Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition.

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

Activation of prostaglandin EP4 receptor attenuates the induction of cyclooxygenase-2 expression by EP2 receptor activation in human amnion fibroblasts: implications for parturition.

Human amnion fibroblasts produce abundant prostaglandin E2 (PGE2), which plays a crucial role in parturition by stimulating not only myometrial contraction and cervical ripening but also the expression of the rate-limiting enzyme in PGE2 synthesis-namely, cyclooxygenase-2 (COX-2). This feed-forward induction of COX-2 expression by PGE2 is mediated via its receptors coupled with the cAMP and PKA pathway and subsequent phosphorylation of the transcription factors cAMP-response element binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3). Although prostaglandin E receptor (EP)-2 and EP4 for PGE2 are coupled with activation of the cAMP and PKA pathway, the exact roles of these 2 receptors in the regulation of COX-2 expression in amnion fibroblasts remain to be determined. Here, we clarify this issue by employing human amnion tissue and fibroblasts with the long-term objective of specific targeting of prostaglandin synthesis in prevention of preterm birth. We find that an EP2 agonist caused long-lasting increases in CREB phosphorylation and COX-2 expression, whereas an EP4 agonist induced only transient increases in CREB phosphorylation and COX-2 expression in amnion fibroblasts. Moreover, only EP2 stimulation increased STAT3 phosphorylation, whereas only EP4 stimulation increased PI3K activity. EP4 antagonist or inhibition of PI3K enhanced the induction of CREB and STAT3 phosphorylation and COX-2 expression by PGE2 or EP2 stimulation, which was attenuated by EP4 overexpression. Of interest, PGE2 and cortisol, both well-demonstrated stimulants of COX-2 expression in amnion fibroblasts, increased EP2 but decreased EP4 receptor expression. Furthermore, increased EP2 but decreased EP4 abundance were observed in amnion tissue at parturition. We conclude that EP2 and EP4 receptors play different roles in the regulation of COX-2 expression in human amnion fibroblasts. EP2 is the dominant PGE2 receptor mediating the induction of COX-2 at parturition, which can be attenuated by simultaneous activation of PI3K coupled to the EP4 receptor.-Lu, J.-W., Wang, W.-S., Zhou, Q., Gan, X.-W., Myatt, L., Sun, K. Activation of prostaglandin EP4 receptor attenuates the induction of cyclooxygenase-2 expression by EP2 receptor activation in human amnion fibroblasts: implications for parturition.

Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

Preterm premature rupture of fetal membranes precedes 30-40% of preterm births. Activation of matrix metalloproteases (MMPs) is the one of the major causes of extracellular matrix (ECM) degradation in membrane rupture. Increased cortisol, regenerated by 11ß-hydroxysteroid dehydrogenase 1 in the amnion at parturition, is known to participate in a number of parturition-pertinent events. However, whether cortisol has a role in the regulation of MMPs in the membranes is not known. Here, we addressed this issue using human amnion tissue, the most tensile layer of the membranes. RNA-sequencing revealed that cortisol induced MMP7 expression dramatically in amnion fibroblasts, which was confirmed by real-time quantitative RT-PCR and Western blotting analysis in cortisol-treated amnion explants and fibroblasts. Measurement of collagen IV α5 chain (COL4A5), a substrate for MMP-7, showed that cortisol reduced its extracellular abundance, which was blocked by an antibody against MMP-7. Moreover, increased MMP-7 but decreased COL4A5 abundance was observed in the amnion tissue following labor-initiated spontaneous rupture of membranes. Mechanistic studies showed that cortisol increased the phosphorylation of c-Jun and the expression of c-Fos, the 2 major components of activated protein 1 (AP-1), respectively. The knocking down of c-Fos or c-Jun significantly attenuated the induction of MMP7 expression by cortisol. Chromatin immunoprecipitation assays showed that cortisol stimulated the enrichment of c-Fos and c-Jun at the AP-1 binding site in the MMP7 promoter. The data suggest that induction of MMP7 by cortisol via AP-1 may be a contributing factor to ECM degradation in membrane rupture at parturition.-Wang, L.-Y., Wang, W.-S., Wang, Y.-W., Lu, J.-W., Lu, Y., Zhang, C.-Y., Li, W.-J., Sun, K., Ying, H. Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

Involvement of serum amyloid A1 in the rupture of fetal membranes through induction of collagen I degradation.

The de novo synthesis of serum amyloid A1 (SAA1) is augmented in human fetal membranes at parturition. However, its role in parturition remains largely unknown. Here, we investigated whether SAA1 was involved in the rupture of fetal membranes, a crucial event in parturition accompanied with extensive degradation of collagens. Results showed that SAA1 decreased both intracellular and extracellular COL1A1 and COL1A2 abundance, the two subunits of collagen I, without affecting their mRNA levels in human amnion fibroblasts. These reductions were completely blocked only with inhibition of both matrix metalloproteases (MMPs) and autophagy. Consistently, SAA1 increased MMP-2/9 abundance and the markers for autophagic activation including autophagy related (ATG) 7 (ATG7) and the microtubule-associated protein light chain 3 ß (LC3B) II/I ratio with the formation of LC3 punctas and autophagic vacuoles in the fibroblasts. Moreover, the autophagic degradation of COL1A1/COL1A2 and activation of MMP-2/9 by SAA1 were blocked by inhibitors for the toll-like receptors 2/4 (TLR2/4) or NF-κB. Finally, reciprocal corresponding changes of SAA1 and collagen I were observed in the amnion following spontaneous rupture of membranes (ROM) at parturition. Conclusively, SAA1 may participate in membrane rupture at parturition by degradating collagen I via both autophagic and MMP pathways. These effects of SAA1 appear to be mediated by the TLR2/4 receptors and the NF-κB pathway.

ADAMTS4 is a crucial proteolytic enzyme for versican cleavage in the amnion at parturition.

Hyalectan cleavage may play an important role in extracellular matrix remodeling. However, the proteolytic enzyme responsible for hyalectan degradation for fetal membrane rupture at parturition remains unknown. Here, we reveal that versican (VCAN) is the major hyalectan in the amnion, where its cleavage increases at parturition with spontaneous rupture of membrane. We further reveal that ADAMTS4 is a crucial proteolytic enzyme for VCAN cleavage in the amnion. Inflammatory factors may enhance VCAN cleavage by inducing ADAMTS4 expression and inhibiting ADAMTS4 endocytosis in amnion fibroblasts. In turn, versikine, the VCAN cleavage product, induces inflammatory factors in amnion fibroblasts, thereby forming a feedforward loop between inflammation and VCAN degradation. Mouse studies show that intra-amniotic injection of ADAMTS4 induces preterm birth along with increased VCAN degradation and proinflammatory factors abundance in the fetal membranes. Conclusively, there is enhanced VCAN cleavage by ADAMTS4 in the amnion at parturition, which can be reenforced by inflammation.

Single cell transcriptomic analysis of human amnion identifies cell-specific signatures associated with membrane rupture and parturition.

BACKGROUND: The human amnion is an intrauterine tissue which is involved in the initiation of parturition. In-depth understanding of gene expression signatures of individual cell types in the amnion with respect to membrane rupture at parturition may help identify crucial initiators of parturition for the development of specific strategies to prevent preterm birth, a leading cause of perinatal mortality. RESULTS: Six major cell types were revealed in human amnion including epithelial cells, fibroblasts and immunocytes as well as three other cell types expressing dual cell markers including epithelial/fibroblast, immune/epithelial and immune/fibroblast markers. The existence of cell types expressing these dual cell markers indicates the presence of epithelial-mesenchymal (EMT), epithelial-immune (EIT) and mesenchymal-immune (MIT) transitions in amnion at parturition. We found that the rupture zone of amnion exhibited some specific increases in subcluster proportions of immune and EMT cells related to extracellular matrix remodeling and inflammation in labor. The non-rupture zone exhibited some common changes in subcluster compositions of epithelial and fibroblast cells with the rupture zone in labor, particularly those related to oxidative stress and apoptosis in epithelial cells and zinc ion transport in fibroblasts. Moreover, we identified that C-C motif chemokine ligand 20 (CCL20) was among the top up-regulated genes in amnion epithelial cells, fibroblasts and immunocytes in the rupture zone at parturition. Studies in pregnant mice showed that administration of CCL20 induced immunocytes infiltration to tissues at the maternal-fetal interface and led to preterm birth. CONCLUSIONS: Apart from the conventional epithelial, fibroblast and immunocytes, human amnion cells may undergo EMT, EIT and FIT in preparation for parturition. Intense inflammation and ECM remodeling are present in the rupture zone, while enhanced apoptosis and oxidative stress in epithelial cells and zinc ion transport in fibroblasts are present in amnion regardless of the rupture zones at parturition. CCL20 derived from the major cell types of the amnion participates in labor onset.

Cortisol Stimulates Local Progesterone Withdrawal Through Induction of AKR1C1 in Human Amnion Fibroblasts at Parturition.

Fetal membrane activation is seen as being one of the crucial triggering components of human parturition. Increased prostaglandin E2 (PGE2) production, a common mediator of labor onset in virtually all species, is recognized as one of the landmark events of membrane activation. Fetal membranes are also equipped with a high capacity of cortisol regeneration by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), and the cortisol regenerated potently induces PGE2 synthesis, an effect normally suppressed by progesterone during gestation. There is no precipitous decline of progesterone synthesis in human parturition. It is intriguing how this suppression is lifted in parturition. Here, we investigated this issue by using human amnion tissue and primary amnion fibroblasts which synthesize the most PGE2 in the fetal membranes. Results showed that the expression of 11ß-HSD1 and aldo-keto reductase family 1 member C1 (AKR1C1), a progesterone-inactivating enzyme, increased in parallel in human amnion tissue with gestational age toward the end of gestation and at parturition. Cortisol induced AKR1C1 expression via the transcription factor CCAAT enhancer binding protein δ (C/EBPδ) in amnion fibroblasts. Inhibition of AKR1C1 not only blocked progesterone catabolism induced by cortisol, but also enhanced the suppression of cortisol-induced cyclooxygenase-2 (COX-2) expression by progesterone in amnion fibroblasts. In conclusion, our results indicate that cortisol regenerated in the fetal membranes triggers local progesterone withdrawal through enhancement of AKR1C1-mediated progesterone catabolism in amnion fibroblasts, so that the suppression of progesterone on the induction of COX-2 expression and PGE2 synthesis by cortisol can be lifted for parturition.

PGE2 vs PGF2α in human parturition.

Prostaglandin E2 (PGE2) and F2α (PGF2α) are the two most prominent prostanoids in parturition. They are involved in cervical ripening, membrane rupture, myometrial contraction and inflammation in gestational tissues. Because multiple receptor subtypes for PGE2 and PGF2α exist, coupled with diverse signaling pathways, the effects of PGE2 and PGF2α depend largely on the spatial and temporal expression of these receptors in intrauterine tissues. It appears that PGE2 and PGF2α play different roles in parturition. PGE2 is probably more important for labor onset, while PGF2α may play a more important role in labor accomplishment, which may be attributed to the differential effects of PGE2 and PGF2α in gestational tissues. PGE2 is more powerful than PGF2α in the induction of cervical ripening. In terms of myometrial contraction, PGE2 produces a biphasic effect with an initial contraction and a following relaxation, while PGF2α consistently stimulates myometrial contraction. In the fetal membranes, both PGE2 and PGF2α appear to be involved in the process of membrane rupture. In addition, PGE2 and PGF2α may also participate in the inflammatory process of intrauterine tissues at parturition by stimulating not only neutrophil influx and cytokine production but also cyclooxygenase-2 expression thereby intensifying their own production. This review summarizes the differential roles of PGE2 and PGF2α in parturition with respect to their production and expression of receptor subtypes in gestational tissues. Dissecting the specific mechanisms underlying the effects of PGE2 and PGF2α in parturition may assist in developing specific therapeutic targets for preterm and post-term birth.

C/EBPδ drives key endocrine signals in the human amnion at parturition.

Amnion-derived prostaglandin E2 (PGE2) and cortisol are key to labor onset. Identification of a common transcription factor driving the expression of both cyclooxygenase-2 (COX-2) and 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), the key enzymes in their production, may hold the key to the treatment of pre-term labor. Here, we have found that the CCAAT enhancer binding protein δ (C/EBPδ) is such a transcription factor which underlies the feed-forward induction of COX-2 and 11ß-HSD1 expression by their own products PGE2 and cortisol in human amnion fibroblasts so that their production would be ensured in the amnion for the onset of labor. Moreover, the abundance of C/EBPδ in the amnion increases along with COX-2 and 11ß-HSD1 at term and further increases at parturition. Knockout of C/EBPδ in mice delays the onset of labor further supporting the concept. In conclusion, C/EBPδ pathway may be speculated to serve as a potential pharmaceutical target in the amnion for treatment of pre-term labor.

De novo Synthesis of SAA1 in the Placenta Participates in Parturition.

Serum amyloid A1 (SAA1) is an acute phase protein produced mainly by the liver to participate in immunomodulation in both sterile and non-sterile inflammation. However, non-hepatic tissues can also synthesize SAA1. It remains to be determined whether SAA1 synthesized locally in the placenta participates in parturition via eliciting inflammatory reactions. In this study, we investigated this issue by using human placenta and a mouse model. We found that SAA1 mRNA and protein were present in human placental villous trophoblasts, which was increased upon syncytialization as well as treatments with lipopolysaccharides (LPS), tumor necrosis factor-α (TNF-α), and cortisol. Moreover, significant increases in SAA1 abundance were observed in the placental tissue or in the maternal blood in spontaneous deliveries without infection at term and in preterm birth with histological chorioamnionitis. Serum amyloid A1 treatment significantly increased parturition-pertinent inflammatory gene expression including interleukin-1ß (IL-1ß), IL-8, TNF-α, and cyclooxygenase-2 (COX-2), along with increased PGF2α production in syncytiotrophoblasts. Mouse study showed that SAA1 was present in the placental junctional zone and yolk sac membrane, which was increased following intraperitoneal administration of LPS. Intraperitoneal injection of SAA1 not only induced preterm birth but also increased the abundance of IL-1ß, TNF-α, and COX-2 in the mouse placenta. Conclusively, SAA1 can be synthesized in the human placenta, which is increased upon trophoblast syncytialization. Parturition is accompanied with increased SAA1 abundance in the placenta. Serum amyloid A1 may participate in parturition in the presence and absence of infection by inducing the expression of inflammatory genes in the placenta.

Involvement of STAT3 in the synergistic induction of 11ß-HSD1 by SAA1 and cortisol in human amnion fibroblasts.

PROBLEM: Cortisol, which is regenerated from biologically inactive cortisone by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in human fetal membranes, may play an important role in human parturition. Recently, we have demonstrated that human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute-phase protein of inflammation, and SAA1 may be engaged in multiple actions associated with human parturition. It remains to be determined whether SAA1 can interact with cortisol in the regulation of 11ß-HSD1 in the fetal membranes. METHOD OF STUDY: In the current study, we examined the regulation of 11ß-HSD1 expression by SAA1, and the interaction between SAA1 and cortisol in the regulation of 11ß-HSD1 expression in primary human amnion fibroblasts and amnion tissue. RESULTS: Either SAA1 or cortisol induced 11ß-HSD1 expression in a concentration-dependent manner. Combination of SAA1 and cortisol synergistically enhanced 11ß-HSD1 expression. Mechanism studies revealed that SAA1 and cortisol induced the phosphorylation of the transcription factor STAT3 in a sequential order with the induction by SAA1 preceding the induction by cortisol. Furthermore, the induction of 11ß-HSD1 expression by either SAA1 or cortisol or combination of SAA1 and cortisol was blocked by STAT3 inhibition with its antagonist S3I-201 or siRNA-mediated knockdown. CONCLUSION: This study has demonstrated that SAA1 and cortisol can reinforce each other in the induction of 11ß-HSD1 expression through sequential phosphorylation of STAT3. The synergistic enhancement of 11ß-HSD1 expression by SAA1 and cortisol may lead to excessive cortisol accumulation in the fetal membranes at parturition.

Enhancement of cortisol-induced SAA1 transcription by SAA1 in the human amnion.

Our previous studies have demonstrated that human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein of inflammation, wherein SAA1 may participate in parturition by inducing a number of inflammation mediators including interleukine-1ß, interleukine-6 and prostaglandin E2. However, the regulation of SAA1 expression in the fetal membranes remains largely unknown. In the current study, we examined the regulation of SAA1 expression by cortisol, a crucial steroid produced locally in the fetal membranes at parturition, and the interaction between cortisol and SAA1 in the feed-forward induction of SAA1 expression in human amnion fibroblasts. Results showed that cortisol-induced SAA1 expression in a concentration-dependent manner, which was greatly enhanced by SAA1 despite modest induction of SAA1 expression by itself. Mechanism studies revealed that the induction of SAA1 expression by cortisol and SAA1 was blocked by either the transcription factor STAT3 antagonist AZD0530 or siRNA-mediated knockdown of STAT3. Furthermore, cortisol- and SAA1-induced STAT3 phosphorylation in a sequential order with the induction by SAA1 preceding the induction by cortisol. However, combination of cortisol and SAA1 failed to further intensify the phosphorylation of STAT3. Consistently, cortisol and SAA1 increased the enrichment of STAT3 at the SAA1 promoter. Taking together, this study has demonstrated that cortisol and SAA1 can reinforce each other in the induction of SAA1 expression through sequential phosphorylation of STAT3. The enhancement of cortisol-induced SAA1 expression by SAA1 may lead to excessive SAA1 accumulation resulting in parturition-associated inflammation in the fetal membranes.

Extracellular matrix remodeling effects of serum amyloid A1 in the human amnion: Implications for fetal membrane rupture.

PROBLEM: Rupture of fetal membranes is a crucial event at parturition, which is preceded by extensive extracellular matrix (ECM) remodeling. Our recent studies have demonstrated that the human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein, and the abundance of SAA1 in the amnion was increased at parturition. However, the exact role of SAA1 in human parturition remains to be established. METHOD OF STUDY: The effects of SAA1 on the abundance of collagenases and lysyl oxidase, the enzyme that cross-links collagens, were investigated in culture primary human amnion fibroblasts and tissue explants with an aim to examine the involvement of SAA1 in the ECM remodeling in the amnion. RESULTS: Serum amyloid A1 (SAA1) time- and dose-dependently increased the abundance of collagenases MMP-1, MMP-8, and MMP-13, while decreased the abundance of lysyl oxidase-like 1 (LOXL1). These effects of SAA1 were attenuated by siRNA-mediated knockdown of the Toll-like receptor (TLR) 4 and its antagonist CLI-095, but not by siRNA-mediated knockdown of TLR2. Furthermore, the inhibitors for NF-κB (JSH-23) and mitogen-activated protein kinases (MAPKs) p38 (SB203580) and JNK (SP600125) could also attenuate the effects of SAA1, while the inhibitor for MAPK ERK1/2 (PD 98059) could block the effects of SAA1 only on MMP-1, MMP-8, and LOXL1 but not on MMP-13. CONCLUSION: These data highlight a possible role for SAA1 in ECM remodeling preceding membrane rupture by regulating the expression of collagenases MMP-1, MMP-8, MMP-13, and LOXL1 through TLR4-mediated activation of the NF-κB and MAPK pathways in amnion fibroblasts.