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. 2012 Aug 23;153(10):4938–4945. doi: 10.1210/en.2012-1441

Cross Talk between PKC and CREB in the Induction of COX-2 by PGF2α in Human Amnion Fibroblasts

C M Guo 1, N Kasaraneni 1, K Sun 1,✉, L Myatt 1,✉
PMCID: PMC3512005  PMID: 22919060

Abstract

Compelling evidence indicates a crucial role of prostaglandin F2α (PGF2α) in parturition. Both the maternal and fetal sides of the fetal membranes synthesize PGF2α, which exerts effects via the prostaglandin F2α receptor (FP) that is coupled to the activation of protein kinase C (PKC). Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step of the inducible synthesis of prostaglandin. Although activation of PKC is known to induce COX-2 expression, it is not clear whether PGF2α can induce COX-2 via FP receptor-coupled PKC activation. COX-2 promoter carries a cAMP-response element (CRE) and phosphorylation of CRE binding protein 1 (CREB1) is associated with COX-2 expression in human amnion fibroblasts. We demonstrated that human amnion fibroblasts produced PGF2α and expressed FP receptor. PGF2α increased COX-2 expression and CREB1 phosphorylation, which could be blocked by either the FP receptor antagonist AL8810 or PKC inhibitor Ro31-7549. The PKC activator, phorbol-12-myristate-13-acetate (PMA), could mimic the induction of COX-2 and CREB1 phosphorylation. The induction of COX-2 by PGF2α and PMA could be attenuated by the small interfering RNA-mediated knockdown of CREB1 expression or overexpressing dominant-negative CREB1. A chromatin immunoprecipitation assay showed that the binding of CREB1 to the COX-2 promoter was increased by PGF2α and PMA in amnion fibroblasts. In conclusion, we provide evidence that PGF2α induces COX-2 expression via the FP receptor and phosphorylates CREB1 by PKC, thus increasing CREB1 binding to the COX-2 promoter and the expression of COX-2 in human amnion fibroblasts. This feed-forward loop may be crucial for the production of prostaglandins in the fetal membranes prior to the onset of labor.

A large body of evidence indicates a role for prostaglandin (PG) F2α in parturition (1). PGF2α concentration is increased in the amniotic fluid and on the maternal side of the fetal membranes, and PGF2α receptor (FP) density is increased in the myometrium toward the end of pregnancy (2–4). Exogenous PGF2α has been shown to induce labor (5, 6), whereas FP knockout mice never go into labor (7). In addition to the stimulation of myometrial contractility and enhancement of cervical ripening, PGF2α also plays important roles in the fetal-maternal interface at the onset of parturition by inducing the expression of matrix metalloproteinases (MMP) such as MMP-2 and MMP-9 and inhibiting their naturally occurring inhibitor tissue inhibitor of metalloproteinase-1 in human term decidua, thus accelerating the breakdown of collagen and the rupture of membranes (8). PGF2α also potentiates the conversion of biologically inactive metabolite cortisone to active cortisol by stimulating its regenerating enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in chorionic trophoblasts (9). In contrast to its inhibitory effect on the production of prostaglandins in most tissues, cortisol stimulates the expression of cytosolic phospholipase A2 and cyclooxygenase-2 (COX-2), the key enzymes involved in prostaglandin synthesis, thus increasing the production of prostaglandins in human fetal membranes (10–14). In addition to PGF2α, cortisol itself also induces the expression of 11β-HSD1 in the fetal membranes (15, 16). Therefore, the interactions of cortisol, PGF2α, 11β-HSD1, cytosolic phospholipase A2, and COX-2 may form a feed-forward loop in the fetal membranes reinforcing the regeneration of cortisol and the production of prostaglandin toward the end of gestation (17). The fetal membranes, particularly the amnion fibroblasts, are generally considered as a major source of PGE2, whereas the decidual stromal cells are regarded as a major source of PGF2α toward the end of pregnancy (18, 19). However, contradictory to this dogma, the amnion, when separated from chorion/decidua, is able to secret PGF2α though at a level about 3-fold less than the chorion/decidua (3). The amnion also expresses all three enzymes with prostaglandin F synthase activity, i.e. aldo-keto reductase (AKR) family 1 member C3 and B1 (AKR1C3, AKR1B1), which are enzymes responsible for the formation of PGF2α PGH2, and carbonyl reductase 1, which converts PGE2 to PGF2α (20, 21).

PGF2α exerts its effects through a FP receptor, which is coupled to the activation of phosphoinositol turnover, calcium mobilization, and activation of protein kinase C (PKC). Activation of PKC has been shown to be associated with increased COX-2 expression and PGE2 output, and inhibition of PKC suppressed glucocorticoid-induction of PGE2 synthesis in preparations of mixed amnion cells (22–24). Using purified amnion fibroblasts, we have demonstrated that glucocorticoids increase the expression of COX-2 by activation of the cAMP/protein kinase A (PKA) pathway, which leads to the phosphorylation of the cAMP-response element binding protein (CREB) and the subsequent binding of CREB to the COX-2 promoter (11, 12). In addition to PKA, PKC has been shown to be able to phosphorylate CREB (25–27). Based on the evidence presented above, we postulate that activation of the PKC pathway by PGF2α via the FP receptor would phosphorylate CREB, thereby increasing the transcription of COX-2 in amnion fibroblasts, which would further strengthen the feed-forward loop with regard to the production of prostaglandin in the fetal membranes before the onset of labor. To address these hypotheses, we investigated the involvement of PGF2α, the FP receptor, and the cross talk between PKC and CREB in the induction of COX-2 expression in cultured primary human amnion fibroblasts.

Materials and Methods

Human amnion fibroblast cell culture

Human amnion fibroblasts were prepared from the fetal membranes collected at term from 33 pregnant women not in labor and delivered by elective cesarean section under a protocol approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio. Patient exclusion criteria include fetal-maternal complications, treatment with steroids or other antiinflammatory agents, and clinical indication of inflammation. Amnion was peeled from the chorion/decidua and was digested with 0.125% trypsin (Sigma, St. Louis, MO) twice for 0.5 h each. The tissue was washed vigorously with PBS three times to remove the residual epithelial cells. The remaining amnion tissue was digested with 0.1% collagenase type I (Roche, Indianapolis, IN) for 1 h. After centrifugation the fibroblasts were collected for culture in DMEM (Life Technologies, Inc., Grand Island, NY) containing 10% newborn calf serum (NCS) (Life Technologies) and antibiotic-antimycotic (Life Technologies). The identity of fibroblasts has been previously verified, and more than 95% of the cells are fibroblasts (10).

Measurement of PGE2 and PGF2α production in cultured human amnion fibroblasts

On the third day of the amnion fibroblast culture, the cells were incubated in NCS-free medium for 24 h. Trypan blue exclusion assay showed no significant change in cell viability after 24 h of incubation in serum-free medium. The medium was collected for PGE2 and PGF2α measurement with enzyme immunoassay kits (Cayman Chemicals, Ann Arbor, MI).

Detection of FP with PCR, Western blotting, and immunocytochemistry in human amnion fibroblasts

On the third day of cell culture, total RNA and protein were extracted from the cells using a RNeasy miniextraction kit (QIAGEN, Germantown, MD) and radioimmunoprecipitation assay buffer (Millipore, Temecula, CA), respectively. After revers transcription, the FP mRNA was dectected using PCR with a set of paired primers designed to produce a 274-bp product. The FP primer sequences are: forward, 5′-AGGCGTCGAGGACCTGGTGT-3′, reverse, 5′-TGGCCATTGTAACCAGAAATGGGC-3′. To control sampling errors, PCR for the housekeep gene β-actin was performed on the samples with the primer sequences as follows: forward, 5′-TCCTCCTGAGCGC AAGTACTCT-3′, reverse, 5′-GCTCAGTAACAGTCCGCCTAGAA-3′. The PCR products were visualized under UV light after electrophoresis in 1.0% agarose gel.

The expression of the FP protein was examined after a standard Western blotting protocol. Briefly, 25 μg protein of each sample was electrophoresed in a 4–20% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and transferred to the nitrocellulose blot (Bio-Rad Laboratories). After blocking, the blot was probed with 1:500 dilution of rabbit antibody raised against a peptide mapping with N-terminal FP of human origin (Cayman Chemicals) overnight. After incubation with an antirabbit IgG antibody conjugated with horseradish peroxidase (Sigma), the enhanced chemiluminescence detection system (Millipore) was used to detect the bands. To control sampling errors, the level of β-actin protein in these samples was also examined with 1:5000 dilution of mouse antibody against β-actin (Sigma) in the same blot.

Immunocytochemical staining for FP was carried out on amnion fibroblasts after 3 d culture using the avidin-biotin-peroxidase method following a protocol provided by the manufacturer (Vector ABC; Vector Laboratories, Burlingame, CA). The FP antibody (Cayman Chemicals) at 1:500 dilution was applied as primary antibody. After incubation with the biotinylated secondary antibody and Elite ABC reagent, the color reaction was developed using 3-amino-9-ethyl carbazole (red color). Cells were counterstained with Carazzi's hematoxylin. To confirm the specificity of immunocytochemical staining, cells were also stained with preimmune serum instead of primary antibody.

Treatment of human amnion fibroblasts with PGF2α and determination of COX-2 mRNA and protein levels

On the third day of cell culture, the culture medium was changed to NCS-free medium. The cells were treated with PGF2α (10−9 to 10−5 m) for 24 h. At the end of the above time period, total RNA or protein was extracted from the cells. After RT of the RNA, COX-2 mRNA level was measured with quantitative real-time PCR (qRT-PCR) with the primer sequences as follows: forward, 5′-TGTGCAACACTTG AGTGGCT-3′, and reverse, 5′-ACTTTCTGTACTGCGGGTG-3′. To control sampling errors, qRT-PCR for 18S RNA was performed on each sample with the primer sequences as follows: forward, 5′-GTAACCCGTTGAACCCCATT-3′, and reverse, 5′-CCATCCAATC GGTAGTAGCG-3′. The reaction solution consisted of 2.0 μl diluted cDNA, 0.2 μm of each paired primer, and power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). The annealing temperature was set at 61 C. 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 18S RNA in each sample was obtained to normalize the expression of the target gene. COX-2 protein level was analyzed with Western blotting with 1:500 dilution of mouse antibody against COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA). To control sampling error, the same blots were probed with mouse antibody against β-actin (Sigma). The bands were visualized using a G Box iChemi chemiluminescence image capture system (Syngene, Cambridge, UK). The ratio of band intensities of target protein over β-actin was obtained as indication of the target protein levels.

Determination of COX-2, CREB1, and phosphorylated CREB1 (p-CREB1) protein levels after PGF2α or phorbol-12-myristate-13-acetate (PMA) treatment with or without inhibition of the signaling pathways

Based on the study of the dose-dependent effect of PGF2α on COX-2 expression, the concentration of PGF2α that caused maximal effect was used in the following studies. The cells were treated with PGF2α (10−6 m) in the presence and absence of the PGF2α receptor antagonist (AL8810, 10−5 m, Cayman Chemicals) or the PKC inhibitor (Ro31-7549, 10−5 m; Cayman Chemicals) for 24 h to determine the COX-2 protein level and for 12 h to determine the p-CREB1 level. To determine the effect of the PKC pathway activation on CREB1 phosphorylation, the cells were treated with PKC agonist phorbol myristate acetate (10−11 to 10−7 m PMA; Sigma) for 12 h. The role of CREB1 in the regulation of COX-2 expression by PGF2α and PKC activation was further examined with transfection of dominant-negative CREB1 plasmid (dnCREB1) (1 μg), in which Ser133 is replaced with Ala (kindly provided by Dr. M. R. Montminy, The Salk Institute, La Jolla, CA) or small interfering RNA (siRNA) against CREB1 (25 nm; Invitrogen, Austin, TX). The cells were transfected on the second day of culture with the above plasmid or siRNA using Lipofectamine LTX or Lipofectamine RNAiMAX (Invitrogen) for 24 h. After removing the transfection reagents, the transfected cells were treated with PGF2α (1 μm) or PMA (0.1 μm) 1 d later.

After the above treatments, total protein was extracted from the cells using radioimmunoprecipitation assay buffer containing phosphatase inhibitor. COX-2, CREB1, p-CREB1, and β-actin protein levels were measured by Western blotting with 1:500 dilution of mouse antibody against COX-2 or rabbit antibody against CREB1 or rabbit antibody against p-CREB1 (all, Santa Cruz Biotechnology) overnight. The bands were visualized using a G Box iChemi chemiluminescence image capture system (Syngene). The ratio of band intensities of target protein over β-actin was obtained as indication of the target protein levels.

Chromatin immunoprecipitation (ChIP) assay demonstrating the binding of CREB1 to COX-2 promoter upon PGF2α and PMA treatment in human amnion fibroblasts

Primary human amnion fibroblasts were prepared as described above. After incubation in DMEM containing 10% newborn bovine serum for 3 d, the cells were treated with PGF2α (1 μm) or PMA (0.1 μm) in medium without normal bovine serum for 12 h. Upon termination of treatment, a ChIP assay was conducted using a kit from Upstate Biotechnology (Millipore) and a method modified from the manufacturer's protocol. Briefly, the cells were fixed with 1% formaldehyde to cross-link the transcription factors to chromatin DNA. After washing with PBS, the cells were incubated with glycine and then scraped off the dish in PBS containing protease inhibitor cocktail. After spinning down, the cells were resuspended with 1% sodium dodecyl sulfate lysis buffer supplemented with protease inhibitor. The chromatin DNA was sheared by sonication to produce DNA fragments approximately 500-1000 bp. The same amounts of sheared DNA were used for subsequent immunoprecipitation with mouse antibody against human Polymerase II (Millipore) or rabbit antibody against human CREB1 (Santa Cruz Biotechnology) or p-CREB1 (Cell Signaling, Danvers, MA). Precipitation with preimmune mouse or rabbit IgG (Millipore) serves as negative control. The immunoprecipitate was then incubated with protein A agarose/salmon sperm DNA (Millipore), and the antibody/protein/DNA/agarose complex was washed adequately and collected for subsequent reverse cross-linking by sodium chloride. The same amount of sheared DNA without antibody precipitation was also processed for reverse cross-linking and served as positive input control. The sheared DNA recovered from reverse cross-linking was extracted with DNA extraction kit (QIAGEN) for further analysis with qRT-PCR. The primers used for qRT-PCR span the cAMP-response element (CRE) in the promoter region (−66 to +75 bp) as we have reported (11), which gives rise to a 150-bp product. The aligning positions and sequences of the primers are illustrated elsewhere (see Fig. 5A). The amount of precipitated DNA was calculated from the cycle threshold values of the qRT-PCR amplification curve, and the percentage of antibody-precipitated DNA to input DNA was obtained for statistical analysis.

Fig. 5.

Fig. 5.

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A, Illustration of the alignment positions and sequences of the primers used for qRT-PCR in ChIP assay. B and C, Increased enrichment of p-CREB1 and RNA polymerase II (Pol II) to COX-2 promoter by PGF2α (10−6 m) or PMA (10−7 m) treatment of human amnion fibroblasts as revealed by ChIP (n = 3). *, P < 0.05 vs. control.

Statistical analysis

All data are reported as mean ± sem. A paired Student's t test or one-way ANOVA test followed by the Student-Newman-Keuls test was used to assess significant differences where appropriate using SPSS software version 12.0 (SPSS Inc., Chicago, IL). Significance was set at P < 0.05.

Results

Production of prostaglandin and expression of FP receptor in human amnion fibroblasts

Enzyme immunoassay showed that the human amnion fibroblasts produced both PGE2 and PGF2α, but the level of PGF2α in the culture medium was about 6-fold lower than that of PGE2 (Fig. 1A) To examine whether human amnion fibroblasts express FP receptor mRNA and protein, PCR, Western blotting and immunocytochemical staining were used. PCR and Western blotting detected both FP mRNA and protein in human amnion fibroblasts (Fig. 1, B and C). Western blotting revealed a single band of an expected 55 kDa, suggesting the high specificity of the primary antibody used in this study. Immunocytochemical staining using the same primary antibody supported the expression of FP receptor protein in human amnion fibroblasts (Fig. 1D).

Fig. 1.

Fig. 1.

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A, PGE2 and PGF2α production in human amnion fibroblasts cultured for 24 h (n = 5). *, P < 0.05 vs. PGE2. B and C, Demonstration of FP mRNA and protein in human amnion fibroblasts with PCR and Western blotting. D, Immunocytohistochemical staining of FP in human amnion fibroblasts.

Effect of PGF2α on COX-2 expression in human amnion fibroblasts

PGF2α (10−9 to 10−5 m) increased COX-2 mRNA and protein levels in a concentration-dependent manner, and significant increases in COX-2 mRNA and protein were observed with PGF2α concentrations at 10−8 m and higher (Fig. 2, A and B). Maximal induction of COX-2 expression by PGF2α was approximately 10−6 m. To address whether PGF2α-induced expression of COX-2 was dependent on the activation of FP receptor and PKC pathway activation, the cells were treated with PGF2α in the presence and absence of FP receptor antagonist AL8810 and PKC antagonist Ro31-7549. Both AL8810 (10−5 m) and Ro31–7549 (10−5 m) significantly attenuated the induction of COX-2 by PGF2α (10−6 m) (Fig. 2, C and D).

Fig. 2.

Fig. 2.

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Effect of PGF2α on the expression of COX-2 in human amnion fibroblasts. A and B, Dose-dependent induction of COX-2 mRNA and protein expression by PGF2α in human amnion fibroblasts. C and D, The FP receptor antagonist AL8810 (10−5 m) and PKC inhibitor Ro31-7549 (Ro, 10−5 m) attenuated the induction of COX-2 by PGF2α (10−6 m) (n = 3–7). *, P < 0.05 vs. control; #, P < 0.05 vs. PGF2α.

Effect of PGF2α and PMA on the p-CREB1 in human amnion fibroblasts

To determine whether CREB1 is involved in the regulation of COX-2 expression by PGF2α and activation of PKC pathway, we first examined the effect of PGF2α and PMA on the phosphorylation of CREB1. PGF2α (10−9 to 10−5 m) and PMA (10−11 to 10−7 m) increased the level of p-CREB1 but not total CREB1 in a concentration-dependent manner (Fig. 3, A and B). We further investigated whether this effect of PGF2α could be blocked by the FP receptor antagonist and the PKC inhibitor and found that the induction of p-CREB1 by PGF2α (10−6 m) was significantly attenuated by either FP receptor antagonist AL8810 (10−5 m) or PKC inhibitor Ro31–7549 (10−5 m) (Fig. 3, C and D).

Fig. 3.

Fig. 3.

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A and B, Effect of PGF2α and PMA on the p-CREB1 in human amnion fibroblasts. C and D, FP receptor antagonist AL8810 (10−5 m) and PKC inhibitor Ro31-7549 (Ro, 10−5 m) decreased the induction of p-CREB1 by PGF2α (10−6 m) (n = 3–7). *, P < 0.05 vs. control; #, P < 0.05, ##, P < 0.01 vs. PGF2α.

Effect of dnCREB1 and siRNA-mediated knock-down of CREB1 on the induction of COX-2 by PGF2α and PMA in human amnion fibroblasts

Transfection of the cells with the vector carrying dnCREB1 significantly attenuated the induction of COX-2 protein expression by either PGF2α (10−6 m) or PMA (10−7 m) (Fig. 4A). In parallel to dnCREB1 data, transfection of the cells with siRNA against CREB1 significantly attenuated the induction of COX-2 protein expression by either PGF2α (10−6 m) or PMA (10−7 m) (Fig. 4B).

Fig. 4.

Fig. 4.

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A, Effect of transfection of dn-CREB1 plasmid on the induction of COX-2 by PGF2α (10−6 m) and PMA (10−7 m) in human amnion fibroblasts. B, Effect of siRNA-mediated knockdown of CREB1 expression on the induction of COX-2 by PGF2α (10−6 m) and PMA (10−7 m) in human amnion fibroblasts (n = 3–4). *, P < 0.05, **, P < 0.01 vs. control; #, P < 0.05 vs. PGF2α and PMA, respectively.

Enhancement of CREB1 binding to the COX2 promoter by PGF2α and PMA in human amnion fibroblasts

Chromatin immunoprecipitation assay revealed significant increases in the enrichment of p-CREB1 and polymerase II to the COX-2 promoter carrying the sequence of CRE by PGF2α (10−6 m) and PMA (10−7 m) treatment (Fig. 5).

Discussion

Because administration of PGF2α initiates but the FP antagonist or FP knockout delays parturition (5–7, 28, 29), studying the effect of PGF2α on the intrauterine tissues before the onset of labor has received significant attention with an aim to develop an effective strategy to control preterm labor. We demonstrated in this study that FP mRNA and protein are clearly demonstrable in human amnion fibroblasts, suggesting this cell type can also be a target of PGF2α action in addition to the demonstrated PGF2α targets such as myometrium, cervix, decidua, and chorion (8, 9, 30, 31). We have previously shown that the cultured amnion fibroblasts prepared from term pregnancy secretes 50 times more PGE2 per cell than the amnion epithelial cells, suggesting the amnion fibroblasts are one of the major sources of prostaglandins toward the end of pregnancy (10). Although the decidua tissue has been regarded as the major source of PGF2α at the maternal/fetal interface at the end of pregnancy, the amnion has also been shown to be able to produce PGF2α (3, 18, 21). By separating the amnion fibroblasts from the epithelial cells, we demonstrated that the amnion fibroblasts indeed produced PGF2α but at a level about 6-fold lower than that of PGE2, supporting a previous study using amnion tissue slices (18). Despite the lower level of PGF2α, we found that it nevertheless increased COX-2 expression via the FP receptor in the amnion fibroblasts, strongly suggesting a paracrine or autocrine role of PGF2α in the feed-forward regulation of COX-2 expression and prostaglandin production in the amnion, which provides further evidence of a role for PGF2α in addition to promoting myometrial contraction, cervical ripening, and membrane rupture in parturition (1, 8).

It is well established that the FP receptor is coupled to the increased phosphoinositol turnover, calcium mobilization, and ultimate activation of PKC. Elevated intracellular calcium levels were found to contribute to the increase of the availability of endogenous arachidonate for subsequent conversion to PGE2 (32). In addition, several lines of evidence also link increased COX-2 expression with activation of PKC in the amnion cells (22, 23). Our study provides further support for the stimulation of COX-2 expression via PKC activation and that this pathway is used by PGF2α in the feed-forward induction of COX-2 in the amnion fibroblasts. Previous studies demonstrated that activation of PKC increases the de novo synthesis of COX-2 in a RNA synthesis-dependent manner in amnion cells (22, 23). As a proinflammatory gene, the COX-2 promoter carries the sequence that binds nuclear factor-κB (NFκB), the major transcription factor mediating the expression of inflammatory genes (33), with transcription of COX-2 gene being under the tight control of NFκB (34). However, we demonstrated that the COX-2 promoter also carries a CRE, which is located downstream to the NFκB binding element, and this CRE was found to mediate a paradoxical stimulation of COX-2 expression by cortisol in human amnion fibroblasts (11). CRE is known to bind the transcription factor CREB1 phosphorylated at the serine residue (Ser133).

cAMP-dependent PKA is normally regarded as the primary stimulator of the phosphorylation of CREB1 at Ser133 (35). However, other protein kinases including calmodulin-dependent protein kinase II, Akt, and PKC have also been shown to phosphorylate and activate CREB1 (25–27, 36, 37). We verified the ability of PKC to phosphorylate CREB1 in human amnion fibroblasts in this study. Because dnCREB1 was able to attenuate the induction of COX-2 by PGF2α and PMA and PGF2α and PMA promoted the binding of CREB1 to COX-2 promoter, we believe that the induction of COX-2 by PGF2α and PMA is, at least in part, mediated by CREB1 in human amnion fibroblasts. However, PKC has also been reported to activate NFκB in different cell types (38), and this effect appear to be mediated by particular PKC isoforms such as PKCδ and PKCϵ (38). Further studies should be conducted to investigate whether these PKC isoforms are expressed in human amnion fibroblasts and the activation of NFκB by PKC is also involved in the induction of COX-2 expression by PGF2α via the FP receptor.

Previous studies have demonstrated that the amnion produces more PGE2 than PGF2α (18), which this study confirms. There are at least four distinct PGE2 receptor subtypes EP1-EP4. EP1 and EP3, like FP, increase the turnover of phosphoinositol and calcium mobilization and PKC activation, but EP3 may also be coupled to the inhibition of adenylyl cyclase and decrease of cAMP level (39). In contrast, EP2 and EP4 are coupled to the stimulation of adenylyl cyclase and increase of cAMP level (39). Of interest, all of the four PGE2 receptor subtypes have been identified in human amnion (40), suggesting PGE2 can also exert paracrine or autocrine effect in the amnion. However, because different EP receptor subtypes are coupled to different signaling pathways, the net effect of PGE2 may depend on the amount of PGE2 produced and the abundance and affinity of the EP receptor subtypes expressed in the amnion, thus complicating the situation of PGE2. Our preliminary study showed that the effect of PGE2 on COX-2 expression varied in amnion fibroblasts prepared from different patients (data not shown), which is obviously worth further investigation in the future.

In conclusion, we have demonstrated that human amnion fibroblasts produce PGF2α and express the FP receptor. PGF2α may exert a paracrine or autocrine feed-forward induction of COX-2 expression via FP receptor-coupled activation of PKC and subsequent phosphorylation of CREB1. These findings revealed a novel target of PGF2α at the onset of labor, which may be implicated in the initiation of parturition and preterm labor.

Acknowledgments

This work was supported by National Institutes of Health Grant R01 HD 31514, Natural Science Foundation of China Grant 30911120485, and National Key Basic Research Program of China Grant 2011CB944403.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
AKR
Aldo-keto reductase
ChIP
chromatin immunoprecipitation
COX-2
cyclooxygenase-2
CRE
cAMP-response element
CREB
cAMP-response element binding protein
dnCREB1
dominant-negative CREB1 plasmid
FP
PGF2α receptor
11β-HSD1
11β-hydroxysteroid dehydrogenase 1
MMP
matrix metalloproteinase
NCS
newborn calf serum
NF-κB
nuclear factor-κB
p-CREB1
phosphorylated CREB1
PG
prostaglandin
PKA
protein kinase A
PKC
protein kinase C
PMA
phorbol-12-myristate-13-acetate
qRT-PCR
quantitative real-time PCR
siRNA
small interfering RNA.

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