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AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in human amnion fibroblasts

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Science Signaling  21 Nov 2017:
Vol. 10, Issue 506, eaac6160
DOI: 10.1126/scisignal.aac6160

PKA labors in the nucleus

Stress hormone signaling is associated with the induction of labor in pregnancy. The stress hormone cortisol stimulates the production of the enzyme COX-2 in fetal membrane cells through induction of the transcription factor CREB. COX-2 produces prostaglandins, which generate positive feedback on the cells to produce more. Lu et al. found that the selective localization of the kinase PKA in the nucleus is critical to this mechanism in the fetal membranes. In human amnion fibroblasts taken from normal “term”-labor deliveries, compared to those from cesarean sections, the localization of PKA in the nucleus by interaction with AKAP95 enabled it to phosphorylate and activate CREB. These findings reveal additional molecular targets through which clinicians might induce labor or prevent it from initiating prematurely. The nuclear localization of PKA-AKAP95 introduces a new subcellular role for PKA signaling, potentially restricted to these highly specialized cells.

Abstract

Phosphorylation of the transcription factors cyclic adenosine monophosphate response element–binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3) by protein kinase A (PKA) is required for the cortisol-induced production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human amnion fibroblasts, which critically mediates human parturition (labor). We found that PKA was confined in the nucleus by A-kinase–anchoring protein 95 (AKAP95) in amnion fibroblasts and that this localization was key to the cortisol-induced expression of PTGS2, the gene encoding COX-2. Cortisol increased the abundance of nuclear PKA by stimulating the expression of the gene encoding AKAP95. Knockdown of AKAP95 not only reduced the amounts of nuclear PKA and phosphorylated CREB but also attenuated the induction of PTGS2 expression in primary human amnion fibroblasts treated with cortisol, whereas the phosphorylation of STAT3 in response to cortisol was not affected. The abundances of AKAP95, phosphorylated CREB, and COX-2 were markedly increased in human amnion tissue after labor compared to those in amnion tissues from cesarean sections without labor. These results highlight an essential role for PKA that is anchored in the nucleus by AKAP95 in the phosphorylation of CREB and the consequent induction of COX-2 expression by cortisol in amnion fibroblasts, which may be important in human parturition.

INTRODUCTION

Prostaglandins E2 (PGE2) and F (PGF) enhance myometrial contractility, induce cervical dilatation, promote fetal membrane rupture, and provoke placental separation (1, 2). Increased local PGE2 and PGF production in the intrauterine tissues is recognized as the crucial common event in the initiation of parturition in many species including humans (35). Although almost all intrauterine tissues including the placenta, fetal membranes, decidua, and myometrium are capable of synthesizing PGE2 and PGF, the fetal membranes and decidua are recognized as the major source for PGE2 and PGF, respectively, toward the end of pregnancy (6, 7). In the fetal membranes, the amnion fibroblasts are the most important source for PGE2 (8, 9). More critically, glucocorticoids, which trigger parturition (10, 11), stimulate PGE2 synthesis through the induction of PTGS2 (9, 1216), the gene encoding cyclooxygenase-2 (COX-2), which catalyzes a key step in the synthesis of prostaglandins (17), in amnion fibroblasts. This induction of PTGS2 by glucocorticoids is in marked contrast to the inhibition of PTGS2 expression by glucocorticoids in most other tissues (18). Because cortisol (also called Kendall’s compound F) regenerated by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) can induce HSD11B1, the gene encoding 11β-HSD1 expression in amnion fibroblasts (19, 20), this paradoxical induction of COX-2 by glucocorticoids is believed to be a component of the key feed-forward events in the initiation of parturition (11, 21, 22). Further mechanistic studies have revealed that the induction of PTGS2 by glucocorticoids in amnion fibroblasts involves phosphorylation of at least two transcription factors, the cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB) (14) and the signal transducer and activator of transcription 3 (STAT3) (15), downstream of the cAMP–protein kinase A (PKA) pathway either directly or indirectly. Phosphorylated CREB and STAT3 interact at the cAMP response element (CRE) of the PTGS2 promoter to stimulate transcription (15).

PKA is a heterotetrameric protein complex consisting of two regulatory (R) and two catalytic (C) subunits (23). When the cAMP-PKA pathway is activated, cAMP binds cooperatively to the R subunits of the PKA holoenzyme, thereby releasing the C subunits so that they can phosphorylate target proteins (24). There are four PKA R subunits (RIα, RIβ, RIIα, and RIIβ) and three C subunits (Cα, Cβ, and Cγ) (25, 26). Because PKA mediates many discrete physiological responses after cAMP engagement in a given cell (27), mechanisms must exist to specify the responses to a particular stimulus. PKA can be compartmentalized at discrete subcellular locations by the interaction of R subunits with A-kinase–anchoring proteins (AKAPs), thereby limiting the access of PKA to a subset of substrates or stimuli (26, 28, 29). AKAPs are a group of structurally diverse proteins, each having a distinct subcellular location (30, 31). AKAP95 is the only AKAP that is specific to the nucleus (30, 32), and AKAP79 anchors PKA in the plasma membrane (33, 34). AKAP95 binds almost exclusively to the RIIα subunit, whereas AKAP79 binds to both RIIα and RIIβ subunits (35). Despite accumulating evidence showing that PKA RIIα can be localized inside the nucleus in several cell types (32, 36, 37), there is also a study demonstrating that PKA RIIα is excluded from the nucleus during interphase and the interaction between PKA RIIα and AKAP95 occurs only during mitosis in the cytoplasm when the nuclear envelope dissolves (38). It is thus necessary to examine whether AKAP95 can anchor PKA RIIα inside the nucleus in the human amnion fibroblast cell. Hence, it is crucial to determine which pool of PKA is involved in the paradoxical induction of COX-2 by glucocorticoids in human amnion fibroblasts. Elucidation of these issues may provide a more specific interventional strategy to block synthesis of prostaglandins in the amnion when the fetus is threatened by preterm birth, a leading cause of neonatal death (39). We hypothesized that the phosphorylation of CREB and STAT3 and subsequent induction of COX-2 by cortisol might be conferred by PKA anchored in a particular subcellular fraction in human amnion fibroblasts. Here, we addressed this question in primary human amnion fibroblasts and human amnion tissue.

RESULTS

Distribution of AKAP95 and AKAP79 in human fetal membranes and amnion fibroblasts

Immunohistochemical staining of human fetal membranes revealed the presence of AKAP95 and AKAP79 in the amnion epithelial and fibroblast cells as well as in the chorionic trophoblast cells, with the strongest staining observed in the amnion fibroblasts (Fig. 1, A to D). Western blotting analysis of fractionated protein extracts from cultured amnion fibroblasts revealed that AKAP95 was detected exclusively in the nucleus as marked by lamin A/C, whereas AKAP79 was detected mainly in the membrane and organelle protein fraction as labeled with calnexin but not in the nuclear fraction (Fig. 1E). These results suggested that AKAP95 and AKAP79 were abundant in human amnion fibroblasts and differentially distributed in the subcellular compartments.

Fig. 1 The distribution of AKAP95 and AKAP79 in human fetal membranes and amnion fibroblasts.

(A to D) Immunohistochemical staining of AKAP95 (A and B) and AKAP79 (C and D) in human fetal membranes. ae, amnion epithelial cells; af, amnion fibroblasts; ct, chorion trophoblasts. (E) Detection of AKAP95 and AKAP79 proteins in different subcellular fractions of human amnion fibroblasts by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calnexin, lamin A/C, and vimentin are markers for the cytoplasm, membrane and organelles, nucleus, and cytoskeleton, respectively. n = 2 experiments with samples from different patients. Scale bars, 50 μm.

Role of AKAP79 and AKAP95 in the induction of COX-2 by cortisol and the cAMP-PKA pathway in human amnion fibroblasts

Exposing cultured amnion fibroblasts to either PKI, a peptide that inhibits the catalytic activity of PKA (40), or Ht31, a peptide that nonselectively inhibits the interaction between the RII subunits of PKA and AKAP proteins (41), attenuated the induction of both PTGS2 transcripts (hereafter referred to as COX-2 mRNA) expression and COX-2 protein abundance by cortisol (Fig. 2, A and B) when compared with cells that were exposed to mutant PKI (m-PKI) or Ht31 (Ht31C), the control peptides for PKI and Ht31, respectively (40, 41). This suggests that the anchoring of PKA by AKAPs plays a crucial role in cortisol-mediated induction of COX-2. To address the contributions of AKAP79 and AKAP95, we knocked down these AKAPs using small interfering RNAs (siRNAs). Knockdown of AKAP79 had no effect on the induction of COX-2 mRNA or protein by either cortisol or forskolin (FSK), an activator of the cAMP-generating enzyme adenylyl cyclase (Fig. 2, C and D). However, siRNA-mediated knockdown of AKAP95 significantly attenuated the increases in COX-2 mRNA and protein abundance induced by either cortisol or FSK or dibutyryl cAMP (dbcAMP), a cell-permeable cAMP analog in amnion fibroblasts (Fig. 2, E to G). Consistent with these results, the increase in PGE2 production induced by cortisol was also significantly attenuated by knocking down AKAP95 (Fig. 2H). These data suggest that AKAP95, but not AKAP79, is involved in the induction of COX-2 and PGE2 synthesis by cortisol in human amnion fibroblasts.

Fig. 2 Involvement of AKAP79 and AKAP95 in the induction of COX-2 expression by cortisol (F) and activation of the cAMP pathway in human amnion fibroblasts.

(A) Quantitative Western blots and quantitative polymerase chain reaction (qPCR) analysis showing the abundance of COX-2 mRNA and protein in human amnion fibroblasts treated with cortisol (“F,” for Kendall’s compound F) in the presence of PKI or control peptide m-PKI. (B) Quantification of COX-2 mRNA and protein in cortisol-treated human amnion fibroblasts in the presence of Ht31 or Ht31C. (C and D) Quantification of COX-2 mRNA and protein in cortisol-treated (C) or FSK-treated (D) human amnion fibroblasts transfected with scrambled (−) or AKAP79-targeted (+) siRNA. (E to G) Quantification of COX-2 mRNA and protein in cortisol-treated (E), FSK-treated (F), and dbcAMP-treated (G) human amnion fibroblasts transfected with scrambled (−) or AKAP95-targeted (+) siRNA. (H) Quantification of PGE2 in cortisol-treated human amnion fibroblasts in the presence of absence of siRNA-targeting AKAP95. *P < 0.05, **P < 0.01, ***P < 0.001 against control with m-PKI, Ht31C, or scrambled siRNA; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to cells treated with cortisol, FSK, or dbcAMP [by one-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test]. Data are means ± SEM of three to five experiments, with representative blots.

Involvement of AKAP95 in the phosphorylation of CREB but not STAT3 in response to cortisol in human amnion fibroblasts

Fluorescence microscopic examination of the immunofluorescence staining of amnion fibroblasts confirmed that AKAP95 was located within the nucleus (Fig. 3A). Confocal microscopic examination revealed that PKA RIIα can also be localized inside the nucleus, and the costaining of PKA RIIα with Golgin-97, the Golgi marker, was found only in the perinuclear region but not in the nucleus (Fig. 3B). Confocal z-stacks (movie S1) also showed that PKA RIIα is not colocalized with Golgin-97 in the nucleus. Colocalization analysis showed that the Manders’ overlap coefficient (R) was 0.75, which indicates that 75% of PKA RIIα colocalized with Golgin-97 and 25% of PKA is not colocalized with Golgin-97. Quantitative Western blotting analysis revealed that siRNA-mediated knockdown of AKAP95 significantly decreased the abundance of not only the RIIα subunit but also the Cα subunit in the nucleus with a concurrent significant increase in the Cα subunit in the cytoplasm in amnion fibroblasts (Fig. 3, C and D). Western blotting analysis of the subcellular protein fractions and immunofluorescence staining of amnion fibroblasts demonstrated that both total and phosphorylated CREB were located exclusively in the nucleus, whereas total and phosphorylated STAT3 were found in both the cytoplasm and nucleus (Fig. 3, E to I). It appeared that total STAT3 was more abundant in the cytoplasm than in the nucleus, whereas phosphorylated STAT3 was more abundant in the nucleus than in the cytoplasm (Fig. 3, E, H, and I). Consistent with our previous findings (14, 15), treatment of amnion fibroblasts with cortisol increased the phosphorylation of CREB and STAT3, and the phosphorylation of CREB, but not STAT3, was blocked by either Ht31 or siRNA-mediated knockdown of AKAP95 (Fig. 4, A to D). Likewise, FSK and dbcAMP also increased the phosphorylation of CREB and STAT3, and siRNA-mediated knockdown of AKAP95 only attenuated the phosphorylation of CREB but not STAT3 (Fig. 4, E to H). These results suggest that AKAP95 plays an essential role in the phosphorylation of CREB by anchoring PKA in the nucleus in amnion fibroblasts.

Fig. 3 The distribution of AKAP95, PKA RIIα, total and phosphorylated CREB, and STAT3 in human amnion fibroblasts.

(A) Fluorescence microscopy images showing immunofluorescence staining of AKAP95 (green) in human amnion fibroblasts [marked by vimentin staining (red) and nuclear stain DAPI (4′,6-diamidino-2-phenylindole) (blue)]. (B) Confocal microscopy images showing immunofluorescence colocalization of PKA RIIα (green) and the Golgi apparatus marker Golgin-97 (red). Nuclei were stained with DAPI (blue). (C and D) Effects of siRNA-mediated knockdown of AKAP95 on the abundance of PKA RIIα and Cα in the cytoplasm and nucleus of human amnion fibroblasts. GAPDH and lamin A/C served as markers for the cytoplasm and nucleus, respectively. *P < 0.05 compared to cells transfected with scrambled siRNA (by paired Student’s t test). Data are means ± SEM of four experiments, with representative blots. (E) Total and phosphorylated (p) CREB and STAT3 abundance in the cytoplasmic and nuclear fractions in human amnion fibroblasts from two patients. (F to I) Fluorescence microscopy images showing immunofluorescence staining of total or phosphorylated CREB [red; (F) and (G), respectively] and STAT3 [green; (H) and (I), respectively] in human amnion fibroblasts. Nuclei were stained with DAPI (blue). Images are representative of two experiments. Scale bars, 25 μm.

Fig. 4 Involvement of AKAP95 in the phosphorylation of CREB but not STAT3 in response to cortisol (F) and activation of the cAMP pathway in human amnion fibroblasts.

(A to D) Effects of Ht31 and siRNA-mediated knockdown of AKAP95 on the phosphorylation of CREB at Ser133 and STAT3 at Tyr705 in response to cortisol. (E to H) Effects of siRNA-mediated knockdown of AKAP95 on the phosphorylation of CREB at Ser133 and STAT3 at Tyr705 in response to FSK or dbcAMP. *P < 0.05, **P < 0.01, ***P < 0.001 against control with Ht31C or scrambled siRNA; #P < 0.05, ##P < 0.01, ###P < 0.001 against cortisol-, FSK-, or dbcAMP-treated cells (by one-way ANOVA followed by the Newman-Keuls multiple comparison test). Data are means ± SEM from three to five experiments, with representative blots.

Effect of cortisol on AKAP95 abundance in human amnion fibroblasts

Treatment of amnion fibroblasts with increasing concentrations of cortisol (0.01, 0.1, and 1 μM) increased AKAP95 mRNA and protein abundance in a concentration-dependent manner with significant changes at 1 μM cortisol (Fig. 5A). The same range of cortisol concentrations had no effect on the total abundance of PKA RIIα mRNA or protein in amnion fibroblasts (Fig. 5B). However, cortisol significantly reduced the abundance of PKA RIIα and Cα protein in the cytoplasm and increased the abundance of PKA RIIα and Cα protein in the nucleus (Fig. 5, C and D). These data suggest that cortisol increases the translocation of PKA RIIα and Cα from the cytoplasm to the nucleus in amnion fibroblasts by stimulating AKAP95 expression.

Fig. 5 The effects of cortisol (F) on AKAP95, PKA RIIα, and Cα protein abundance in human amnion fibroblasts.

(A and B) Concentration-dependent effects of cortisol on cellular AKAP95, PKA RIIα mRNA, and protein abundance in human amnion fibroblasts (by one-way ANOVA followed by the Newman-Keuls multiple comparison test). (C and D) Effects of cortisol on the abundance of PKA RIIα and Cα in the cytoplasm and nucleus of human amnion fibroblasts. GAPDH and lamin A/C are markers for cytoplasm and nucleus, respectively. *P < 0.05, **P < 0.01 against control cells without cortisol (by paired Student’s t test). Data are means ± SEM from four to five experiments, with representative blots.

Changes in AKAP95, phosphorylated CREB, and COX-2 abundance in human amnion tissue at parturition

On the basis of the in vitro findings that cortisol increases the phosphorylation of CREB and subsequent COX-2 expression by stimulating AKAP95 expression, we examined whether there were corresponding changes in the abundance of AKAP95, phosphorylated CREB, and COX-2 in the amniotic tissue at parturition. Quantitative Western blotting revealed that there were significant increases in the abundance of AKAP95, COX-2, and phosphorylated CREB but not of total CREB (Fig. 6, A to E) in the amnion tissue collected at term after spontaneous labor (also referred to as “term labor”) as compared with the tissue collected at elective cesarean section without labor (also referred to as “term non-labor”). Given our previous findings that the abundances of 11β-HSD1 and cortisol are increased in the amnion tissue at parturition (15, 4244), these data suggest that the increases in AKAP95, phosphorylated CREB, and COX-2 abundance may be ascribed, at least in part, to the increased cortisol concentration in the amnion tissue at parturition.

Fig. 6 Changes in the abundance of AKAP95, COX-2, and phosphorylated and total CREB in human amnion tissue at parturition.

(A) Western blots showing the abundance of AKAP95, COX-2, and phosphorylated and total CREB protein in human amnion tissue collected upon cesarean section without labor at term [term non-labor (TNL); n = 6] and upon delivery after spontaneous labor [term labor (TL); n = 6]. (B to E) Quantification (means ± SEM) of the Western blotting assays represented in (A). *P < 0.05, **P < 0.01 against TNL (by unpaired Student’s t test).

DISCUSSION

Our data suggest that nuclear AKAP95-anchored PKA RIIα is crucial for the phosphorylation of CREB and subsequent transcriptional induction of COX-2 and production of PGE2 in response to cortisol in human amnion fibroblasts. Thus, this study uncovered a role for AKAP95 in human amnion fibroblasts in the initiation of labor and parturition. It also reveals a physiological context in which nuclear-localized PKA has a functional role.

Cortisol concentrations in the amniotic fluid and maternal circulation increase gradually toward the end of gestation (45, 46). Because of the feed-forward induction of 11β-HSD1 by cortisol (1921, 47), the abundance of cortisol in the amnion tissue at labor is further increased and can reach a concentration of about 4.5 ng/mg (15, 42, 43), which is much higher than the cortisol concentrations in the amniotic fluid and maternal circulation. Because the concentrations of cortisol used in this study are within the concentration range detected at parturition in the amnion, we believed that the observed effects of cortisol in this study are physiologically relevant to parturition.

It is generally accepted that the actions of glucocorticoids are mediated by the intracellular GR (48), and the activation of the cAMP-PKA-CREB pathway is coupled with the membrane stimulatory G protein (Gs)–coupled receptors (49). Previous studies have demonstrated that cortisol can activate the cAMP-PKA-CREB pathway indirectly at least through two mechanisms. On the one hand, glucocorticoids may also stimulate the production of factors that their receptors are coupled with the Gs protein, thereby leading to more ligands available to activate the downstream cAMP-PKA-CREB pathway. In human amnion fibroblasts, glucocorticoids potently induce COX-2 as well as the cytosolic phospholipase A2 (cPLA2) (14, 50), the two rate-limiting enzymes in the synthesis of PGE2. Because both EP2 and EP4 receptors for PGE2 are coupled with the Gs protein (51), the increased production of PGE2 under glucocorticoid stimulation can inevitably activate the cAMP-PKA-CREB pathway. As demonstrated in our previous studies, cortisol treatment of amnion fibroblasts can increase the intracellular abundance of cAMP (15) and phosphorylation of CREB (14, 15), which can be blocked by antagonists to EP2 and EP4 receptors (15). On the other hand, the expression of the stimulatory subunit of Gs protein, which mediates the activation of adenylyl cyclase, is inducible under glucocorticoid stimulation in several cell types (5254) including human amnion fibroblasts (50), thus facilitating activation of the cAMP-PKA-CREB pathway by those factors that their receptors are coupled with Gs protein. In addition, we have demonstrated that, in the presence of cortisol, the activated GR can interact with CREB and STAT3 at the promoter of the COX-2 gene to amplify the transcriptional effects of CREB and STAT3 (15), which may explain why the application of FSK or dbcAMP to cells induced smaller increases in the abundance of COX-2 mRNA than that of cortisol.

The specificity of the responses elicited by activation of the cAMP-PKA pathway in a given cell has puzzled scientists for decades. It has been suggested that PKA may be anchored in different subcellular compartments such that only a limited subset of substrates are available for PKA, thus ensuring the specificity of activation of the cAMP-PKA pathway (29, 55). Taggart et al. (55) suggested that this compartmentalized distribution of PKA may account for the switch from myometrial quiescence to myometrial activation by prostaglandin I2 (PGI2), which uses cAMP in its signaling activity toward the end of gestation. It is now recognized that the AKAP proteins may undertake such a role by anchoring PKA at the distinct subcellular sites (56, 57). Findings in the present study, as well as in previous studies (32, 36, 37), endorse such a role for AKAP95 in the partitioning of PKA in the nucleus. This study revealed that cortisol increased the expression of AKAP95, which may account for the increased PKA abundance and subsequent CREB phosphorylation in the nucleus. However, there is also a report showing that PKA RII is segregated from the nuclear AKAP95 in HeLa cells when the nuclear membrane is intact during the interphase, and the interaction between AKAP95 and PKA RII occurs in the cytoplasm only when the nuclear membrane disintegrates during mitosis (38). At present, we do not know what causes these discrepancies. We speculate that there might be cell-specific scenarios in terms of subcellular localization of PKA RII and interaction between AKAP95 and PKA RII.

Our previous work showed that activation of the cAMP-PKA pathway by cortisol via PGE2 increases the phosphorylation of not only CREB but also STAT3 and that both transcription factors are required for the transcription of PTGS2 induced by cortisol in human amnion fibroblasts (14, 15). Despite the important role of STAT3 in the induction of COX-2 mRNA expression by cortisol in human amnion fibroblasts (15), our results indicated that the phosphorylation of STAT3 was not affected by AKAP95 knockdown, suggesting that the site of STAT3 phosphorylation by PKA is more likely in the cytoplasm rather than in the nucleus. The nuclear distribution of STAT3 may reflect the nuclear translocation of phosphorylated STAT3 under the stimulation of basal PGE2 production. Because knockdown of AKAP79 failed to affect the induction of COX-2 by cortisol, we speculate that STAT3 may be phosphorylated by cytoplasmic PKA anchored by other members of the AKAP family (30) or by soluble PKA in the cytoplasm (58).

Increased AKAP95 abundance was observed not only in amnion fibroblasts upon cortisol treatment in vitro but also in the amnion tissue obtained after spontaneous labor, suggesting that the increased expression of AKAP95 is associated with the labor process. Consistently, the abundance of phosphorylated CREB and COX-2 was also increased in the amnion tissues collected from spontaneous labor. We believe that the increased abundance of AKAP95, phosphorylated CREB, and COX-2 in the amnion tissue at parturition is tightly correlated. All these changes may be initiated by increasing regeneration of cortisol by 11β-HSD1 in the amnion toward the end of gestation, because cortisol induces the abundance not only of 11β-HSD1 (19, 20) but also of AKAP95, COX-2, and cPLA2 in the amnion fibroblasts. Because PGE2 plays a pivotal role in parturition (1, 2), we believe that all these events elicited by cortisol in the amnion may play a crucial role in human parturition and activation of this cascade before term may provoke preterm birth.

In conclusion, we have demonstrated that cortisol increases the abundance of PKA anchored in the nucleus by inducing AKAP95 expression, thereby enhancing the phosphorylation of CREB in the nucleus and subsequent transcription of PTGS2 (and subsequent synthesis of COX-2), leading to increased production of PGE2 in human amnion fibroblasts (Fig. 7).

Fig. 7 A working model illustrating the role of AKAP95 in cortisol-induced PTGS2 expression in human amnion fibroblasts.

By inducing AKAP95 expression, cortisol increases the abundance of PKA anchored in the nucleus, thereby enhancing the activation of nuclear PKA by the cAMP pathway that is coupled with the PGE2 receptor. Consequently, the phosphorylation of nuclear CREB is increased. In contrast, the phosphorylation of STAT3 may occur in the cytoplasm. Phosphorylated CREB, STAT3, and activated glucocorticoid receptor (GR) interact at the PTGS2 promoter, thereby leading to the increased PTGS2 expression and subsequent PGE2 production in human amnion fibroblasts. Gs, stimulatory G protein; AC, adenylate cyclase; ATP, adenosine 5′-triphosphate.

MATERIALS AND METHODS

Human fetal membrane collection

Human fetal membranes (n = 72) were obtained from uncomplicated term (37 to 40 weeks) pregnancies after elective Cesarean section without labor (TNL) or after spontaneous labor (TL) with written informed consent from the participant patients under a protocol approved by the Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University (No. [2013]N025). Pregnancies with complications such as preeclampsia, fetal growth restriction, gestational diabetes, and chorioamnionitis were excluded from this study. The fetal membranes from TNL patients were fixed in 10% paraformaldehyde for immunohistochemical staining of AKAP95 and AKAP79. After peeling off the chorion, the amnion tissues from both TNL (n = 6) and TL (n = 6) patients were snap-frozen in liquid nitrogen for extraction of protein for Western blotting analysis of changes of AKAP95, CREB, and COX-2 proteins in parturition. The amnion tissues from TNL patients were processed for amnion fibroblast isolation to study the role of AKAP proteins in the induction of COX-2. Detailed methodology was described as follows.

Immunohistochemical and immunofluorescence staining

Tissue sections were cut from paraffin-embedded fetal membranes collected from TNL patients, and the isolated amnion fibroblasts were fixed in 4% paraformaldehyde 3 days after plating. The avidin-biotin-peroxidase method was used after a protocol provided by the manufacturer (Vector Laboratories). After blocking with preimmune serum, tissue sections were incubated with primary antibodies against AKAP95 (Santa Cruz Biotechnology) and AKAP79 (Santa Cruz Biotechnology) at a dilution of 1:100, and the cultured cells were incubated with antibodies against AKAP95 (Santa Cruz Biotechnology), the RIIα subunit of PKA (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), Golgin-97 (Cell Signaling Technology), total and phosphorylated CREB (Cell Signaling Technology), and STAT3 (Cell Signaling Technology) overnight at 4°C. After washing with phosphate-buffered saline (PBS), the tissue sections were incubated with secondary antibodies conjugated with biotinylated horseradish peroxidase H, and red color reaction was developed using the substrate 3-amino-9-ethylcarbazole (Vector Laboratories). The cells were permeabilized with 0.4% Triton X-100 before primary antibody application. After primary antibody application, cells were incubated with Alexa Fluor 488–labeled anti-rabbit (green color; 1:100) immunoglobulin G (IgG) or Alexa Fluor 594–labeled anti-mouse (red color; 1:100) IgG (Proteintech) in darkness at room temperature for 2 hours. Nuclei were counterstained with DAPI (blue color; 1 μg/ml). The stained slides were coverslipped with mounting medium and examined using a fluorescence microscope (Carl Zeiss). Confocal imaging was performed on a Leica TCS SP8 MP confocal microscope system equipped with a 100× oil objective. Confocal z-stacks were collected with a spacing of 0.4 μm in the z axis through 5.6-μm thickness of the cell. Colocalization between PKA RIIα and Golgin-97 was analyzed using a LAS AF software, and the Manders’ overlap coefficient (R) was calculated.

Extraction of protein from the amnion tissue

Tissue pieces were cut from the amnion within 5 cm of the spontaneous (TL) or artificial (TNL) membrane rupture sites and ground in liquid nitrogen. The ground tissue was homogenized and lysed in an ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Active Motif) containing a protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Active Motif) and centrifuged at 12,000 rpm for 10 min at 4°C. Protein in the supernatant was collected for Western blotting analysis.

Extraction of subcellular and total cell protein

Proteins from four different subcellular compartments were extracted using a ProteoExtract Subcellular Proteome Extraction kit (Merck Millipore). Cultured cells were scratched in PBS containing 5 mM EDTA. Cytosolic and nuclear fractions were extracted using cytosolic buffer [10 mM Hepes (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1 mM NaF, and 1 mM glycerol phosphate] and nuclear buffer [20 mM Hepes (pH 7.5), 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM NaF, and 1 mM glycerol phosphate], respectively. Total protein was extracted from the treated cells using an ice-cold RIPA lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor.

Isolation and culture of primary human amnion fibroblasts

Primary human amnion fibroblasts were isolated from TNL amnion. Briefly, the amnion tissue was digested twice with 0.125% trypsin (Life Technologies Inc.) for 20 min at 37°C and then washed vigorously with PBS to remove epithelial cells. The remaining amniotic tissue was digested with 0.1% collagenase (Sigma) for 25 min at 37°C to release the fibroblasts from the mesenchymal tissue. The fibroblasts in the digestion medium were collected by centrifugation at 2400 rpm for 10 min. The isolated amnion fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) plus antibiotics (Life Technologies Inc.) at 37°C and 5% CO2/95% air. Immunocytochemical staining with mesenchymal maker vimentin showed that more than 95% of the isolated cells were vimentin-positive.

Transfection of siRNA in amnion fibroblasts with electroporation

After isolation, the amnion fibroblasts were resuspended and transfected with 50 nM siRNA (GenePharma Co.) against AKAP95 (5′-GCAUCUGCAAAGCAAAUUUTT-3′) or AKAP79 (5′-GCAACAAAGGCUAAGUCAATT-3′) or randomly scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) in Opti-MEM (Life Technologies) in 2-mm gap cuvettes. Fibroblasts were electroporated at 175 V for 5 ms using a NEPA21 electroporator (Nepa Gene). After dilution with DMEM containing 10% FBS and antibiotics, the cells were transferred to a six-well cell culture plate to incubate for 48 hours and then treated with different regimens of reagents. After treatment, the cells were used for RNA and protein extraction, and the medium was collected for PGE2 measurement. The efficiency of knockdown was assessed in each experiment by measuring the target protein with Western blotting, which was 78% and 76% on the average for AKAP95 and AKAP79, respectively (fig. S1).

Treatment of human amnion cells

The amnion fibroblasts were cultured for 3 days before treatments. The cells were treated in phenol red/FBS–free culture medium. To examine the involvement of AKAP95 and AKAP79 in the regulation of COX-2 expression, amnion fibroblasts were treated with cortisol (F, 1 μM, Sigma), FSK (100 μM, Sigma), or dbcAMP (100 μM, Sigma) for 12 hours in the presence or absence of a PKA activity inhibitory peptide (PKI, TTYADFIASGRTGRRNAIHD, 20 μM, Promega) or a PKA-AKAP–anchoring inhibitory peptide (Ht31, DLIEEAASRIVDAVIEQVKAAGAY, 10 μM, Promega) or siRNA-mediated knockdown of either AKAP95 or AKAP79. m-PKI (TTYADAIASGATGAANAIHD, 20 μM, ChinaPeptides) and Ht31 (Ht31C, DLIEEAASRPVDAVPEQVKAAGAY, 10 μM, Promega) serve as controls for PKI and Ht31 studies, respectively. To study whether AKAP95 is involved in the phosphorylation of CREB and STAT3, amnion fibroblasts were treated with cortisol (1 μM, Sigma) for 2 hours or FSK (100 μM, Sigma) or dbcAMP (100 μM, Sigma) for 1 hour in the presence of Ht31 or siRNA-mediated knockdown of AKAP95. To determine whether cortisol affects the abundance of AKAP95 and PKA RIIα, amnion fibroblasts were treated with cortisol (0.01, 0.1, and 1 μM) for 12 hours. After treatment, total mRNA and protein were extracted for analysis with quantitative real-time PCR (qRT-PCR) or Western blotting.

Quantification of mRNA abundance with qRT-PCR

Total cellular RNA was extracted using a commercial kit (Omega Bio-Tek) following a protocol provided by the manufacturer. mRNA was reverse-transcribed to complementary DNA (cDNA) using a PrimeScript RT Master Mix Perfect Real Time kit (TaKaRa). The amounts of AKAP95, PKA RIIα, and COX-2 mRNA were determined with qRT-PCR using the above transcribed cDNA and SYBR Premix Ex Taq (TaKaRa). The annealing temperature was set at 61°C. The absolute mRNA in each sample was calculated according to a standard curve set up using serial dilutions of known amounts of PCR products against corresponding cycle threshold (Ct) values. The housekeeping gene GAPDH was amplified in parallel as an internal loading control. The primer sequences used for amplifying AKAP95, PKA RIIα, COX-2, and GAPDH were as follows: AKAP95, 5′-AGACCCTGCGGTTCATAAGC-3′ (forward) and 5′-TCCATCAATTCCTGACGCCG-3′ (reverse); PKA RIIα, 5′-AACCCGCTCTGTTGGTCAAT-3′ (forward) and 5′-ACAATGGTAGCAGCTCTCGG-3′ (reverse); COX-2 (PTGS2), 5′-TGTGCAACACTTGAGTGGCT-3′ (forward) and 5′-ACTTTCTGTACTGCGGGTG-3′ (reverse); GAPDH, 5′-CCCCTCTGCTGATGCCCCCA-3′ (forward) and 5′-TGACCTTGGCCAGGGGTGCT-3′ (reverse). The ratio of the target gene over GAPDH in each sample was obtained as an indication of the target gene expression.

Western blotting

The abundance of AKAP95, AKAP79, PKA RIIα, PKA Cα, COX-2, total CREB, phosphorylated CREB at Ser133, total STAT3, and phosphorylated STAT3 at Tyr705 was determined using a standard Western blotting protocol. Briefly, after determination of protein concentration with Bradford assays, 30 μg of protein of each sample was electrophoresed in 9% SDS-polyacrylamide gel and transferred to the nitrocellulose membrane. After blocking with 5% nonfat milk, the membrane was incubated with antibodies against AKAP95 (1:500; Santa Cruz Biotechnology), AKAP79 (1:500; Santa Cruz Biotechnology), PKA RIIα (1:500; Santa Cruz Biotechnology), PKA Cα (1:1000; Cell Signaling Technology), COX-2 (1:500; Santa Cruz Biotechnology), total CREB (1:1000; Cell Signaling Technology), phosphorylated CREB at Ser133 (1:1000; Cell Signaling Technology), total STAT3 (1:1000; Cell Signaling Technology), and phosphorylated STAT3 at Tyr705 (1:1000; Cell Signaling Technology) overnight at 4°C. After washing with 1× Tween/tris-buffered salt solution, the membrane was incubated with the appropriate secondary antibody conjugated with horseradish peroxidase (Sigma) for 1 hour. The enhanced chemiluminescent detection system (Millipore) was used to detect the bands with peroxidase activity. To control sampling error, the same blot was also probed for GAPDH (1:10,000; Proteintech) and lamin A/C (1:5000; Cell Signaling Technology) as an internal loading control. The bands were visualized using a G:BOX iChemi Chemiluminescence Image Capture system (Syngene). The ratio of band intensities of AKAP95, AKAP79, PKA RIIα, PKA Cα, COX-2, total CREB, phosphorylated CREB at Ser133, total STAT3, and phosphorylated STAT3 at Tyr705 over GAPDH for cytoplasmic protein or lamin A/C for nuclear protein was obtained as a measure of target protein abundance, respectively. Ratios of phosphorylated CREB and STAT3 over total CREB and STAT3 were also obtained for the analysis of protein phosphorylation.

Measurements of PGE2 with enzyme immunoassay

PGE2 in the culture medium collected from cultured amnion fibroblasts treated with or without cortisol (1 μM, 12 hours) in the presence or absence of siRNA-mediated knockdown of AKAP95 was measured with enzyme immunoassay kits (Cayman Chemicals) according to the manufacturer’s protocol.

Statistical analysis

All data are reported as means ± SEM. The number of each study in amnion fibroblasts represents separate experiments using amnion fibroblasts prepared from different pregnancies. Statistical analysis was performed with paired or unpaired Student’s t test or one-way ANOVA followed by the Newman-Keuls multiple comparison test where appropriate. Significance was set at P < 0.05.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/10/506/eaac6160/DC1

Fig. S1. The efficiency of siRNA-mediated knockdown of AKAP79 and AKAP95 in human amnion fibroblasts.

Movie S1. Confocal z-stack imaging of the colocalization of PKA RIIα and Golgin-97 in human amnion fibroblasts.

REFERENCES AND NOTES

Funding: This work was supported by the National Natural Science Foundation of China (grant 81330018) and the National Key R&D Program of China (grant 2017YFC1001403). Author contributions: J.L. and K.S. designed the project. J.L., Y.M., and C.Z. performed experiments. J.L., L.W., Y.W., and H.Y. collected samples from patients and analyzed clinical information. J.L., W.W., and K.S. analyzed the data. J.L., L.M., and K.S. wrote the manuscript. Competing interests: The authors declare that they have no competing interests.
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