Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

Estradiol (E2), a sex steroid hormone molecule, plays a key role in regulating the actin and shape dynamics of cells in a multitude of normal and pathophysiological conditions. While cytoskeletal rearrangements, membrane dynamics, and cellular protrusions are intimately involved in cell motility and invasiveness, little is known about the impact of E2 on these processes in estrogen-dependent epithelial cells. In this study, we quantified the impact of E2 on cell shape and actin dynamics in 12Z human endometriotic epithelial cells transfected with LifeAct-GFP and observed with lattice lightsheet microscopy, a new imaging technique fast enough to capture 3D dynamics on second timescales. E2, when applied for 24 hours, significantly decreased cell circularity, solidity, and rate of change of circularity, indicating a transition to a more elongated and less variable morphology. 24-hour E2 treatment also induced the formation of large membrane protrusions reminiscent of invadopodia and led to a more disordered flow of actin within those protrusions. However, these effects were not seen after 15 minutes of E2 treatment, suggesting that longer-term signaling is required to drive these structural changes. Together, these results suggest that E2 modulates actin polymerization and membrane protrusion dynamics in endometriotic epithelial cells and may prime them for cell invasion. This work highlights a role for hormonal signaling in mediating cytoskeletal plasticity and migratory cell phenotypes.

Identification of Cells with Colony-Forming Activity, Self-Renewal Capacity, and Multipotency in Ovarian Endometriosis

PPAR-gamma ligand activation decreases p450 aromatase gene expression in human endometriotic epithelial and stromal cells in vitro

PPAR-gamma activation inhibits proliferation of endometriotic epithelial and stromal cells and is pro-apoptotic in endometriotic stromal cells in vitro

Immunocytochemical markerprofile of endometriotic epithelial, endometrial epithelial, and mesothelial cells: a comparative study

ObjectiveTo study the molecular mechanisms responsible for fibrosis in endometriosis by investigating whether the protein expression levels of sphingosine-1-phosphate receptor 3 (S1PR3), one of the five specific receptors of the bioactive sphingolipid sphingosine 1-phosphate (S1P), correlated with fibrosis extent in endometriotic lesions and which are the cellular mechanisms involved in this process. DesignCase-control laboratory study and cultured endometriotic cells. SubjectsUniversity research institute and university hospital. Patient(s)A total of 33 women, with and without endometriosis, were included in the study. Interventions(s)Endometriotic lesions were obtained from women with endometriosis (endometrioma [OMA], n=8; deep infiltrating endometriosis [DIE], n=15, (urological n=5, gastrointestinal n=6, posterior n=4)) and control endometrium from healthy women, n=10, by means of laparoscopic/hysteroscopic surgery. The expression of S1PR3 was evaluated by immunohistochemistry and the extent of fibrosis by Masson’s trichrome staining. Human cultured epithelial endometriotic 12Z cells were used to evaluate the mechanisms involved in the profibrotic effect of S1PR3 activation. Main Outcome Measure(s)The expression of S1PR3 in endometriotic lesions positively correlated to endometriosis-associated fibrosis. In addition, S1P induced epithelial-mesenchymal transition (EMT)/fibrosis in epithelial endometriotic cells. By RNA interference and pharmacological approaches, the profibrotic effect of S1P was shown to rely on S1PR3, thus unveiling the molecular mechanism implicated in the profibrotic action of the bioactive sphingolipid. Result(s)The protein expression levels of S1PR3 resulted significantly augmented in the glandular sections of OMA and DIE of different localizations in respect to control endometrium and positively correlated with the extent of fibrosis. S1P was shown to have a crucial role in the onset of fibrosis in epithelial endometriotic cells, stimulating the expression of EMT and fibrotic markers. Genetic approaches highlighted that S1PR3 mediates the fibrotic effect of S1P. Downstream of S1PR3, ezrin and ERK1/2 signaling were found critically implicated in the EMT/fibrosis elicited by S1P. Conclusion(s)The S1PR3 receptor may represent a possible innovative pharmacological target for endometriosis.

Basal release of urokinase plasminogen activator, plasminogen activator inhibitor-1, and soluble plasminogen activator receptor from separated and cultured endometriotic and endometrial stromal and epithelial cells

Endometriosis is a debilitating disease affects up to 20% of reproductive age women characterized by the presence of functional endometrial glandular epithelium and stroma out side the uterine cavity. The pathophysiology of endometriosis remains as enigma in reproductive medicine. Accumulating evidence indicates that steroids, growth factors, and cytokines, and prostaglandins may promote establishment and maintenance of endometriosis. Treatment options include oral contraceptives, aromatase inhibitors, androgenic agents, and gonadotrophin releasing hormone analogues and nonsteroidal anti-inflammatory drugs and surgical removal. But the recurrence rate is up to 70% after cessation of therapy. Prostaglandin E2 (PGE2) promotes cell proliferation, migration, invasion, angiogenesis, anti-apoptosis, pain and immunomodulation. PGE2 mediated effects are primarily mediated through G-protein coupled membrane receptors designated EP that includes EP1, EP2, EP3 and EP4. EP2 and EP4 receptors are coupled to adenyl cyclase generating cAMP that in turn activates the protein kinase A signaling pathway. EP1 receptor is coupled to phospholipase C generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium (Ca2+), and diacyl glycerol an activator of protein kinase C. There are several EP3 isoforms exhibiting a wide range of actions from inhibition of cAMP production to increases in Ca2+ and IP3. In this study, we used immortalized human endometriotic epithelial cells (11-Z, 12-Z, 49-Z, and 108-Z) and stromal cells (22-B) as in vitro models to unravel PGE2 signaling in pathophysiology of endometriosis in human. Normal endometrial epithelial and stromal cells were used as control. We investigated: (1) PGE2 production; (2) expression profiles of EP1, EP2, EP3, and EP4 receptors and (3) role of EP receptors in endometriotic cell proliferation, migration and invasion, and unraveled the underlined mechanisms. We used RT-PCR, western blot, zymography, matrigel migration and invasion assays, and ELISA. Endometriotic cells produced 8–10 fold more PGE2 than normal endometrial cells at basal condition. EP2 and EP4 receptor mRNAs and proteins were abundantly expressed whereas EP1 and EP3 receptor mRNAs and proteins are barely detectable in endometriotic epithelial and stromal cells. Inhibition of either EP2 or EP4 decreased (60%) the endometriotic epithelial and stromal cell proliferation, migration and invasion. Combined inhibition of EP2 and EP4 synergistically decreased (80–85%) endometriotic epithelial and stromal cell proliferation, migration and invasion. Inhibition of EP1 receptor did not have any effect. Inhibition of EP2 and/or EP4 decreased the expression and phosphorylation of Bcl2 protein. Inhibition of EP2 and/or EP4 decreased MMP2 and MMP9 activities. These results suggest that EP2 and/or EP4 mediated cell proliferation and invasion might be mediated respectively through Bcl2 and MMP2 and MMP9 pathways in endometriotic epithelial and stromal cells. Although endometriosis is not a malignancy its pathophysiology seems to be analogous to the mechanisms of cancer metastasis. In conclusion, these endometriotic epithelial and stromal cells can be used as ideal model to study the cellcell communications and interactions in the establishment and maintenance of endometriosis in human. Targeting PGE2 receptors and downstream signaling might be a novel therapy for endometriosis in human. (poster)

Background: The nuclear factor-ĸB (NF-ĸB) pathway activates many of the target genes that are critical to the initiation and establishment of endometriosis. We sought to examine the potential application of pyrrolidine dithiocarbamate (PDTC), a potent NF-ĸB inhibitor, in the treatment of endometriosis. Methods: The phosphorylation of IĸB, expression of nuclear p65 protein and NF-ĸB DNA binding in endometriotic epithelial cells (EECs), endometriotic eutopic epithelial cells (EuECs) and normal epithelial cells (NECs) were detected by Western blot analysis and electrophoretic mobility shift assay. Cyclooxgenase-2 (COX-2) gene and protein expressions in EECs were measured by RT-PCR and Western blot analysis. Prostaglandin E₂ (PGE₂) production of EECs was measured by ELISA. Results: PDTC in the absence or presence of tumor necrosing factor-α (TNF-α) showed stronger inhibitory effects on IĸB phosphorylation, expression of nuclear p65 protein and NF-ĸB DNA-binding activity in EECs than in EuECs or NECs. Pretreatment of EECs with PDTC resulted in a dose-dependent reduction in the TNF-α-induced expressions of COX-2 at gene and protein levels, as well as a reduction of PGE₂ synthesis. Conclusion: PDTC may represent a novel therapeutic strategy for treatment of endometriosis.

In normal endometrium, stromal factors regulate the growth of epithelial cells. However, epithelial cells in endometriotic lesions display increased proliferation and decreased apoptosis. This work tested the hypothesis that in endometriosis stromal cells lose the ability to regulate survival signaling and cell growth in epithelial cells. Primary normal, endometriotic eutopic and ectopic epithelial cells were cultured in the presence of medium conditioned by normal, eutopic and ectopic endometriotic endometrial stromal cells. Endometriotic epithelial cells showed higher Survivin expression than normal epithelial cells. Conditioned medium (CM) from normal or eutopic endometriotic stromal cells significantly inhibited the Survivin expression and AKt phosphorylation in normal or eutopic endometriotic epithelial cells. However, CM from ectopic endometriotic stromal cells did not have an inhibitory effect on normal or ectopic endometriotic epithelial cells. Inhibition of AKt phosphorylation and Survivin expression in normal or eutopic endometriotic epithelial cells in the presence of stromal factors from normal or eutopic endometriotic stromal cells was enhanced by progesterone, whereas progesterone had little effect in the presence of stromal factors from ectopic endometriotic stromal cells. The inability of ectopic endometriotic stromal cells to regulated PI3K/AKt/Survivin signaling and mediate the progesterone response in endometriotic epithelial cells may facilitate epithelial cell proliferation in endometriosis and promote the survival of endometriotic lesions.

Accumulating evidence has suggested an interaction between endometriotic cells and macrophages in the endometriotic microenvironment and the potential role of this interaction in the pathogenesis of endometriosis. However, how endometriotic cells communicate with macrophages to influence their function is poorly understood. In the present study, we found that the mRNA expression and production of CC chemokine ligand 2 (CCL2) were much higher in human endometriotic epithelial cells (11Z and 12Z) than those in human endometrial epithelial cells (HES). The inhibition of CCL2 action using neutralizing antibodies substantially suppressed macrophage migration induced by endometriotic epithelial cells. The endometriosis-associated macrophages (EAMs), which are the macrophages that are stimulated by the conditioned medium (CM) of human endometriotic cells, highly expressed the M2 phenotype markers (MRC1 and TREM2). In addition, the CM of EAMs significantly increased cell migration in 12Z cells, but no significant change was observed in cell growth. RT-PCR and antibody array analyses revealed that EAMs highly express and produce interleukin (IL) 6 compared to macrophages stimulated by the CM of HES cells. Moreover, the EAM-CM-induced migration and MMP2/9 expression in endometriotic cells were significantly attenuated by IL6 signaling inhibition. These results suggest a reciprocal activation of macrophages and endometriotic cells via the soluble factors CCL2 and IL6, which may contribute to the development of endometriosis.

Gene expression profiles and functional characterization of human immortalized endometriotic epithelial and stromal cells

BackgroundMelatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear. Here, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic endometrial epithelial cells and explored the pathway that might be involved.MethodsThis hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the expression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by CCK8 assays.ResultsCompared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of Notch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by specific inhibitor blocked 17β-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells.ConclusionsOur data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present in endometriotic endometrium. Melatonin may block 17β-estradiol-induced migration, invasion and EMT in normal and endometriotic epithelial cells by upregulating Numb expression and decreasing the activity of the Notch signaling pathway.

TGF-β/snail-mediated epithelial-to-mesenchymal transition disrupts estradiol metabolism through suppressing the HSD17B2 expression in endometriotic epithelial cells.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion, migration, and invasion of human endometriotic cells