The role of Prokineticin 1 in endometrial function

Abstract

The Endometrium is a dynamic tissue which undergoes cyclical growth in preparation for pregnancy and regression in the absence of pregnancy. In the midsecretory phase of the menstrual cycle, also known as the window of implantation, the endometrium prepares for the implantation of an embryo by undergoing predecidual changes, leukocyte recruitment and increasing the secretory capacity of the epithelial cells. In pregnancy, the embryo secretes human chorionic gonadotropin (hCG), which maintains progesterone production and has direct effects on the endometrium. Prokineticin 1 (PROK1) is elevated during this transient period and may be a mediator of endometrial receptivity.Prokineticins (PROK1 and PROK2) are pleiotropic proteins with multiple functions including gastrointestinal tract contractility, angiogenesis in the normal and pathological ovary, testis and prostate, noiciceptive sensitisation, transmission of the circadian rhythm of the suprachiasmatic nucleus and immune cell activation. The receptors for the prokineticins (PROKR1 and PROKR2) are two closely related Gprotein coupled receptors. Signalling via these receptors is linked to PLC-ß activation, inositol phosphate mobilisation, ERK 1/2 and Akt phosphorylation and nitric oxide production.Investigation of the temporal pattern of expression of PROK1 and PROKR1 in pregnant and non-pregnant endometrium has shown (a) both factors to be expressed in the normal cycling endometrium with an elevation in PROK1 expression during the secretory phase of the cycle and (b) further elevation of both factors in the pregnant decidua when compared with the non-pregnant endometrium. Expression of PROK1 was down-regulated in endometrial cancer tissue compared with secretory phase endometrium, suggesting PROK1-PROKR1 is not involved in the pathogenesis of this disease. PROK1 and PROKR1 localise to the glandular epithelium, stroma and vasculature of the non-pregnant and pregnant endometrium. Additionally, PROK1 expression was localised in macrophages and uterine natural killer cells within the stromal compartment.In order to investigate signalling and the role of PROK1 in endometrial epithelial cells, an endometrial epithelial cell line (Ishikawa cells) stably expressing PROKR1 was utilised. PROK1-PROKR1 interaction, using this cell line, induced a signalling cascade involving phosphorylation of cSrc, epidermal growth factor receptor (EGFR) and ERK 1/2. This cascade to ERK 1/2 phosphorylation was dependent on activation of Gq protein, PLC-ß and Ca²⁺ as well as phosphorylation of cSrc and EGFR and activation of the small GTPase Ras.Gene array analysis was subsequently conducted using RNA extracted from PROKR1 Ishikawa cells treated with vehicle or 40nM PROK1 for 8 hours. Gene array analysis was conducted using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 and the ABI 1700 v.2 Applied Biosystems Human Genome Survey microarrays. A total of 277 genes were differentially expressed in response to PROK1 (226 genes were up-regulated and 51 genes down-regulated). A number of these genes have suggested roles in implantation. These include: cyclooxygenase-2 (COX-2), leukaemia inhibitory factor (LIF), Interleukin (IL)-6, IL-11 and Heparin bound-EGF. Two genes, COX-2 and LIF were selected for further investigation in this thesis. In the human endometrium and first trimester decidua, expression of COX-2 and LIF co-localise with PROKR1 to the glandular epithelium and stromal cells. PROK1 induces mRNA expression of both of these factors in a time dependent manner in the PROKR1 Ishikawa cell line and first trimester human decidua. Moreover, prostaglandin production and LIF protein secretion was elevated in response to treatment with PROK1. Using specific inhibitors of cell signalling, the expression of COX-2 and LIF in response to PROK1 was dependent on activation of the Gq-PLC-ß-cSrc-EGFR-MEK signalling pathway. Finally, the expression of PROK1 and LIF in the human endometrium can be mediated by embryonic hCG secretion. Treatment of PROKR1 Ishikawa cells and first trimester decidua with 1IU hCG results in sequential increase in PROK1 and LIF expression.Collectively these data strongly suggest that PROK1-PROKR1 signalling is important (a) in the preparation of the human endometrium for pregnancy by regulating expression of implantation related genes and (b) may perform further functions in early pregnancy under the influence of hCG.