akap95-mediated nuclear anchoring of pka mediates cortisol ... · further mechanistic studies have...

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

LABOR

1Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, ShanghaiJiao Tong University, Shanghai 200135, P. R. China. 2Shanghai Key Laboratory forAssisted Reproduction and Reproductive Genetics, Shanghai 200135, P. R. China.3Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine,Shanghai 201204, P. R. China. 4Department of Obstetrics and Gynecology, OregonHealth and Science University, Portland, OR 97239, USA.*Corresponding author. Email: [email protected]

Lu et al., Sci. Signal. 10, eaac6160 (2017) 21 November 2017

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AKAP95-mediated nuclear anchoringof PKA mediates cortisol-induced PTGS2expression in human amnion fibroblastsJiangwen Lu,1,2 Wangsheng Wang,1,2 Yabing Mi,1,2 Chuyue Zhang,1,2 Hao Ying,3 Luyao Wang,3

Yawei Wang,3 Leslie Myatt,4 Kang Sun1,2*

Phosphorylation of the transcription factors cyclic adenosine monophosphate response element–binding pro-tein (CREB) and signal transducer and activator of transcription 3 (STAT3) by protein kinase A (PKA) is requiredfor the cortisol-induced production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human amnionfibroblasts, which critically mediates human parturition (labor). We found that PKA was confined in the nucleusby 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 bystimulating the expression of the gene encoding AKAP95. Knockdown of AKAP95 not only reduced the amountsof nuclear PKA and phosphorylated CREB but also attenuated the induction of PTGS2 expression in primary humanamnion fibroblasts treated with cortisol, whereas the phosphorylation of STAT3 in response to cortisol was notaffected. The abundances of AKAP95, phosphorylated CREB, and COX-2 were markedly increased in human amniontissue after labor compared to those in amnion tissues from cesarean sections without labor. These results highlightan essential role for PKA that is anchored in the nucleus by AKAP95 in the phosphorylation of CREB and the conse-quent induction of COX-2 expression by cortisol in amnion fibroblasts, which may be important in human parturition.

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INTRODUCTIONProstaglandins E2 (PGE2) and F2a (PGF2a) enhance myometrial con-tractility, induce cervical dilatation, promote fetal membrane rupture,and provoke placental separation (1, 2). Increased local PGE2 andPGF2a production in the intrauterine tissues is recognized as the crucialcommon event in the initiation of parturition inmany species includinghumans (3–5). Although almost all intrauterine tissues including theplacenta, fetal membranes, decidua, and myometrium are capable ofsynthesizing PGE2 and PGF2a, the fetal membranes and decidua arerecognized as the major source for PGE2 and PGF2a, respectively,toward the end of pregnancy (6, 7). In the fetal membranes, the amnionfibroblasts are themost important source for PGE2 (8, 9).More critically,glucocorticoids, which trigger parturition (10, 11), stimulate PGE2synthesis through the induction ofPTGS2 (9, 12–16), the gene encodingcyclooxygenase-2 (COX-2), which catalyzes a key step in the synthesisof prostaglandins (17), in amnion fibroblasts. This induction of PTGS2by glucocorticoids is in marked contrast to the inhibition of PTGS2 ex-pression by glucocorticoids in most other tissues (18). Because cortisol(also called Kendall’s compound F) regenerated by 11b-hydroxysteroiddehydrogenase 1 (11b-HSD1) can induceHSD11B1, the gene encoding11b-HSD1 expression in amnion fibroblasts (19, 20), this paradoxicalinduction of COX-2 by glucocorticoids is believed to be a componentof the key feed-forward events in the initiation of parturition (11, 21, 22).Further mechanistic studies have revealed that the induction of PTGS2by glucocorticoids in amnion fibroblasts involves phosphorylationof at least two transcription factors, the cyclic adenosine monophos-phate (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 di-rectly or indirectly. Phosphorylated CREB and STAT3 interact at thecAMP response element (CRE) of the PTGS2 promoter to stimulatetranscription (15).

PKA is a heterotetrameric protein complex consisting of two regu-latory (R) and two catalytic (C) subunits (23). When the cAMP-PKApathway is activated, cAMP binds cooperatively to the R subunits of thePKA holoenzyme, thereby releasing the C subunits so that they canphosphorylate target proteins (24). There are four PKA R subunits(RIa, RIb, RIIa, and RIIb) and three C subunits (Ca, Cb, and Cg)(25, 26). Because PKA mediates many discrete physiological responsesafter cAMP engagement in a given cell (27), mechanisms must exist tospecify the responses to a particular stimulus. PKAcan be compartmen-talized at discrete subcellular locations by the interaction of R subunitswithA-kinase–anchoring proteins (AKAPs), thereby limiting the accessof PKA to a subset of substrates or stimuli (26, 28, 29). AKAPs are agroup of structurally diverse proteins, each having a distinct subcellularlocation (30, 31). AKAP95 is the only AKAP that is specific to thenucleus (30, 32), and AKAP79 anchors PKA in the plasma membrane(33, 34). AKAP95 binds almost exclusively to the RIIa subunit, whereasAKAP79 binds to both RIIa and RIIb subunits (35). Despite accumu-lating evidence showing that PKA RIIa can be localized inside the nu-cleus in several cell types (32, 36, 37), there is also a study demonstratingthat PKA RIIa is excluded from the nucleus during interphase and theinteraction between PKARIIa andAKAP95 occurs only duringmitosisin the cytoplasm when the nuclear envelope dissolves (38). It is thusnecessary to examine whether AKAP95 can anchor PKA RIIa insidethe nucleus in the human amnion fibroblast cell. Hence, it is crucialto determine which pool of PKA is involved in the paradoxical induc-tion of COX-2 by glucocorticoids in human amnion fibroblasts. Eluci-dation of these issues may provide a more specific interventionalstrategy to block synthesis of prostaglandins in the amnion when thefetus is threatened by preterm birth, a leading cause of neonatal death

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(39). We hypothesized that the phosphorylation of CREB and STAT3and subsequent induction of COX-2 by cortisol might be conferred byPKA anchored in a particular subcellular fraction in human amnionfibroblasts. Here, we addressed this question in primary human amnionfibroblasts and human amnion tissue.

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RESULTSDistribution of AKAP95 and AKAP79 in human fetalmembranes and amnion fibroblastsImmunohistochemical staining of human fetalmembranes revealed thepresence of AKAP95 and AKAP79 in the amnion epithelial and fibro-blast cells as well as in the chorionic trophoblast cells, with the strongeststaining observed in the amnion fibroblasts (Fig. 1, A to D). Westernblotting analysis of fractionated protein extracts from cultured amnionfibroblasts revealed thatAKAP95was detected exclusively in the nucleusas marked by lamin A/C, whereas AKAP79 was detected mainly in themembrane and organelle protein fraction as labeled with calnexinbut not in the nuclear fraction (Fig. 1E). These results suggested thatAKAP95 and AKAP79 were abundant in human amnion fibroblastsand differentially distributed in the subcellular compartments.


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Role of AKAP79 and AKAP95 in the induction of COX-2 bycortisol and the cAMP-PKA pathway in humanamnion fibroblastsExposing cultured amnion fibroblasts to either PKI, a peptide that in-hibits the catalytic activity of PKA (40), or Ht31, a peptide that non-selectively inhibits the interaction between the RII subunits of PKAand AKAP proteins (41), attenuated the induction of both PTGS2 tran-scripts (hereafter referred to as COX-2 mRNA) expression and COX-2protein abundance by cortisol (Fig. 2, A and B) when compared withcells that were exposed to mutant PKI (m-PKI) or Ht31 (Ht31C), thecontrol peptides for PKI and Ht31, respectively (40, 41). This suggeststhat the anchoring of PKA by AKAPs plays a crucial role in cortisol-mediated induction of COX-2. To address the contributions of AKAP79and AKAP95, we knocked down these AKAPs using small interferingRNAs (siRNAs). Knockdown ofAKAP79 had no effect on the inductionof COX-2 mRNA or protein by either cortisol or forskolin (FSK), anactivator of the cAMP-generating enzyme adenylyl cyclase (Fig. 2, Cand D). However, siRNA-mediated knockdown of AKAP95 signifi-cantly attenuated the increases in COX-2 mRNA and protein abun-dance 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 inducedby cortisol was also significantly attenuated by knocking downAKAP95(Fig. 2H). These data suggest that AKAP95, but not AKAP79, is in-volved in the induction of COX-2 and PGE2 synthesis by cortisol inhuman amnion fibroblasts.

Involvement of AKAP95 in the phosphorylation of CREB butnot STAT3 in response to cortisol in humanamnion fibroblastsFluorescence microscopic examination of the immunofluorescencestaining of amnion fibroblasts confirmed that AKAP95 was locatedwithin the nucleus (Fig. 3A). Confocal microscopic examination re-vealed that PKA RIIa can also be localized inside the nucleus, and thecostaining of PKA RIIa with Golgin-97, the Golgi marker, was foundonly in the perinuclear region but not in the nucleus (Fig. 3B). Confocalz-stacks (movie S1) also showed that PKA RIIa is not colocalized withGolgin-97 in the nucleus. Colocalization analysis showed that theManders’ overlap coefficient (R) was 0.75, which indicates that 75% ofPKA RIIa colocalized with Golgin-97 and 25% of PKA is not coloca-lized with Golgin-97. Quantitative Western blotting analysis revealedthat siRNA-mediated knockdown of AKAP95 significantly decreasedthe abundance of not only the RIIa subunit but also the Ca subunit inthe nucleus with a concurrent significant increase in the Ca subunitin the cytoplasm in amnion fibroblasts (Fig. 3, C and D). Westernblotting analysis of the subcellular protein fractions and immuno-fluorescence staining of amnion fibroblasts demonstrated that bothtotal and phosphorylated CREBwere located exclusively in the nucleus,whereas total and phosphorylated STAT3 were found in both the cyto-plasm and nucleus (Fig. 3, E to I). It appeared that total STAT3 wasmore abundant in the cytoplasm than in the nucleus, whereas phos-phorylated STAT3 was more abundant in the nucleus than in the cyto-plasm (Fig. 3, E,H, and I). Consistentwith our previous findings (14, 15),treatment of amnion fibroblasts with cortisol increased the phosphoryl-ation of CREB and STAT3, and the phosphorylation of CREB, but notSTAT3, was blocked by either Ht31 or siRNA-mediated knockdown ofAKAP95 (Fig. 4, A to D). Likewise, FSK and dbcAMP also increased thephosphorylation of CREB and STAT3, and siRNA-mediated knock-down of AKAP95 only attenuated the phosphorylation of CREB but

Fig. 1. The distribution of AKAP95 and AKAP79 in human fetal membranesand amnion fibroblasts. (A to D) Immunohistochemical staining of AKAP95 (Aand B) and AKAP79 (C and D) in human fetal membranes. ae, amnion epithelialcells; af, amnion fibroblasts; ct, chorion trophoblasts. (E) Detection of AKAP95 andAKAP79 proteins in different subcellular fractions of human amnion fibroblasts byWestern 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 differ-ent patients. Scale bars, 50 mm.

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not STAT3 (Fig. 4, E to H). These results suggest that AKAP95 plays anessential role in the phosphorylation of CREB by anchoring PKA in thenucleus in amnion fibroblasts.

Effect of cortisol on AKAP95 abundance in humanamnion fibroblastsTreatment of amnion fibroblasts with increasing concentrations of cor-tisol (0.01, 0.1, and 1 mM) increasedAKAP95mRNAand protein abun-dance in a concentration-dependentmannerwith significant changes at1 mM cortisol (Fig. 5A). The same range of cortisol concentrations hadno effect on the total abundance of PKA RIIamRNA or protein in am-nion fibroblasts (Fig. 5B). However, cortisol significantly reduced theabundance of PKARIIa andCa protein in the cytoplasm and increasedthe abundance of PKARIIa andCa protein in the nucleus (Fig. 5, C andD). These data suggest that cortisol increases the translocation of PKARIIa andCa from the cytoplasm to the nucleus in amnion fibroblasts bystimulating AKAP95 expression.

Changes in AKAP95, phosphorylated CREB, and COX-2abundance in human amnion tissue at parturitionOn the basis of the in vitro findings that cortisol increases the phospho-rylation of CREB and subsequent COX-2 expression by stimulatingAKAP95 expression, we examined whether there were correspondingchanges in the abundance of AKAP95, phosphorylated CREB, and


COX-2 in the amniotic tissue at parturition. QuantitativeWestern blot-ting revealed that there were significant increases in the abundance ofAKAP95, COX-2, and phosphorylated CREB but not of total CREB(Fig. 6, A to E) in the amnion tissue collected at term after spontaneouslabor (also referred to as “term labor”) as compared with the tissuecollected at elective cesarean section without labor (also referred to as“term non-labor”). Given our previous findings that the abundances of11b-HSD1 and cortisol are increased in the amnion tissue at parturition(15, 42–44), these data suggest that the increases in AKAP95, phos-phorylated CREB, and COX-2 abundance may be ascribed, at leastin part, to the increased cortisol concentration in the amnion tissueat parturition.

DISCUSSIONOur data suggest that nuclear AKAP95-anchored PKA RIIa is crucialfor the phosphorylation ofCREB and subsequent transcriptional induc-tion ofCOX-2 andproduction of PGE2 in response to cortisol in humanamnion fibroblasts. Thus, this study uncovered a role for AKAP95 inhuman amnion fibroblasts in the initiation of labor and parturition. Italso reveals a physiological context in which nuclear-localized PKA hasa functional role.

Cortisol concentrations in the amniotic fluid and maternal circula-tion increase gradually toward the end of gestation (45, 46). Because of

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 amnionfibroblasts. (A) Quantitative Western blots and quantitative polymerase chain reaction (qPCR) analysis showing the abundance of COX-2 mRNA and protein in humanamnion 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 incortisol-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, orscrambled 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 theNewman-Keuls multiple comparison test]. Data are means ± SEM of three to five experiments, with representative blots.

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the feed-forward induction of 11b-HSD1 by cortisol (19–21, 47), theabundance of cortisol in the amnion tissue at labor is further increasedand can reach a concentration of about 4.5 ng/mg (15, 42, 43), which ismuch higher than the cortisol concentrations in the amniotic fluid andmaternal circulation. Because the concentrations of cortisol used in this


study are within the concentration range detected at parturition in theamnion, we believed that the observed effects of cortisol in this study arephysiologically relevant to parturition.

It is generally accepted that the actions of glucocorticoids aremediated by the intracellular GR (48), and the activation of the

Fig. 3. The distribution of AKAP95, PKA RIIa, total and phosphorylated CREB, and STAT3 in human amnion fibroblasts. (A) Fluorescence microscopy imagesshowing 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 RIIa (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 RIIa and Ca in the cytoplasm and nucleus ofhuman amnion fibroblasts. GAPDH and lamin A/C served as markers for the cytoplasm and nucleus, respectively. *P < 0.05 compared to cells transfected withscrambled siRNA (by paired Student’s t test). Data are means ± SEM of four experiments, with representative blots. (E) Total and phosphorylated (p) CREB and STAT3abundance in the cytoplasmic and nuclear fractions in human amnion fibroblasts from two patients. (F to I) Fluorescence microscopy images showing immuno-fluorescence staining of total or phosphorylated CREB [red; (F) and (G), respectively] and STAT3 [green; (H) and (I), respectively] in human amnion fibroblasts. Nucleiwere stained with DAPI (blue). Images are representative of two experiments. Scale bars, 25 mm.

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cAMP-PKA-CREB pathway is coupledwith themembrane stimulatoryG protein (Gs)–coupled receptors (49). Previous studies have demon-strated that cortisol canactivate the cAMP-PKA-CREBpathway indirectlyat least through two mechanisms. On the one hand, glucocorticoids mayalso stimulate the production of factors that their receptors are coupledwith the Gs protein, thereby leading to more ligands available to activatethe downstream cAMP-PKA-CREB pathway. In human amnion fibro-blasts, glucocorticoids potently induce COX-2 as well as the cytosolicphospholipase A2 (cPLA2) (14, 50), the two rate-limiting enzymes inthe synthesis of PGE2. Because both EP2 and EP4 receptors for PGE2are coupled with the Gs protein (51), the increased production of PGE2under glucocorticoid stimulation can inevitably activate the cAMP-PKA-CREB pathway. As demonstrated in our previous studies, cortisoltreatment of amnion fibroblasts can increase the intracellular abundanceof cAMP (15) and phosphorylation of CREB (14, 15), which can beblocked by antagonists to EP2 and EP4 receptors (15). On the other hand,the expressionof the stimulatory subunit ofGsprotein,whichmediates theactivation of adenylyl cyclase, is inducible under glucocorticoid stimula-tion in several cell types (52–54) including human amnion fibroblasts(50), thus facilitating activation of the cAMP-PKA-CREB pathway bythose factors that their receptors are coupled with Gs protein. In addition,we have demonstrated that, in the presence of cortisol, the activated GRcan interact with CREB and STAT3 at the promoter of theCOX-2 gene toamplify the transcriptional effects of CREB and STAT3 (15), which mayexplain why the application of FSK or dbcAMP to cells induced smallerincreases 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 hasbeen suggested that PKAmay be anchored in different subcellular com-partments such that only a limited subset of substrates are available for

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 humanamnion 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 tocortisol. (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.

Fig. 5. The effects of cortisol (F) on AKAP95, PKA RIIa, and Ca protein abun-dance in human amnion fibroblasts. (A and B) Concentration-dependenteffects of cortisol on cellular AKAP95, PKA RIIa mRNA, and protein abundancein human amnion fibroblasts (by one-way ANOVA followed by the Newman-Keulsmultiple comparison test). (C and D) Effects of cortisol on the abundance of PKARIIa and Ca in the cytoplasm and nucleus of human amnion fibroblasts. GAPDHand 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.

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PKA, thus ensuring the specificity of activation of the cAMP-PKApath-way (29, 55). Taggart et al. (55) suggested that this compartmentalizeddistribution of PKA may account for the switch from myometrial qui-escence tomyometrial activation by prostaglandin I2 (PGI2), which usescAMP in its signaling activity toward the end of gestation. It is nowrecognized that the AKAP proteins may undertake such a role byanchoring PKA at the distinct subcellular sites (56, 57). Findings inthe present study, aswell as in previous studies (32, 36, 37), endorse sucha role for AKAP95 in the partitioning of PKA in the nucleus. This studyrevealed that cortisol increased the expression of AKAP95, which mayaccount for the increased PKA abundance and subsequent CREB phos-phorylation in the nucleus. However, there is also a report showing thatPKA RII is segregated from the nuclear AKAP95 in HeLa cells whenthe nuclear membrane is intact during the interphase, and the inter-action betweenAKAP95 andPKARII occurs in the cytoplasmonlywhenthe nuclear membrane disintegrates during mitosis (38). At present, we donot know what causes these discrepancies. We speculate that theremight be cell-specific scenarios in terms of subcellular localizationof PKA RII and interaction between AKAP95 and PKA RII.

Our previous work showed that activation of the cAMP-PKApathway by cortisol via PGE2 increases the phosphorylation of notonly CREB but also STAT3 and that both transcription factors arerequired for the transcription of PTGS2 induced by cortisol in hu-man amnion fibroblasts (14, 15). Despite the important role ofSTAT3 in the induction of COX-2mRNA expression by cortisol in hu-


man amnion fibroblasts (15), our results indicated that the phosphoryl-ation of STAT3 was not affected by AKAP95 knockdown, suggestingthat the site of STAT3 phosphorylation by PKA is more likely in the cy-toplasm rather than in the nucleus. The nuclear distribution of STAT3may reflect the nuclear translocation of phosphorylated STAT3 un-der the stimulation of basal PGE2 production. Because knockdownof AKAP79 failed to affect the induction of COX-2 by cortisol, wespeculate that STAT3 may be phosphorylated by cytoplasmic PKAanchored by other members of the AKAP family (30) or by solublePKA in the cytoplasm (58).

Increased AKAP95 abundance was observed not only in amnionfibroblasts upon cortisol treatment in vitro but also in the amniontissue obtained after spontaneous labor, suggesting that the increasedexpressionofAKAP95 is associatedwith the labor process. Consistently,the abundance of phosphorylated CREB and COX-2 was also increasedin the amnion tissues collected from spontaneous labor.We believe thatthe increased abundance of AKAP95, phosphorylated CREB, andCOX-2 in the amnion tissue at parturition is tightly correlated. Allthese changes may be initiated by increasing regeneration of cortisolby 11b-HSD1 in the amnion toward the end of gestation, because cor-tisol induces the abundance not only of 11b-HSD1 (19, 20) but also ofAKAP95, COX-2, and cPLA2 in the amnion fibroblasts. BecausePGE2 plays a pivotal role in parturition (1, 2), we believe that all theseevents elicited by cortisol in the amnion may play a crucial role in hu-man parturition and activation of this cascade before term may pro-voke preterm birth.

In conclusion, we have demonstrated that cortisol increases theabundance of PKA anchored in the nucleus by inducing AKAP95 ex-pression, thereby enhancing the phosphorylation of CREB in the nucleusand subsequent transcription of PTGS2 (and subsequent synthesis ofCOX-2), leading to increased production of PGE2 in human amnionfibroblasts (Fig. 7).

MATERIALS AND METHODSHuman fetal membrane collectionHuman fetal membranes (n = 72) were obtained from uncomplicatedterm (37 to 40 weeks) pregnancies after elective Cesarean section with-out labor (TNL) or after spontaneous labor (TL) with written informedconsent from the participant patients under a protocol approved by theEthics Committee of Ren Ji Hospital, School ofMedicine, Shanghai JiaoTong University (No. [2013]N025). Pregnancies with complicationssuch as preeclampsia, fetal growth restriction, gestational diabetes,and chorioamnionitis were excluded from this study. The fetal mem-branes from TNL patients were fixed in 10% paraformaldehyde forimmunohistochemical staining of AKAP95 andAKAP79. After peelingoff the chorion, the amnion tissues frombothTNL (n= 6) andTL (n= 6)patients were snap-frozen in liquid nitrogen for extraction of protein forWesternblotting analysis of changes ofAKAP95,CREB, andCOX-2pro-teins in parturition. The amnion tissues from TNL patients were pro-cessed for amnion fibroblast isolation to study the role of AKAPproteins in the induction of COX-2. Detailedmethodologywas describedas follows.

Immunohistochemical and immunofluorescence stainingTissue sections were cut from paraffin-embedded fetal membranescollected from TNL patients, and the isolated amnion fibroblastswere fixed in 4% paraformaldehyde 3 days after plating. The avidin-biotin-peroxidase method was used after a protocol provided by the

Fig. 6. Changes in the abundance of AKAP95, COX-2, and phosphorylatedand total CREB in human amnion tissue at parturition. (A) Western blotsshowing the abundance of AKAP95, COX-2, and phosphorylated and total CREBprotein in human amnion tissue collected upon cesarean section without labor atterm [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 Westernblotting assays represented in (A). *P < 0.05, **P < 0.01 against TNL (by unpairedStudent’s t test).

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manufacturer (Vector Laboratories). After blocking with preimmuneserum, tissue sections were incubated with primary antibodies againstAKAP95 (Santa Cruz Biotechnology) and AKAP79 (Santa Cruz Bio-technology) at a dilution of 1:100, and the cultured cells were incubatedwith antibodies against AKAP95 (Santa Cruz Biotechnology), the RIIasubunit of PKA (Santa Cruz Biotechnology), vimentin (Santa CruzBiotechnology), Golgin-97 (Cell Signaling Technology), total andphosphorylated CREB (Cell Signaling Technology), and STAT3(Cell Signaling Technology) overnight at 4°C. After washing withphosphate-buffered saline (PBS), the tissue sections were incubatedwith secondary antibodies conjugated with biotinylated horseradishperoxidase H, and red color reaction was developed using the sub-strate 3-amino-9-ethylcarbazole (Vector Laboratories). The cells werepermeabilized with 0.4% Triton X-100 before primary antibodyapplication. After primary antibody application, cells were incubatedwith Alexa Fluor 488–labeled anti-rabbit (green color; 1:100) immu-noglobulinG (IgG) orAlexa 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 mg/ml). Thestained slides were coverslipped withmountingmedium and examinedusing a fluorescence microscope (Carl Zeiss). Confocal imaging wasperformed on a Leica TCS SP8 MP confocal microscope systemequipped with a 100× oil objective. Confocal z-stacks were collected


with a spacing of 0.4 mm in the z axisthrough 5.6-mm thickness of the cell.Colocalization between PKA RIIa andGolgin-97 was analyzed using a LAS AFsoftware, and theManders’ overlap co-efficient (R) was calculated.

Extraction of protein from theamnion tissueTissue pieces were cut from the amnionwithin 5 cm of the spontaneous (TL) orartificial (TNL) membrane rupture sitesandground in liquid nitrogen. The groundtissue was homogenized and lysed in anice-cold radioimmunoprecipitation assay(RIPA) lysis buffer (Active Motif) contain-ing a protease inhibitor cocktail (Sigma)and phosphatase inhibitor (ActiveMotif)and centrifuged at 12,000 rpm for 10minat 4°C. Protein in the supernatant wascollected for Western blotting analysis.

Extraction of subcellular and totalcell proteinProteins from four different subcellularcompartments were extracted using aProteoExtract Subcellular Proteome Ex-traction kit (Merck Millipore). Culturedcells were scratched in PBS containing5 mM EDTA. Cytosolic and nuclear frac-tions were extracted using cytosolic buf-fer [10mMHepes (pH 7.5), 10 mMKCl,1.5 mM MgCl2, 0.5 mM dithiothreitol(DTT), 1 mM NaF, and 1 mM glycerolphosphate] and nuclear buffer [20 mMHepes (pH 7.5), 420 mM NaCl, 1.5 mM

MgCl2, 0.5 mMDTT, 1 mM NaF, and 1 mM glycerol phosphate], re-spectively. Total protein was extracted from the treated cells using anice-cold RIPA lysis buffer containing protease inhibitor cocktail andphosphatase inhibitor.

Isolation and culture of primary human amnion fibroblastsPrimary human amnion fibroblasts were isolated from TNL amnion.Briefly, the amnion tissue was digested twice with 0.125% trypsin (LifeTechnologies Inc.) for 20 min at 37°C and then washed vigorously withPBS to remove epithelial cells. The remaining amniotic tissuewas digestedwith 0.1% collagenase (Sigma) for 25min at 37°C to release the fibroblastsfrom the mesenchymal tissue. The fibroblasts in the digestion mediumwere collected by centrifugation at 2400 rpm for 10min. The isolatedamnion fibroblastswere cultured inDulbecco’smodifiedEagle’smedium(DMEM) containing 10% fetal bovine serum (FBS) plus antibiotics (LifeTechnologies Inc.) at 37°C and 5% CO2/95% air. Immunocytochemicalstainingwithmesenchymalmaker vimentin showed thatmore than 95%of the isolated cells were vimentin-positive.

Transfection of siRNA in amnion fibroblastswith electroporationAfter isolation, the amnion fibroblasts were resuspended and trans-fected with 50 nM siRNA (GenePharma Co.) against AKAP95

Fig. 7. A working model illustrating the role of AKAP95 in cortisol-induced PTGS2 expression in humanamnion fibroblasts. By inducing AKAP95 expression, cortisol increases the abundance of PKA anchored in thenucleus, thereby enhancing the activation of nuclear PKA by the cAMP pathway that is coupled with the PGE2receptor. Consequently, the phosphorylation of nuclear CREB is increased. In contrast, the phosphorylation of STAT3may occur in the cytoplasm. Phosphorylated CREB, STAT3, and activated glucocorticoid receptor (GR) interact at thePTGS2 promoter, thereby leading to the increased PTGS2 expression and subsequent PGE2 production in human amnionfibroblasts. Gs, stimulatory G protein; AC, adenylate cyclase; ATP, adenosine 5′-triphosphate.

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(5′-GCAUCUGCAAAGCAAAUUUTT-3′) or AKAP79 (5′-GCAACA-AAGGCUAAGUCAATT-3′) or randomly scrambled siRNA (5′-UU-CUCCGAACGUGUCACGUTT-3′) in Opti-MEM (Life Technologies)in 2-mm gap cuvettes. Fibroblasts were electroporated at 175 V for 5 msusing a NEPA21 electroporator (Nepa Gene). After dilution withDMEM containing 10% FBS and antibiotics, the cells were transferredto a six-well cell culture plate to incubate for 48 hours and then treatedwith different regimens of reagents. After treatment, the cells were usedfor RNA and protein extraction, and the medium was collected forPGE2 measurement. The efficiency of knockdown was assessed in eachexperiment 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 cellsThe amnion fibroblasts were cultured for 3 days before treatments. Thecells were treated in phenol red/FBS–free culture medium. To examinethe involvement of AKAP95 and AKAP79 in the regulation of COX-2expression, amnion fibroblasts were treated with cortisol (F, 1 mM,Sigma), FSK (100mM,Sigma), or dbcAMP(100mM,Sigma) for 12hoursin the presence or absence of a PKA activity inhibitory peptide (PKI,TTYADFIASGRTGRRNAIHD, 20 mM, Promega) or a PKA-AKAP–anchoring inhibitory peptide (Ht31, DLIEEAASRIVDAVIEQVKAA-GAY, 10 mM, Promega) or siRNA-mediated knockdown of eitherAKAP95 or AKAP79. m-PKI (TTYADAIASGATGAANAIHD, 20 mM,ChinaPeptides) andHt31(Ht31C,DLIEEAASRPVDAVPEQVKAAGAY,10 mM, Promega) serve as controls for PKI and Ht31 studies, respec-tively. To study whether AKAP95 is involved in the phosphorylationof CREB and STAT3, amnion fibroblasts were treated with cortisol(1 mM,Sigma) for 2 hours or FSK (100 mM,Sigma) or dbcAMP (100 mM,Sigma) for 1 hour in the presence of Ht31 or siRNA-mediated knock-down of AKAP95. To determine whether cortisol affects the abundanceof AKAP95 and PKARIIa, amnion fibroblasts were treated with cortisol(0.01, 0.1, and 1 mM) for 12 hours. After treatment, total mRNA andprotein were extracted for analysis with quantitative real-time PCR(qRT-PCR) or Western blotting.

Quantification of mRNA abundance with qRT-PCRTotal cellular RNA was extracted using a commercial kit (OmegaBio-Tek) following a protocol provided by the manufacturer. mRNA wasreverse-transcribed to complementaryDNA(cDNA)using aPrimeScriptRTMasterMixPerfect RealTimekit (TaKaRa). The amounts ofAKAP95,PKA RIIa, and COX-2 mRNA were determined with qRT-PCR usingthe above transcribed cDNA and SYBR Premix Ex Taq (TaKaRa). Theannealing temperature was set at 61°C. The absolute mRNA in eachsample was calculated according to a standard curve set up using serialdilutions of known amounts of PCR products against correspondingcycle threshold (Ct) values. The housekeeping geneGAPDHwas ampli-fied in parallel as an internal loading control. The primer sequencesused for amplifying AKAP95, PKA RIIa, COX-2, and GAPDH wereas follows: AKAP95, 5′-AGACCCTGCGGTTCATAAGC-3′ (forward)and 5′-TCCATCAATTCCTGACGCCG-3′ (reverse); PKA RIIa, 5′-AACCCGCTCTGTTGGTCAAT-3′ (forward) and 5′-ACAATGG-TAGCAGCTCTCGG-3′ (reverse); COX-2 (PTGS2), 5′-TGTGCAA-CACTTGAGTGGCT-3′ (forward) and 5′-ACTTTCTGTACTGCGGGTG-3′ (reverse); GAPDH, 5′-CCCCTCTGCTGATGCCCCCA-3′ (forward)and 5′-TGACCTTGGCCAGGGGTGCT-3′ (reverse). The ratio of thetarget gene over GAPDH in each sample was obtained as an indicationof the target gene expression.


Western blottingThe abundance of AKAP95, AKAP79, PKA RIIa, PKA Ca, COX-2,total CREB, phosphorylated CREB at Ser133, total STAT3, and phos-phorylated STAT3 at Tyr705 was determined using a standard Westernblotting protocol. Briefly, after determination of protein concentrationwith Bradford assays, 30 mg of protein of each sample was electro-phoresed in 9% SDS-polyacrylamide gel and transferred to the nitro-cellulose membrane. After blocking with 5% nonfat milk, the membranewas incubated with antibodies against AKAP95 (1:500; Santa CruzBiotechnology), AKAP79 (1:500; Santa Cruz Biotechnology), PKA RIIa(1:500; Santa Cruz Biotechnology), PKA Ca (1:1000; Cell SignalingTechnology), 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 Sig-naling 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 withthe appropriate secondary antibody conjugated with horseradishperoxidase (Sigma) for 1 hour. The enhanced chemiluminescent detec-tion system (Millipore) was used to detect the bands with peroxidaseactivity. To control sampling error, the same blot was also probed forGAPDH (1:10,000; Proteintech) and lamin A/C (1:5000; Cell Sig-naling Technology) as an internal loading control. The bands werevisualized using a G:BOX iChemi Chemiluminescence Image Capturesystem (Syngene). The ratio of band intensities of AKAP95, AKAP79,PKA RIIa, PKA Ca, COX-2, total CREB, phosphorylated CREB atSer133, total STAT3, and phosphorylated STAT3 at Tyr705 over GAPDHfor cytoplasmic protein or lamin A/C for nuclear protein was obtainedas a measure of target protein abundance, respectively. Ratios of phos-phorylated CREB and STAT3 over total CREB and STAT3 were alsoobtained for the analysis of protein phosphorylation.

Measurements of PGE2 with enzyme immunoassayPGE2 in the culturemedium collected from cultured amnion fibroblaststreated with or without cortisol (1 mM, 12 hours) in the presence orabsence of siRNA-mediated knockdown of AKAP95 was measuredwith enzyme immunoassay kits (Cayman Chemicals) according tothe manufacturer’s protocol.

Statistical analysisAll data are reported as means ± SEM. The number of each study inamnion fibroblasts represents separate experiments using amnionfibroblasts prepared from different pregnancies. Statistical analysiswas performed with paired or unpaired Student’s t test or one-wayANOVA followed by the Newman-Keuls multiple comparison testwhere appropriate. Significance was set at P < 0.05.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/506/eaac6160/DC1Fig. S1. The efficiency of siRNA-mediated knockdown of AKAP79 and AKAP95 in humanamnion fibroblasts.Movie S1. Confocal z-stack imaging of the colocalization of PKA RIIa and Golgin-97 in humanamnion fibroblasts.

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AcknowledgmentsFunding: 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. performedexperiments. J.L., L.W., Y.W., and H.Y. collected samples from patients and analyzed clinicalinformation. 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.

Submitted 13 December 2016Accepted 19 October 2017Published 21 November 201710.1126/scisignal.aac6160

Citation: J. Lu, W. Wang, Y. Mi, C. Zhang, H. Ying, L. Wang, Y. Wang, L. Myatt, K. Sun, AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in humanamnion fibroblasts. Sci. Signal. 10, eaac6160 (2017).

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human amnion fibroblasts expression inPTGS2AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced

Jiangwen Lu, Wangsheng Wang, Yabing Mi, Chuyue Zhang, Hao Ying, Luyao Wang, Yawei Wang, Leslie Myatt and Kang Sun

DOI: 10.1126/scisignal.aac6160 (506), eaac6160.10Sci. Signal.

restricted to these highly specialized cells.prematurely. The nuclear localization of PKA-AKAP95 introduces a new subcellular role for PKA signaling, potentially findings reveal additional molecular targets through which clinicians might induce labor or prevent it from initiatinglocalization of PKA in the nucleus by interaction with AKAP95 enabled it to phosphorylate and activate CREB. These human amnion fibroblasts taken from normal ''term''-labor deliveries, compared to those from cesarean sections, thethat the selective localization of the kinase PKA in the nucleus is critical to this mechanism in the fetal membranes. In

. foundet alCREB. COX-2 produces prostaglandins, which generate positive feedback on the cells to produce more. Lu stimulates the production of the enzyme COX-2 in fetal membrane cells through induction of the transcription factor

Stress hormone signaling is associated with the induction of labor in pregnancy. The stress hormone cortisolPKA labors in the nucleus

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