Prokineticin 1 induces Dickkopf 1 expression and regulates cell proliferation and decidualization in the human endometrium

Prokineticin 1 (PROK1) signalling via prokineticin receptor 1 (PROKR1) regulates the expression of several genes with important roles in endometrial receptivity and implantation. This study investigated PROK1 regulation of Dickkopf 1 (DKK1) expression, a negative regulator of canonical Wnt signalling, and its function in the non-pregnant endometrium and first trimester decidua. DKK1 mRNA expression is elevated during the mid-secretory phase of the menstrual cycle and expression increases further in first trimester decidua. DKK1 protein expression is localized to glandular epithelial and stromal cells during the proliferative, early- and mid-secretory phases, whereas expression is confined to the stroma in the late-secretory phase and first trimester decidua. PROK1 induces the expression of DKK1 in endometrial epithelial cells stably expressing PROKR1 and in first trimester decidua explants, via a Gq-calcium-calcineurin-nuclear factor of activated T-cells-mediated pathway. Endometrial epithelial cell proliferation is negatively regulated by PROK1-PROKR1 signalling. We demonstrate that this effect on cell proliferation occurs via DKK1 expression, as siRNA targeted against DKK1 reduces the PROK1-induced decrease in proliferation. Furthermore, decidualization of primary human endometrial stromal cells with progesterone and cyclic adenosine monophosphate is inhibited by miRNA knock down of PROK1 or DKK1. These data demonstrate important roles for PROK1 and DKK1 during endometrial receptivity and early pregnancy, which include regulation of endometrial cell proliferation and decidualization.     

Expression of integrin alpha5 and integrin beta4 and their extracellular ligands fibronectin and laminin in human decidua during early pregnancy and its sex steroid-mediated regulation

The reorganization of the human endometrium is termed decidualization, which includes endometrial cell proliferation, differentiation, integrin switching and extracellular matrix (ECM) remodeling during early pregnancy. The present study aimed to investigate distribution patterns, staining intensity and sex steroid-mediated regulation of integrin alpha5 (CD49e), integrin beta4 (CD49f) expression and their ligands fibronectin and laminin during decidualization. Human tissue samples were evaluated in two groups, those collected in early days and those collected in advanced days of the first trimester. Correlating immunostaining was found between laminin and integrin beta4, and between fibronectin and integrin alpha5. The expression of fibronectin was higher than that of laminin in the early days (p < 0.05). Temporal and spatial immunostaining of integrin beta4 and alpha5 in the apical pole of luminal and glandular cells was observed as pregnancy progressed (p < 0.05). In vitro results showed that human chorionic gonadotropin (hCG) stimulated laminin expression, downregulated integrin beta4 expression, whereas estradiol decreased fibronectin expression by Ishikawa cells. hCG suppressed fibronectin expression in endometrial stromal cells in culture. Our results suggest that fibronectin is responsible for induction of decidual cell differentiation, and different temporal and spatial expression of the integrins may play a role in implantation. Our in vitro results suggest that regulation of extracellular matrix remodeling and integrin switching are at least partially regulated by reproductive hormones.     

Expression of beta hCG and alpha CG mRNA and hCG hormone in human decidual tissue in patients during tubal pregnancy

We recently showed that endometrial tissue produces hCG during the secretory phase of the menstrual cycle. Based on these findings, we hypothesized that the decidua should also be able to secrete hCG. We examined the decidualized endometrium of patients with extrauterine pregnancies. Decidual specimens were obtained for mRNA extraction and paraffin embedding from 24 patients that were between weeks 6-11 of tubal pregnancy. Tissues were evaluated and classified into one of three groups based on the endometrial differentiation that took place prior to conception: (A) high secretory transformation, (B) diminished transformation with restricted decidualization and (C) inferior endometrial proliferation. Decidual gland hCG secretion was demonstrated immunohistochemically and by Western blotting. Serum hCG levels were higher (P < 0.0001) in patients from group A than group C. mRNA expression of both the beta subunit (beta-hCG) and alpha subunit (alpha-CG) was determined by RT-PCR. Furthermore, the specificity of beta-hCG amplification was confirmed by restriction enzymes. beta-LH amplification was not found. Moreover, the degree of endometrial transformation and the level of decidualization was found to correlate with hCG hormone staining and beta-hCG mRNA expression. hCG protein in the decidua was present in the glands of the compact layer and in the spongy layer, and was more pronounced in previously transformed high secretory endometrium than in inferior endometrium. In conclusion, this study provides the first evidence that hCG is produced in the decidua of patients during extrauterine pregnancies and might play a possible paracrine role.     

Endothelin and dopamine release

Endothelins and endothelin receptors are widespread in the brain. There is increasing evidence that endothelins play a role in brain mechanisms associated with behaviour and neuroendocrine regulation as well as cardiovascular control. We review the evidence for an interaction of endothelin with brain dopaminergic mechanisms. Our work has shown that particularly endothelin-1 and ET(B) receptors are present at significant levels in typical brain dopaminergic regions such as the striatum. Moreover, lesion studies showed that ET(B) receptors are present on dopaminergic neuronal terminals in striatum and studies with local administration of endothelins into the ventral striatum showed that activation of these receptors causes dopamine release, as measured both with in vivo voltammetry and behavioural methods. While several previous studies have focussed on the possible role of very high levels of endothelins in ischemic and pathological mechanisms in the brain, possibly mediated by ET(A) receptors, we propose that physiological levels of these peptides play an important role in normal brain function, at least partly by interacting with dopamine release through ET(B) receptors.     

Expression of intermediate filament in endometrial glands changes with the onset of pregnancy and in endometriosis.

Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermediate filament proteins, cytokeratin and vimentin, in human endometrium and first trimester decidua and in ectopic endometrium from women with endometriosis. Stromal elements, including vascular endothelial cells, were consistently vimentin-positive and cytokeratin-negative. Surface and glandular epithelial cells of human endometrium co-expressed vimentin and cytokeratin during all stages of the menstrual cycle, but failed to express vimentin after the onset of pregnancy. This suggests that intermediate filaments, and especially vimentin, may have a role to play in the proliferation and/or differentiation of the endometrial glands during decidualization. Ectopic endometrium showed a staining pattern similar to normal endometrium.     

Effects of endothelin ETA receptor antagonism on granulocyte and lymphocyte accumulation in LPS-induced inflammation

Endothelin peptides play active roles in different aspects of inflammation. This study investigates the contribution of endogenous endothelins to lipopolysaccharide (LPS) pulmonary inflammation by assessing the influence of ET(A) receptor antagonism on leukocyte accumulation, granulocyte adhesion molecule expression, and chemokine/cytokine modulation. Local pretreatment with BQ-123 or A-127722 (150 pmol), two selective and chemically unrelated endothelin ET(A) receptor antagonists, inhibits neutrophil and eosinophil accumulation in LPS-induced pleurisy at 24 h but not neutrophil migration at 4 h. The effect of endothelin antagonism on neutrophil accumulation at 24 h was concomitant with inhibition of eosinophil and CD4 and CD8 T lymphocyte influx. It is surprising that the ET(A) receptor blockade did not inhibit the accumulation of gammadelta T lymphocytes, cells that are important for granulocyte recruitment in this model. Blockade of ET(A) receptors did not influence the expression of adhesion molecules (CD11b, CD49d) on granulocytes but abrogated the increase in tumor necrosis factor alpha levels 4 h after LPS stimulation and also markedly inhibited increases in levels of interleukin-6 and keratinocyte-derived chemokine/CXC chemokine ligand 1 but not eotaxin/chemokine ligand 11. Thus, acting via ET(A) receptors, endogenous endothelins play an important role in early cytokine/chemokine production and on granulocyte and lymphocyte mobilization in LPS-induced pleurisy.     

Localization of fibrillin-1 in human endometrium and decidua during the menstrual cycle and pregnancy

We have examined the existence and distribution of fibrillin-1 within the endometrium and decidua to ascertain the effect of decidualization upon its synthesis, and its relationship to other extracellular matrix proteins. Formalin-fixed, paraffin-embedded tissues were stained using monoclonal antibodies to collagen IV, elastin, fibrillin-1 and laminin, by immunocytochemistry. Fibrillin-1 was present throughout the menstrual cycle and appeared to be cell-associated. Similar staining for fibrillin-1 was detected within the Fallopian tube of ectopic pregnancy material. In first trimester, second trimester and term decidua, staining for fibrillin-1 was more intense than that during the menstrual cycle and was detected as a thick layer encapsulating decidual cells, but also in fibrillar form inter-connecting these cells. In comparison with the association of collagen IV and laminin with all basal laminae, fibrillin-1 was absent from the vascular and glandular elements of pregnancy endometrium. Elastin was absent in all tissues examined. Hence, fibrillin-1 exists within the uterus in association with basal lamina components rather than with elastin- containing fibrils, where its presence in the endometrium and decidua could be of functional significance.      

Differential effects of interleukin-1beta and transforming growth factor-beta1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro

BACKGROUND: The pro-inflammatory cytokine, interleukin-1 beta (IL-1beta) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. METHODS: The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1beta and TGF-beta1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. RESULTS: ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1beta increased whereas TGF-beta1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. CONCLUSION: IL-1beta and TGF-beta1 differentially regulate ADAMTS-1 expression in human decidual stromal cells.     

Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception

Inhibins and activins are dimeric hormones which share common subunits and which have diverse endocrine and paracrine roles in regulating reproductive function. Endometrial expression of inhibin alpha, ssA and ssB subunits was examined by immunohistochemistry and in-situ hybridization, across the menstrual cycle and in early pregnancy. All three subunits were found to be expressed in endometrium, primarily by glandular epithelium in the early stages of the cycle. Following the onset of decidualization, expression of alpha, ssA and ssB subunits was up-regulated in decidualized stromal cells. A marked down-regulation of alpha subunit was detected in glandular epithelium, whilst expression of ssA and ssB subunits was maintained. This pattern was consistent in decidua from early pregnancy and additionally in endometrium from women using progestin-only contraceptives, either subdermal implants (Norplant((R))) or levonorgestrel-releasing intrauterine systems (Lng-IUS). Immunostaining was also observed for both ssA and ssB subunits in subpopulations of endometrial leukocytes, identified to be distinct subsets of macrophages, neutrophils and mast cells. Potential paracrine roles for activins may be envisaged in facilitating tissue remodelling during decidualization, in tissue repair following menstruation, and additionally in modulating premenstrual inflammatory events.     

Alteration of the immune cell profiles in the pathophysiology of tubal ectopic pregnancy

Tubal ectopic pregnancy (TEP) refers to implantation of conceptus in the fallopian tube. It makes up over 98% of ectopic pregnancy (EP), which is the leading cause of maternal morbidity and mortality in the first trimester of pregnancy. Immune cells at the maternal‐fetal interface play important roles in the process of embryo implantation, stroma decidualization, and early placental development. Alterations in the composition, phenotype, and activity of the immune cells in the fallopian tubes contribute toward the onset of TEP. In this review, we compare the leukocytic proportions in decidua of normal pregnancy, and in decidua and fallopian tubes of TEP. The possible functions of these immune cells in the pathophysiology of TEP are also discussed.     

Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization

The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.     

Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukaemia inhibitory factor and prolactin

Interleukin-11 (IL-11) is crucial in the decidualization response of the uterine stroma to the implanting blastocyst in the mouse. This study examined the localization and expression of IL-11 in human endometrium throughout the menstrual cycle and of prolactin and leukaemia inhibitory factor (LIF) in secretory phase endometrium. The mRNA expression of IL-11 receptor alpha and the signalling component, gp130, in endometrial tissue were also determined. Immunoreactive IL-11 was highest in the secretory phase and present in decidualized stromal cells, glandular epithelial cells, endothelial and smooth muscle cells, and the mRNA expression was verified by in-situ hybridization. Decidual cells showed the most intense staining. IL-11 receptor alpha and gp130 mRNA were detected throughout the cycle with minimal variation. Expression of IL-11 mRNA and protein preceded that of prolactin. While immunoreactive prolactin was found in stromal, decidual and glandular epithelial cells, prolactin mRNA was confined to decidual cells. In contrast, endometrial LIF expression preceded IL-11 but was largely confined to the glandular epithelium. The sequence of appearance of LIF, IL-11 and prolactin suggests a synchronized role for each in the differentiation of the endometrium. The cyclical changes and cell type specific expression of IL-11 suggests a potential role in the decidualization of stromal cells.     

Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.

In addition to their vasoactive action, endothelins are potent peptides in the regulation of both cell proliferation and the turnover of extracellular matrix. Using immunohistochemical, autoradiographic, and molecular analyses, we have studied the localization and expression of endothelin-1 and endothelin A (ETA) and B (ETB) receptors in scleroderma-associated fibrotic lung disease. Increased ET-1 immunoreactivity was found in sclerotic tissue compared with control and was associated with the vasculature, pulmonary interstitium, and bronchial and alveolar epithelium. Microautoradiographic analysis after 125I-labeled ET-1 binding showed a two- to threefold increase in the expression of total ET-1 receptors in scleroderma lung tissue localized to the alveolar epithelium and the pulmonary interstitium which was composed of mainly fibroblastic cells with macrophages and some microvessels. RNAse protection assay revealed significantly reduced ETA receptor and slightly raised ETB message levels in systemic sclerosis lung. Surface expression of functional ET receptors was examined by targeted receptor blocking using mixed and receptor-subtype-selective ligands. A consistent decrease in ETA receptor binding sites was noted primarily within the interstitium and vasculature, in contrast to a slight increase in ETB receptors. Elevated ET-1 and the cell-specific pattern of endothelin receptor expression suggest that the endothelins may represent important mediators that influence the pathology of scleroderma-associated lung disease and other fibrotic conditions.     

Proto-oncogenes c-jun and c-fos are down-regulated in human endometrium during pregnancy: relationship to oestrogen receptor status

Oestrogen is the major stimulatory factor in endometrial cellproliferation. Animal and in-vitro studies have shown that proto-oncogenesc-fos and c-jun are regulated by oestrogen receptor (ER) complex.We have previously shown by Northern blot analysis that proto-oncogenesc-fos and c-jun are strongly expressed in human proliferativeand early to mid-secretory endometrium. In this study, we examinedthe expression of the messenger RNA (mRNA) of the nuclear proto-oncogenesc-fos and c-jun in 10 early (6–10 weeks) and 20 term (30–40weeks) pregnancy decidua by Northern blotting. In order to investigatethe relationship between ER and these proto-oncogenes, the ERand progesterone receptors (PR) were identified in the sametissue samples by immunohistochemistry. When using 30-mer oligonucleotideprobes, hardly any signals for c-fos and c-jun could be identifiedeither in early or in late pregnancy decidua. Nuclear ER stainingwas intense in the epithelium and stroma of proliferative andearly to mid-secretory endometrium but was sparsely scatteredin stroma and lacking in epithelium during early pregnancy.In late pregnancy decidua, no positive ER staining was detectable.PR were present in abundance both in endometrial epitheliumand stroma in proliferative and early secretory phase, and clearpositive staining remained in stromal cells in late secretoryphase and throughout pregnancy. The temporal association betweenimmunoreactive ERs and the expression of c-fos and c-jun mRNAsuggests that the activation of both proto-oncogenes is ER-mediatedin human endometrium. The down-regulation of ER is one possibleexplanation for the repression of these immediate early genesduring pregnancy. c-fos/c-jun/decidua/oestrogen receptor     

Pregnancy associated endometriosis with pronounced stromal myxoid change

A case of endometriosis presenting as a mass in the groin of a pregnant woman is described. The mass increased in size during the pregnancy and the radiological features were suspicious of malignancy. Histological examination showed atrophic glands set in an abundant stroma. This was not typical of normal endometrial stroma but had a pronounced myxoid appearance with areas of decidualization. The atypical site of the endometriosis together with the unusual stromal changes resulted in diagnostic confusion. Although stromal decidualization is well recognized in endometriosis in pregnancy, pronounced myxoid change appears unusual. The myxoid change in this case may be a degenerative phenomenon related to pregnancy.