48 results found
Lai PF, Lei K, Zhan X, et al., 2021, Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues., PLoS One, Vol: 16, Pages: 1-22, ISSN: 1932-6203
High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue ('bulk') RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.
Yulia A, Singh N, Varley AJ, et al., 2021, PKA and AKIP1 interact to mediate cAMP-driven COX-2 expression: A potentially pivotal interaction in preterm and term labour, PLoS One, Vol: 16, Pages: 1-21, ISSN: 1932-6203
Previously, we showed that cAMP increased COX-2 expression in myometrial cells via MAPK. Here, we have extended these observations, using primary myometrial cell cultures to show that the cAMP agonist, forskolin, enhances IL-1β-driven COX-2 expression. We then explored the role of A-kinase interacting protein (AKIP1), which modulates the effect of PKA on p65 activation. AKIP1 knockdown reversed the effect of forskolin, such that its addition inhibited IL-1β-induced COX-2 mRNA expression and reduced the IL-1β-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1β increased IκBα mRNA expression suggesting that in the context of inflammation and in the presence of AKIP1, cAMP enhances p65 activation. AKIP1 knockdown reversed these changes. Interestingly, AKIP1 knockdown had minimal effect on the ability of forskolin to repress either basal OTR expression or IL-1β-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1β, but not stretch and was repressed by cAMP. The mRNA expression of AKIP1 increased in early labour in tandem with an increase in COX-2 mRNA and protein. AKIP1 protein levels were also increased with inflammation and stretch-induced preterm labour. Our results identify a second important cAMP effector-switch occurring at term in human myometrium and suggest that a hitherto unrecognized interaction may exist between AKIP1, NFκB and AP-1. These data add to the proposition that cAMP acts as a key regulator of human myometrial contractility.
Yulia A, Varley AJ, Singh N, et al., 2020, The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression, PLoS One, Vol: 15, Pages: 1-17, ISSN: 1932-6203
Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1β to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1β-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1β-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1β-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1β alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.
Yulia A, Varley AJ, Singh N, et al., 2020, Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour, PLoS One, Vol: 15, Pages: 1-15, ISSN: 1932-6203
We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined.Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour.These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.
Stanfield Z, Lai PF, Lei K, et al., 2019, Corrigendum: Myometrial transcriptional signatures of human parturition, Frontiers in Genetics, Vol: 10, ISSN: 1664-8021
A Corrigendum onMyometrial Transcriptional Signatures of Human Parturitionby Stanfield, Z., Lai, P. F., Lei, K., Johnson, M. R., Blanks, A. M., Romero, R., et al. (2019). Front. Genet. 10:185. doi: 10.3389/fgene.2019.00185“Pei F. Lai” and “Kaiyu Lei” were not included as authors in the published article. Due to the addition of authors, the list of affiliations had been updated accordingly. The corrected Author Contributions Statement appears below.
Varley A, Yulia A, Lei K, et al., 2019, Novel and Transformative Changes in the cAMP Effector Pathway in Three Distinct Types of Preterm Labour., 66th Annual Scientific Meeting of the Society-for-Reproductive-Investigation (SRI), Publisher: SAGE PUBLICATIONS INC, Pages: 219A-220A, ISSN: 1933-7191
Yu W, Xu L, Lei K, et al., 2018, Effect of crystallization of settled aluminum hydroxide precipitate on "dissolved Al", WATER RESEARCH, Vol: 143, Pages: 346-354, ISSN: 0043-1354
Edey LF, Georgiou H, O'Dea KP, et al., 2017, Progesterone, the maternal immune system and the onset of parturition in the mouse, Biology of Reproduction, Vol: 98, Pages: 376-395, ISSN: 1529-7268
The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2) and the onset of parturition was examined in: 1) naïve mice delivering at term; 2) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and 3) in mice treated with P4 to prevent term parturition.In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43 and COX-2 increased with, but not before, parturition.With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9h post RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition.In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased.These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabour factor synthesis via mRNA dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.
Lei K, Georgiou EX, Yulia A, et al., 2017, Progesterone and the Repression of Myometrial Inflammation: The Roles of MKP-1 and the AP-1 System., 64th Annual Scientific Meeting of the Society-for-Reproductive-Investigation (SRI), Publisher: SAGE PUBLICATIONS INC, Pages: 107A-107A, ISSN: 1933-7191
Yulia A, Singh N, Lei K, et al., 2017, The effects of inflammation and stretch on cyclic AMP levels and pathways components in myometrial tissues, Publisher: WILEY, Pages: 52-52, ISSN: 1470-0328
Georgiou E, Lei K, Lai P, et al., 2016, Progesterone repression of IL-1β action is maintained in human myometrium after the onset of labour, Publisher: WILEY-BLACKWELL, Pages: E12-E13, ISSN: 1470-0328
Georgiou E, Lei K, Singh N, et al., 2016, Chorioamnionitis-induced preterm labour is associated with progesterone receptor cofactor protein changes, Publisher: WILEY-BLACKWELL, Pages: E10-E10, ISSN: 1470-0328
Yulia A, Singh N, Lei K, et al., 2016, Cyclic AMP effectors regulate myometrial oxytocin receptor expression, Endocrinology, Vol: 157, Pages: 4411-4422, ISSN: 1945-7170
The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1β increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1β had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation.
Georgiou EX, Lei K, Lai PF, et al., 2016, The study of progesterone action in human myometrial explants., Molecular Human Reproduction, ISSN: 1460-2407
STUDY HYPOTHESIS: Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition. STUDY FINDING: Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset. WHAT IS KNOWN ALREADY: Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 µM P4 led to a reduction in interleukin-1 (IL-1) β-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset. LIMITATIONS/REASONS FOR CAUTION: There is evidence of basal inflammation in the myometrial explant model. WIDER IMPLICATIONS OF THE FINDINGS: Myomet
Lei K, Georgiou EX, Chen L, et al., 2015, Progesterone and the Repression of Myometrial Inflammation: The Roles of MKP-1 and the AP-1 System, MOLECULAR ENDOCRINOLOGY, Vol: 29, Pages: 1454-1467, ISSN: 0888-8809
Lei K, Sooranna SR, Johnson MR, 2015, Expression data from primary culture human myometrial cells, Genomics Data, Vol: 6, Pages: 182-183, ISSN: 2213-5960
Inflammation plays a central role in many human diseases. Human parturition also resembles an inflammatory reaction, where progesterone (P4) and progesterone receptors (PRs) have already been demonstrated to suppress contraction-associated gene expression. In our previous studies, we have found that the progesterone actions, including progesterone-induced gene expression and progesterone's anti-inflammatory effect, are mediated by PR, GR or both. In this study, we used microarrays (GSE68171) to find P4 and IL-1β responsive genes and IL-1β responsive genes which were repressed by P4. These data may provide a broader view of gene networks and cellular functions regulated by P4 and IL-1β in human myometrial cells. These data will also help us understand the role of PR and GR in human parturition.
Georgiou EX, Lai PF, Yulia A, et al., 2015, Progesterone Repression of IL-1β-Driven COX-2 Expression in Myometrial Explant Cultures, Publisher: SAGE PUBLICATIONS INC, Pages: 134A-134A, ISSN: 1933-7191
Lei K, Sooranna SR, Johnson MR, 2015, P4 Plays Its Anti-inflammatory Role Primarily Via GR and the Inhibition of AP-1 in Human Myometrial Cells, REPRODUCTIVE SCIENCES, Vol: 22, Pages: 136A-136A, ISSN: 1933-7191
Yulia A, Georgiou EX, Lei K, et al., 2015, The Effects of Combinations of cAMP and Progesterone on Progesterone Responsive Genes in Term Human Myometrium, REPRODUCTIVE SCIENCES, Vol: 22, Pages: 142A-142A, ISSN: 1933-7191
Yulia A, Singh N, Lei K, et al., 2014, Molecular Markers of cAMP Related Genes during Gestation and Parturition in Human Myometrium, REPRODUCTIVE SCIENCES, Vol: 21, Pages: 347A-347A, ISSN: 1933-7191
Das A, Lei K, Sooranna SR, et al., 2014, Mechanical Stretch of Human Myometrial Cells in the Absence and Presence of Specific FAK Inhibition., REPRODUCTIVE SCIENCES, Vol: 21, Pages: 139A-140A, ISSN: 1933-7191
Lei K, Chen L, Georgiou EX, et al., 2014, Progesterone Antagonizes IL-1b-Induced COX-2 Expression by Inhibiting AP-1 in Human Myometrial Cells, REPRODUCTIVE SCIENCES, Vol: 21, Pages: 243A-244A, ISSN: 1933-7191
Yulia A, Georgiou EX, Das A, et al., 2014, Enhancement of Anti-Inflammatory Effect by the Combination of Progesterone and cAMP on IL-1b-Stimulated Human Myometrial Cells., REPRODUCTIVE SCIENCES, Vol: 21, Pages: 111A-111A, ISSN: 1933-7191
Singh N, Yulia A, Lei K, et al., 2014, The Molecular Expression of Different Phenotype-Specific Causes of Preterm Labour in Comparison to Term Labour, REPRODUCTIVE SCIENCES, Vol: 21, Pages: 340A-341A, ISSN: 1933-7191
Georgiou EX, Edey LF, Lei K, et al., 2014, RU486-Induced Preterm Labour: A Shifting Paradigm?, REPRODUCTIVE SCIENCES, Vol: 21, Pages: 141A-142A, ISSN: 1933-7191
Chen L, Lei K, Malawana J, et al., 2014, Cyclic AMP enhances progesterone action in human myometrial cells, MOLECULAR AND CELLULAR ENDOCRINOLOGY, Vol: 382, Pages: 334-343, ISSN: 0303-7207
Singh N, Lei K, Sooranna SR, et al., 2013, Molecular Markers of Early and Established Labour in Human Myometrium., 60th Annual Scientific Meeting of the Society-for-Gynecologic-Investigation (SGI), Publisher: SAGE PUBLICATIONS INC, Pages: 300A-300A, ISSN: 1933-7191
Lei K, Sooranna S, Bennett P, et al., 2013, Enhancement of Inflammation-Associated Genes by Progesterone in IL-1β-Stimulated Human Myometrial Cells, 60th Annual Scientific Meeting of the Society-for-Gynecologic-Investigation (SGI), Publisher: SAGE PUBLICATIONS INC, Pages: 161A-162A, ISSN: 1933-7191
Lei K, Chen L, Georgiou EX, et al., 2012, Progesterone acts via the nuclear glucocorticoid receptor to suppress IL-1 beta-Induced COX-2 expression in human term myometrial cells, PLoS ONE, Vol: 7, ISSN: 1932-6203
Progesterone is widely used to prolong gestation in women at risk of preterm labour (PTL), and acts at least in part via theinhibition of inflammatory cytokine-induced prostaglandin synthesis. This study investigates the mechanisms responsiblefor this inhibition in human myometrial cells. We used reporter constructs to demonstrate that interleukin 1beta (IL-1b)inhibits progesterone driven PRE activation via p65 activation and that IL-1b reduced progesterone driven gene expression(FKBP5). Conversely, we found that the activity of a p65-driven NFkB reporter construct was reduced by overexpression ofprogesterone receptor B (PRB) alone and that this was enhanced by the addition of MPA and that both MPA andprogesterone suppressed IL-1b-driven cyclo-oxygenase-2 (COX-2) expression. We found that over-expressed Halo-taggedPRB, but not PRA, bound to p65 and that in IL-1b-treated cells, with no overexpression of either PR or p65, activated p65bound to PR. However, we found that the ability of MPA to repress IL-1b-driven COX-2 expression was not enhanced byoverexpression of either PRB or PRA and that although the combined PR and GR antagonist Ru486 blocked the effects ofprogesterone and MPA, the specific PR antagonist, Org31710, did not, suggesting that progesterone and MPA act via GRand not PR. Knockdown using siRNA confirmed that both MPA and progesterone acted via GR and not PR or AR to repressIL-1b-driven COX-2 expression. We conclude that progesterone acts via GR to repress IL-1b-driven COX-2 activation and thatalthough the interaction between p65 and PRB may be involved in the repression of progesterone driven gene expression itdoes not seem to be responsible for progesterone repression of IL-1b-induced COX-2 expression.
Chen L, Sooranna SR, Lei K, et al., 2012, Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells, Journal of Cellular and Molecular Medicine, Vol: 16, Pages: 1447-1460, ISSN: 1582-4934
Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI2, acting via cAMP/PKA, was found to increase contraction‐associated protein expression in myometrial cells and to promote oxytocin‐driven myometrial contractility. Cyclo‐oxygenase‐2 (COX‐2) is the rate‐limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX‐2 expression, synthesis and activity. Three cAMP agonists (8‐bromo‐cAMP, forskolin and rolipram) increased COX‐2 mRNA expression and further studies confirmed that this was associated with COX‐2 protein synthesis and activity (increased PGE2 and PGI2 in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP‐effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP‐effector PDZ‐GEF1‐2, without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX‐2 expression and that PGE2 acts through EP‐2 to activate MAPK and increase COX‐2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function.
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