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Molecular Mechanisms of Progesterone Withdrawal in Human Uterine Smooth Muscle Cells Kaiyu

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Molecular Mechanisms of Progesterone Withdrawal in Human Uterine Smooth Muscle Cells Kaiyu Molecular Mechanisms of Progesterone Withdrawal in Human Uterine Smooth Muscle Cells Kaiyu Lei July 2011 Institute of Reproductive and Developmental Biology Department of Surgery and Cancer Division of Cancer, Faculty of Medicine Imperial College London This thesis is submitted for the degree of Doctor of Philosophy at Imperial College London - 1 - Dedicated to my beloved parents and wife - 2 - Abstract Progesterone administration reduces the risk of preterm labour in high-risk women with singleton pregnancies but has no effect in women with a multiple pregnancy. However, it should be noted that it is not clear why progesterone is effective in the singleton pregnancies and in addition stretch-induced preterm labour is an attractive but not fully proven explanation. The data from my studies showed that progesterone did not inhibit stretch-induced MAPK activation or gene expression possibly explaining why progesterone is ineffective in the prevention of preterm labour in multiple pregnancies. Although stretch did reduce PR expression in a NF-κB- dependent manner, this was not sufficient to inhibit progesterone action, suggesting that it is not responsible for the functional progesterone withdrawal observed with the onset of human labour. Progesterone is thought to reduce the risk of preterm labour by inhibiting inflammatory cytokine-induced increases in prostaglandin synthesis. Initially, I demonstrated that IL-1 inhibited progesterone-driven PRE activation via p65. Conversely, p65-driven NF-B reporter construct activity was reduced by overexpression of PR-B and this was enhanced by the addition of MPA. Both MPA and progesterone repression of IL-1-driven COX-2 expression was lost by knockdown of GR. Subsequently, a series of in vitro studies suggested that progesterone acted via progesterone-induced and GR-mediated MKP-1 activation to repress IL-1-driven COX-2 and that although the interaction between p65 and PR-B might be involved in the repression of progesterone-driven gene expression, it did not seem to be responsible for progesterone repression of IL-1-induced COX-2 expression. Finally, the cDNA microarray analysis showed that the PR and GR regulated distinct gene networks and cellular functions in the absence or presence of ligand. However, the ability of progesterone to modulate gene expression can be mediated via both PR and GR. These data broaden our view of progesterone action and suggest alternative roles for PR and GR in human parturition. - 3 - Statement of originality All work presented in this thesis was performed by myself unless stated otherwise in the text. - 4 - Acknowledgements I would first like to express my gratitude to my supervisors: Professor Mark Johnson, for giving me this opportunity to complete my PhD study, for your unflagging support and encouragement and for your unlimited ideas; Dr. Suren (Dev) Sooranna, for your guidance and innumerable help and for sharing all my happiness and sadness; Professor Phillip Bennett, for your brilliant advice and comments. I want to thank my labmates who have been studying and working with me over the past three years, especially the Chairman of our Communist Party Branch Dr. Chen (our cell-culture expert, should be called Prof. Chen or 陈老) for your generous help and “telling me off” all the time and the Minister of the Organization Dr. Hua (Juliet or 华老) for spending several nights with me in the animal house without complaining a single word. Many thanks to Ben for your advice in some of my studies and improving my listening with your speedy speaking; to Bronwen for guiding me in the animal work and teaching me lots of slang; to Leila for inspiring me with your hard working and the lovely Swiss dark chocolates; to Jackson for all the efforts you have made and your unlimited questions; to Johann for your suggestion about medical school and showing me what is the real social skill; to Winnie and Natasha for getting us lots of biopsies. I would also like to thank the ladies in Prof. Phillip Bennett’s group, Yooni (the PR pioneer) for showing me all the preliminary data and inviting us to the parties and lunch-time concert; Shirin for giving me your precious constructs and sharing with me your experience as a foreigner; Mandeep for teaching me the electroporation and some very “useful” English words which I could never learn from textbooks; Lynne for influencing me with your constant optimism and teaching me the posh English; Vasso for your enthusiasm and yummy hand-made cakes; Kim for your kindness, diligence and declining my food offer without any hesitation. I am also grateful to Prof. Jan Brosens and Dr. Mark Christian for your insightful ideas and advice; to Sue and Ros for all your help and concern; to my friends: Shadi (Boss II), Mady (professor-to-be), Hiro (Boss I), Keiji (CEO), Marwa (consultant), Giulia (the CHIP expert) and Beatriz (for your generosity and advice); also to those who gave me the possibility to complete this thesis. Meeting you guys was fate, and working with you has always been my great pleasure. - 5 - I also appreciate all the patients who were willing to donate their biopsies and all the doctors and midwives who kindly helped us with tissue collection. Last but not least, I would love to give the final thank to my family: my dear parents, parents-in-law and my beloved wife, for your constant love and support no matter where and no matter when. - 6 - List of Publications and Conference Presentations during PhD Study List of Publications: 1. Lei K, Chen L, Cryar BJ, Hua R, Sooranna SR, Brosens JJ, Bennett PR, Johnson MR. Uterine stretch and progesterone action. J Clin Endocrinol Metab. 2011 Mar 30. [Epub ahead of print] PubMed PMID: 21450990. 2. Chen L, Sooranna SR, Lei K, Kandola, M, Bennett PR, Liang Z, Grammatopoulos D, Johnson MR. Cyclic AMP increases COX-2 expression via mitogen activated kinase in human myometrial cells. J Cell Mol Med. 2011 Aug 19. [Epub ahead of print] PMID:21854542 3. Lei K, Chen L, Khanjani S, Sooranna SR, Brosens JJ, Bennett PR, Johnson MR. Progesterone suppression of interleukin-1β gene expression is mediated via nuclear glucocorticoid receptors and MKP-1 activation in human myometrial cells. [Submitted to EMBO Molecular Medicine]. 4. Al-Sabbagh M, Fusi L, Higham J, Lee Y, Lei K, Hanyaloglu AC, Lam EW, Christian M, Brosens JJ. NADPH Oxidase-Derived Reactive Oxygen Species Mediate Decidualization of Human Endometrial Stromal Cells in Response to Cyclic AMP Signaling. Endocrinology. 2010 Dec 15. [Epub ahead of print] PubMed PMID: 21159852. List of Conference Presentations: 1. Lei K, Sooranna SR, Bennett PR, Johnson MR 2011 Glucocorticoid receptor is a key player that mediates progesterone action in human myometrium. Reprod Sci 18: 157A (oral) 2. Lei K, Chen L, Sooranna SR, Bennett PR, Johnson MR 2011 Mechanical stretch and progesterone action in human myometrium. Reprod Sci 18: 297A (poster) 3. Sooranna SR, Lei K, Bennett PR, Johnson MR 2011 Mechanical stretch of cultured human uterine myocytes leads to increased expression of metallothioneins and heat shock 70kDa proteins. Reprod Sci 18: 299A (poster) 4. Hua R, Lei K, Herbert B, Sooranna SR, Bennett PR, Johnson MR 2011 Uterine chemokine expression in progesterone delayed labour in mice. Reprod Sci 18: 296A (poster) 5. Al-Sabbagh M, Fusi L, Higham J, Lee Y, Lei K, Hanyaloglu AC, Lam E W-F, Christian M, Brosens JJ 2011 NAPDH oxidase-derived reactive oxygen species mediate decidualization of human endometrial stromal cells in response to cyclic AMP signalling. Reprod Sci 18: 162A (oral) - 7 - 6. Lei K, Chen L, Sooranna SR, Bennett PR, Johnson MR 2010 The role of cyclin dependent kinase 2 in stretch-induced progesterone receptor B activation in human myometrium. Reprod Sci 17: 281A (poster) 7. Lei K, Chen L, Sooranna SR, Bennett PR, Johnson MR 2010 The effect of pro-inflammatory transcription factors on progesterone receptor B transactivation in human myometrium. Reprod Sci 17: 281A (poster) 8. Lei K, Chen L, Sooranna SR, Bennett PR, Johnson MR 2010 The effect of cytokines and prostaglandins on nuclear receptor co-regulators in human myometrium. Reprod Sci 17: 285A (poster) 9. Chen L, Lei K, Sooranna SR, Bennett PR, Liang Z, Grammatopoulos DK, Johnson MR 2010 The effect of cAMP on PRE promoter activity in presence or absence of progesterone. Reprod Sci 17: 278A (poster) 10. Khanjani S, Sooranna SR, Lei K, Bennett PR, Johnson MR 2009 Regulation of progesterone receptor by transcriptions factors in human uterine myocytes. Reprod Sci 16: 116A (poster) - 8 - Table of Contents ABSTRACT ............................................................................................................................................ - 3 - STATEMENT OF ORIGINALITY ............................................................................................................... - 4 - ACKNOWLEDGEMENTS ........................................................................................................................ - 5 - LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS DURING PHD STUDY ............................... - 7 - TABLE OF CONTENTS ............................................................................................................................ - 9 - LIST OF FIGURES ................................................................................................................................. - 12 - LIST OF TABLES ................................................................................................................................... - 14 -
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