amnion tissue was washed vigorously with PBS to wash residual epithelial cells off the amnion tissue. The wash solution was then combined with the previous trypsin digestion media. For the preparation of amnion fibroblasts, the remaining amnion tissue was further digested with 0.1% collagenase (Roche, Indianapolis, IN) at 37 C for 1 h. The digestion medium was then collected. Both trypsin (epithelial cells) and collagenase (fibroblasts) digestion media were centrifuged at 2300 rpm. Cell pellets were collected and resuspended in DMEM without phenol red (Sigma). Resuspended cells were loaded onto previously prepared discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradients (5, 20, 40, and 60%), and the gradients were centrifuged at 2500 rpm. A single band of cells around 20% Percoll concentration was collected.

Chorionic trophoblasts were prepared using a modification of Kliman’s method (17). Briefly, the minced chorionic tissue was digested with 0.125% trypsin (Sigma) and 0.1% collagenase (Roche) three times for 60 min. The chorionic cells were loaded onto a stepwise 5–75% Percoll gradients composed of increments of 5% Percoll and centrifuged at 2500 x g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected.

Amniotic epithelial cells, fibroblasts, and chorionic trophoblasts were all plated at a density of 1 x 106 cells per well in 12-well plates in DMEM culture medium (Sigma) containing 10% fetal calf serum (FCS) and antibiotic-antimycotic (Life Technologies, Rockville, MD). The cells were cultured at 37 C in 5% CO2 in air. Cell types prepared using the above methods have all been characterized previously (18, 19).

Detection of SP-A mRNA in cells derived from human fetal membranes

The above prepared cells were cultured for 3 d before extraction of total RNA. After removal of the culture media, cells were scraped into cell lysis buffer (supplied with RNeasy kit; QIAGEN, Valencia, CA). Subsequent extraction and purification of total RNA from the cells was conducted using the RNeasy kit according to the protocol provided by the company.

RNA (1.0 µg) was reverse transcribed with oligo(dT)12–18 primer using the Superscript II kit (Invitrogen, Carlsbad, CA). Reverse-transcription products (cDNA) were used for subsequent PCR. Paired oligonucleotide primers for amplification of human SP-A were designed using Primer Designer (Scientific and Educational Software, Durham, NC) against the sequence downloaded from GenBank. Annealing temperature and PCR amplification cycles were set at 61 C and 60 cycles, respectively. To control sampling errors, PCR for the housekeeping gene ß-actin was performed on each sample. The primer sequences for human SP-A and ß-actin are listed in Table 1Go. The PCR products were analyzed by electrophoresis in 2% agarose gel and sequenced by the DNA Core of the University of Cincinnati.


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  		TABLE 1. Paired oligonucleotide primers used in PCR

 
SP-A purification

Purified human SP-A was kindly provided by Dr. F. McCormack, Department of Internal Medicine, University of Cincinnati, College of Medicine. SP-A was isolated from patients with pulmonary alveolar proteinosis. Briefly, SP-A was purified by the method of Suwabe et al. (20) from the cell-free surfactant pellet of bronchoalveolar lavage by serial sedimentation and resuspension in buffer containing 5 mM Tris, 150 mM NaCl, and 1 mM Ca2+ release by incubation with 2 mM EDTA and adsorption of the recalcified supernatant to mannose-Sepharose affinity columns. SP-A was eluted from the carbohydrate affinity column using 2 mM EDTA. The purified proteins were dialyzed for 2 d against daily changes of 2000 vol of 5 mM Tris (pH 7.4) and 150 mM NaCl and for 1 d against 2000 vol of 5 mM Tris (pH 7.4) and stored at –20 C. To test whether the observed effect of SP-A was due to the possible contamination of endotoxin, some aliquots of SP-A were subjected to heat treatment at 100 C for 30 min followed by quick cooling on ice before application to the cells.

Treatment of chorionic trophoblasts with cortisol and purified SP-A

Chorionic trophoblasts were cultured for 3 d in DMEM containing 10% FCS and then changed to FCS-free medium. Cortisol (10–8, 10–7, or 10–6 M) in the presence or absence of glucocorticoid receptor antagonist RU486 (10–6 M) or SP-A (10, 50, or 100 µg/ml) or heat-inactivated SP-A (100 µg/ml) was added into the medium. The cells were incubated with the above treatments for 24 h, and then the media were collected for subsequent PGE2 measurement and cells were collected either for total RNA extraction or for protein extraction for the measurement of SP-A and prostaglandin-synthesizing enzyme expression.

Measurements of SP-A and prostaglandin-synthesizing enzyme mRNA levels with real-time PCR

To measure SP-A, cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX2), and microsomal prostaglandin E synthase (mPGES) mRNA levels in response to cortisol or SP-A treatment, quantitative real-time (QRT) PCR analysis was carried out with oligonucleotide primers listed in Table 1Go. To control sampling errors, QRT PCR for the housekeeping gene ß-actin was routinely performed on each sample.

QRT PCR solution consisted of 2.0 µl diluted RT-PCR product, 0.2 µM of each paired primer, and power SYBR Green PCR master mix (Applied Biosystems, Foster, CA). The annealing temperature was set at 61 C, and amplification cycles were set at 45–60 cycles. The absolute mRNA levels in each sample were calculated according to a standard curve set up using serial dilutions of known amounts of specific templates against corresponding cycle threshold values. Then the ratio of the target gene over ß-actin in each sample was obtained to normalize the expression of the target gene. The specificity of the primers was verified by examining the melting curve as well as sequencing of the PCR products.

Measurement of SP-A and COX2 protein levels with Western blotting analysis

For protein extraction, cells were scraped off the plate into the cell lysis buffer in the presence of protease inhibitors. The cell lysate was then passed through a 20-gauge needle and centrifuged at 12,000 x g at 4 C. The supernatant was collected for protein analysis.

Samples containing the same amount of protein were electrophoresed on precast 12% Tris-glycine gel (Invitrogen). The protein bands were then transferred electrophoretically to a nitrocellulose membrane, and standard protocol for Western blotting was followed with mouse monoclonal anti-SP-A antibody (Lab Vision) and anti-COX2 antibody (Oxford Biomedical Research, Oxford, MI) as primary antibodies and horseradish peroxidase-conjugated donkey antimouse IgG as secondary antibody. The enhanced chemiluminescence detection system (Amersham, Piscataway, NJ) was used to detect the bands with peroxidase activity. After stripping the blot with Re-Blot Plus stripping solution (Chemicon, Temecula, CA), the blot was reprobed with antihuman ß-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and the corresponding secondary antibody. The ß-actin bands were detected in the same way as described above.

Measurements of PGE2 level with RIA

To measure PGE2 level in cultured media of chorionic trophoblasts, aliquots of collected media were incubated with 10,000 cpm [3H]PGE2 (Amersham Life Science, Arlington Heights, IL) and anti-PGE2 antibody at 4 C overnight. Subsequently, 0.2 ml 12% bovine {gamma}-globulin and 0.5 ml 40% polyethylene glycol were added into the reaction mixture to precipitate antibody-bound PGE2 in the reaction mixture. The antibody-bound and unbound [3H]PGE2 were separated by centrifugation at 2000 x g for 15 min. The pellet containing the bound [3H]PGE2 was counted using a liquid scintillation counter. The concentration of PGE2 in the sample was calculated from a standard curve of known concentrations of PGE2 standard (16–2000 pg/100 µl).

All data are reported as mean ± SEM. Paired Student’s t tests were used to assess significant differences between groups. Significance was set at P < 0.05. The values for n refer to the number of experiments performed with cells prepared from different patients.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution and expression of SP-A in human fetal membranes

Immunohistochemistry showed that positive immunostaining for SP-A was found in both layers of human fetal membranes with stronger staining in the epithelium (Fig. 1AGo). In the epithelium, the distribution of SP-A appeared diffuse across the whole layer with occasional punctate staining within cytoplasm and some strong staining on the apical membrane facing the amniotic cavity (Fig. 1BGo). The fibroblasts in the mesenchymal layer underneath the epithelium and the whole layer of chorionic trophoblasts also showed positive immunostaining of SP-A (Fig. 1Go, C and D). In agreement with the immunohistochemical staining, PCR also revealed specific amplified SP-A mRNA products in all the cell types examined including amnion epithelial cells, amnion fibroblasts, and chorionic trophoblasts (Fig 2Go). However, relatively weaker bands of amplified PCR products were observed in amnion epithelial cells than fibroblasts and chorionic trophoblasts (Fig. 2Go). The sequence of the PCR products has a full alignment to the SP-A sequence in GenBank (data not shown).


Figure 1
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  		FIG. 1. Immunohistochemical staining of SP-A in human fetal membranes. Cryosections were stained with ABC method with aminoethylcarbazole (red) as substrate for peroxidase and counterstained with hematoxylin. Arrows indicate amnion fibroblasts. A, Whole layers of fetal membranes; B, amnion epithelium; C, amnion fibroblasts; D, chorionic trophoblasts. Magnification, 60 x 10 (A) and 100 x 10 (B–D).

 

Figure 2
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  		FIG. 2. Agarose gel electrophoresis of the SP-A and ß-actin RT-PCR products. ct, Chorionic trophoblasts; af, amnion fibroblasts; ae, amnion epithelial cells.

 
Effect of cortisol on SP-A mRNA and protein expression in cultured human chorionic trophoblasts

Treatment of cultured human chorionic trophoblasts with cortisol at 10–7 and 10–6 but not 10–8 M induced the expression of SP-A mRNA (Fig. 3BGo), and this induction was blocked by the glucocorticoid receptor antagonist RU486 (10–6 M, Fig. 3CGo). Likewise, SP-A protein expression in the chorionic trophoblasts was also induced by cortisol treatment (Fig. 3AGo).


Figure 3
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  		FIG. 3. Change of SP-A protein (A) and mRNA expression (B and C) in the cultured human chorionic trophoblasts by cortisol (F, 10–8 to 10–6 M) treatments with or without glucocorticoid receptor antagonist RU486 (RU, 10–6 M). *, P < 0.05 vs. vehicle control (ctr); #, P < 0.05 vs. cortisol (F). n = 4.

 
Effect of SP-A on prostaglandin-synthesizing enzyme expressions and PGE2 release in cultured human chorionic trophoblasts

QRT PCR showed that treatment of cultured human chorionic trophoblasts with SP-A induced COX2 mRNA expression in a dose-dependent manner (10, 50, and 100 µg/ml), but cPLA2 and mPGES mRNA expression were not affected (Figs. 4Go and 5Go). In agreement with QRT PCR results, Western blotting analysis also showed a dose-dependent induction of COX2 protein expression by SP-A (Fig. 4Go). PGE2 release was also dose-dependently increased in cultured chorionic trophoblasts by SP-A (10, 50, and 100 µg/ml) (Fig. 4Go). However, heat-inactivated SP-A (100 µg/ml) no longer induced COX2 mRNA expression and PGE2 release in chorionic trophoblasts, suggesting that the effect of SP-A was mediated by the protein component rather than the possible endotoxin contamination (Fig. 4Go).


Figure 4
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  		FIG. 4. Effects of SP-A on COX2 expression and PGE2 release in cultured human chorionic trophoblasts. Top, Effects of SP-A on COX2 protein expression; middle, effects of SP-A on COX2 mRNA expression and PGE2 release (n = 4); bottom, effect of heat-inactivated SP-A (100 C, 30 min) on COX2 mRNA expression and PGE2 release (n = 3). *, P < 0.05; **, P < 0.01 vs. vehicle control.

 

Figure 5
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  		FIG. 5. Effects of SP-A on cPLA2 and mPGES mRNA expression in cultured human chorionic trophoblasts as measured with real-time PCR. n = 4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it has long been demonstrated with immunohistochemistry that positive staining for SP-A was present in human amniotic epithelium and chorio-decidual layers (5), whether SP-A in the fetal membranes is derived from the sharp rise of SP-A level in the amniotic fluid in the last trimester (2, 3) or is synthesized locally remains unclear. Madsen et al. (21) using RT-PCR showed that there is no expression of SP-A mRNA in human placental tissue. Newman et al. (6) observed that the presence of SP-B on the surface of amnion epithelium was possibly due to the adsorption of the hydrophobic surfactant apoprotein to the mucosal surface of fetal membranes. Although unlike other surfactant proteins, SP-A has been reported to be hydrophilic rather than hydrophobic (22), these observations raised the possibility of absorption or adsorption of SP-A into the fetal membranes rather than local synthesis. We found in this study that both SP-A protein and mRNA were present in amniotic epithelial cells, fibroblasts, and chorionic trophoblasts, suggesting local synthesis of SP-A exists in all these three cell types of the fetal membranes. However, uptake of SP-A from the amniotic fluid by cells in the fetal membranes cannot be totally ruled out because the most intensive staining of SP-A protein was observed in the amnion epithelium, especially the apical membrane facing the amniotic fluid, but the expression of SP-A mRNA in amniotic epithelial cells appears to be the lowest among the three cell types examined. Moreover, droplet-like staining of SP-A was also observed within the cytoplasm of epithelial cells, and whether this pattern of staining represents endocytosis of SP-A from the amniotic fluid needs to be determined. On the other hand, this pattern of staining may also be an indication of a secretory process of SP-A from the fetal membranes into amniotic fluid.

Synthetic glucocorticoids have long been used by obstetricians to reduce the incidence of respiratory distress syndrome of the newborn in preterm labor. It is believed that glucocorticoids accelerate fetal lung maturation by inducing differentiation of epithelial cells into type II alveolar cells and the synthesis of both surfactant glycerophospholipids and surfactant-associated protein by the type II alveolar cells (10, 11). However, conflicting results were obtained regarding the effect of glucocorticoids on SP-A synthesis in cultured human fetal lung explant (23, 24, 25). It has been reported that at low concentration (≤10–8 M), dexamethasone stimulated SP-A expression, whereas at high concentration (≥10–7 M), dexamethasone inhibited SP-A expression (23). Cortisol, the endogenous glucocorticoid, is much less potent than dexamethasone. Previous studies showed that the induction of SP-A mRNA by cortisol was maximal at 0.3 x 10–6 M, and stimulation was lost at 10–5 M (23). This paradoxical inhibition of SP-A expression was reported to be due to the decreased stability of SP-A mRNA by a higher concentration of glucocorticoids (26, 27). In this study, we observed that cortisol significantly stimulated SP-A expression in chorionic trophoblasts at 10–7 and 10–6 M, and this effect could be blocked by RU486, suggesting the involvement of glucocorticoid receptor in this effect. Because RU486 alone had no significant effect on SP-A expression, it appears unlikely that the blockade of cortisol action by RU486 is due to the blockade of the effect of endogenous progesterone produced by chorionic trophoblasts.

Human fetal membranes, especially the chorionic trophoblasts, express abundant 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), the enzyme responsible for regeneration of cortisol from cortisone (28). The expression of 11ß-HSD1 in the fetal membranes not only increases with gestational age (29) but is also synergistically induced by glucocorticoids and proinflammatory cytokines (30, 31). This unique feature of 11ß-HSD1 expression in the fetal membranes could be supplemental to the human fetal adrenal glands because the predominant product of human fetal adrenal glands is dehydroepiandrosterone rather than glucocorticoids (32). Nonetheless, compared with other animals, the cortisol concentration in the human fetus rises more slowly and to a more modest extent at the end of gestation. In contrast to the cortisol level in the human fetus, the cortisol level in amniotic fluid rises by 10-fold, reaching micromolar concentration at late gestation (33). Early work by Murphy (34) showed that the cortisol/cortisone ratio of amniotic fluid is considerably higher than that of cord serum, indicating that the chorionic membrane plays a role as an important extraadrenal source of fetal cortisol in human amniotic fluid. Carson et al. (35) demonstrated that isotope-labeled cortisol injected into amniotic fluid could be traced in several fetal tissues such as lung, liver, and adrenal glands, further suggesting cortisol derived from the fetal membranes may be of critical importance for human fetal lung maturation. Both animal and human studies have shown that cortisol has an important role in regulating surfactant synthesis in the fetal lung (10, 11, 24). In this study, we provided evidence for the de novo synthesis of SP-A in the fetal membranes. Consistent with the induction of SP-A expression by cortisol in the fetal lung, we found that SP-A expression in the fetal membranes was also stimulated by cortisol within the physiological range achieved in the amniotic fluid in late gestation, which suggests that cortisol derived from the fetal membranes could be important for the induction of SP-A expression both in the fetal lung and fetal membranes.

It has been very well recognized that the human fetal membranes are the major sources for prostaglandins (PGE2 and PGF2{alpha}) at the end of gestation (15, 16). Activation of prostaglandin synthesis in human fetal membranes appears to be one of the key events leading to parturition in both term and preterm labor (15, 16). Accumulating evidence points to a central role for nuclear factor {kappa}B (NF-{kappa}B) in the activation of prostaglandin synthesis in the fetal membranes (36). It has been reported that SP-A plays important roles in the regulation of immune function in the fetal lung, including stimulating proinflammatory cytokine expression and activation of the Toll-like receptors (TRL) (37). SP-A has been shown to bind to TLR2 and TRL4 (38, 39). The intracellular signaling pathways of both TLR2 and TRL4 lead to NF-{kappa}B activation. Recent work by Condon et al. (14) demonstrated that injection of SP-A into mouse amniotic fluid caused preterm labor, which was blocked by injection of the NF-{kappa}B inhibitor SN50. These findings strongly suggest that prostaglandin-synthesizing enzymes in the fetal membranes are possible targets for SP-A. Evidence has been obtained from early studies that phospholipid content in surfactant provides a source of arachidonate that can be used by the amnion for prostaglandin synthesis (12). Here we demonstrated that the apoprotein component of surfactant, SP-A, dose-dependently stimulated COX2 but not cPLA2 and mPGES expression in chorionic trophoblasts. COX2 catalyzes the rate-limiting reaction of the formation of intermediate product PGH2 from arachidonic acid in the biosynthesis of prostaglandins. As a consequence of increased COX2 expression, PGE2 release from chorionic trophoblasts was also dose-dependently increased by SP-A. Based upon these findings, we speculate that, together with SP-A derived from fetal lung via amniotic fluid, SP-A synthesized locally in the fetal membranes may participate in the initiation of parturition by stimulating prostaglandin synthesis in the fetal membranes at the end of gestation, which may parallel the increased expression of 11ß-HSD1 in the fetal membranes and the consequent dramatic increase of cortisol level in the amniotic fluid by the third trimester.

In conclusion, this study provides evidence that SP-A can be synthesized locally in the fetal membranes and the expression of SP-A in the fetal membranes can be stimulated by cortisol. SP-A simulates PGE2 release via inducing COX2 expression in the chorionic trophoblast. These findings may provide an important link between the endocrine signal from the maturing fetus and the activation of prostaglandin synthesis in the fetal membranes that ultimately leads to parturition.


   Acknowledgments
 
We are grateful to Dr. F. X. McCormack, Department of Internal Medicine, University of Cincinnati, College of Medicine, for his critical discussions and Ms. Jan Paul for her secretarial assistance.


   Footnotes
 
This work was supported by NIH RO1 HD31514-10 and NSFC 30570680 and 30470655.

First Published Online September 26, 2006

Abbreviations: COX2, Cyclooxygenase 2; cPLA2, cytosolic phospholipase A2; FCS, fetal calf serum; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; mPGES, microsomal prostaglandin E synthase; NF-{kappa}B, nuclear factor {kappa}B; PGE2, prostglandin E2; QRT, quantitative real-time; SP-A, surfactant protein A; TRL, Toll-like receptors.

Received July 11, 2006.

Accepted September 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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