Endometrial cancer is the most common malignancy of the female genital tract. Progesterone (P4) has been used for several decades in endometrial cancer treatment, especially in women who wish to retain fertility. However, it is unpredictable which patients will respond to P4 treatment and which may have a P4-resistant cancer. Therefore, identifying the mechanism of P4 resistance is essential to improve the therapies for endometrial cancer. Mitogen-inducible gene 6 (Mig-6) is a critical mediator of progesterone receptor (PGR) action in the uterus. In order to study the function of Mig-6 in P4 resistance, we generated a mouse model in which we specifically ablated Mig-6 in uterine epithelial cells using Sprr2f-cre mice (Sprr2f(cre+) Mig-6(f/f)). Female mutant mice develop endometrial hyperplasia due to aberrant phosphorylation of signal transducers and activators of transcription 3 (STAT3) and proliferation of the endometrial epithelial cells. The results from our immunoprecipitation and cell culture experiments showed that MIG-6 inhibited phosphorylation of STAT3 via protein interactions. Our previous study showed P4 resistance in mice with Mig-6 ablation in Pgr-positive cells (Pgr(cre/+) Mig-6(f/f)). However, Sprr2f(cre+) Mig-6(f/f) mice were P4-responsive. P4 treatment significantly decreased STAT3 phosphorylation and epithelial proliferation in the uterus of mutant mice. We showed that Mig-6 has an important function of tumor suppressor via inhibition of STAT3 phosphorylation in uterine epithelial cells, and the antitumor effects of P4 are mediated by the endometrial stroma. These data help to develop a new signaling pathway in the regulation of steroid hormones in the uterus, and to overcome P4 resistance in human reproductive diseases, such as endometrial cancer.
Uterine epithelial cells are unique cells in that they are both epithelial in the typical barrier sense but in many mammalian species, they characteristically allow the blastocyst to penetrate them from the apical surface. Here we examine how these cells subserve both functions and in particular we synthesize recent evidence on focal adhesions and how these membrane structures contribute to uterine receptivity for blastocyst implantation. Focal adhesions emerge as a dynamic new player in the ‘plasma membrane transformation’ of early pregnancy and uterine receptivity in that they disassemble at the time of implantation in common with many other structures on the basolateral plasma membrane of these cells.
Glycogen content in mink uterine glandular and luminal epithelia (GE and LE) is maximal during estrus and is depleted before implantation while embryos are in diapause. Uterine glycogen synthesis is stimulated by estradiol (E ) while its mobilization is induced by progesterone (P ). Nevertheless, treatment of an immortalized mink uterine epithelial cell line (GMMe) with E did not affect glycogen production. Interestingly, insulin alone significantly increased synthesis of the nutrient and glycogen content in response to insulin + E was greater than for insulin alone. Our objectives were to determine: 1) If insulin receptor protein (INSR) is expressed by mink uterine GE and LE and if the amount differs between estrus, diapause and pregnancy; 2) if E , P or insulin regulate insulin receptor gene ( ) expression by GMMe cells, and 3) if E and P act independently to regulate glycogen metabolism by GMMe cells and/or if their effects are mediated in part through the actions of insulin. The mean (±S.E.) percent INSR content of uterine epithelia was greatest during diapause (GE: 15.65 ± 0.06, LE:16.56 ± 1.25), much less during pregnancy (GE: 2.53 ± 0.60, LE:2.25 ± 0.32) and barely detectable in estrus (GE: 0.03 ± 0.01, LE:0.02 ± 0.01). Glycogen concentrations in GMMe cells increased 10-fold in response to insulin and 20-fold with insulin + E when compared to controls. Expression of was increased 2-fold by insulin and insulin + E when compared to controls and there was no difference between the two hormone treatments, indicating that E does not increase expression in insulin-treated cells. To simulate E -priming, cells were treated with Insulin + E for 24 h, followed by the same hormones + P for the second 24 h (Insulin + E → P ) which resulted in and glycogen levels not different from controls. Similarly, cells treated with Insulin + P resulted in glycogen concentrations not different from controls. We conclude that the glycogenic actions of E on GMMe cells are due to increased responsiveness of the cells to insulin, but not as a result of up-regulation of the insulin receptor. Glycogen mobilization in response to P was the result of decreased glycogenesis and increased glycogenolysis occurring concomitantly with reduced expression. Mink uterine glycogen metabolism appears to be regulated in a reproductive cycle-dependent manner in part as a result of the actions of E and P on cellular responsiveness to insulin.
The objective of this study was to examine the expression of toll-like receptors (TLRs) by the uterine epithelial cell line ECC-1 and to determine if stimulation of the expressed TLRs induces changes in cytokine and/or chemokine secretion. The expression of TLR1 to TLR9 by ECC-1 cells was demonstrated by reverse transcription polymerase chain reaction, with only TLR10 not being expressed. Stimulation of ECC-1 cells using agonists to TLR2, TLR4 and TLR5 induced the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), as well as the pro-inflammatory cytokine IL-6, and occurred in a dose-dependent manner. In response to zymosan and flagellin, pathogen-associated molecular patterns (PAMP) that are recognized by TLR2 and TLR5 respectively, ECC-1 cells secreted significantly more IL-8, MCP-1 and IL-6 than in response to other TLR agonists. In contrast, agonists to TLR3, TLR7, and TLR9 had no effect on the secretion of the 13 cytokines or chemokines analysed. These results indicate that uterine epithelial cells are important sentinels of the innate immune system. Further it indicates that all but one of the known TLRs are expressed by ECC-1 cells and that stimulation through specific TLRs mediates changes in the expression of key chemokines and pro-inflammatory cytokines that aid in the defence of the uterus against potential pathogens.
Glycogen synthesis by mink uterine epithelial cells is stimulated by estradiol (E-2) during estrus, although the mechanism/s through which the steroid promotes glycogen accumulation are unknown. Our aim was to determine if insulin is required for E-2 induced glycogen synthesis by an immortalized mink uterine epithelial cell line (GMMe). We show that the cells expressed the genes for glycogen metabolizing enzymes (hexokinase 1, glucose-6-phosphatase 3, glycogen synthase 1, and glycogen phosphorylase-muscle), receptors for insulin, insulin-like growth factor 1 and E-2 (Esr1). Interestingly, treatment of cells with E-2 alone failed to stimulate glycogen production, whereas supraphysiological concentrations of insulin (50 g/ml) only, significantly increased glycogen content. Moreover, insulin + E-2 increased glycogen content when compared to insulin alone (p < 0.05), an affect that was blocked when cells were treated with the pure E-2 receptor antagonist ICI 182,780. Glycogen synthesis in response to insulin was significantly inhibited when cells were pre-treated with picropodophyllotoxin, an IGF1R antagonist. Treatment of cells with LY294002, a phosphatidylinositol 3-kinase (PI3K) antagonist, blocked insulin's effects on glycogen production whereas treatment with U0126, an inhibitor of mitogen activated kinase-kinase (MEK1/2) was without effect. These findings suggest to us that the affects of E-2 on glycogen synthesis by GMMe cells is mediated through Esr1 and increased responsiveness of the cells to insulin. Because picropodophylotoxin blocked the effects of insulin on glycogen production, and both insulin and IGF1 act through PI3K, it is possible that IGF1 plays a role in glycogen production by these cells.
Preservation of a pathogen free uterine environment is critical for maintaining healthy swine herds with high reproductive performance. Considering that uterine epithelial cells are the most numerous and thus likely point of cellular contact for pathogens in the uterus, we hypothesize that these cells may be critical for activating the immune system to clear uterine infections. Although uterine epithelial cells have not been well characterized in pigs, studies in several other species have shown that these cells express several pattern recognition receptors (PRR) and thus may act as sentinels for the uterine immune response. To characterize PRR expression in the porcine uterine epithelia, we used laser-capture microdissection to isolate epithelial cells lining the porcine uterus to quantify mRNA expression levels for select PRRs. As well, primary uterine epithelial cells (UECs) were isolated, cultured, polarized and PRR expression was quantified. Immunohistofluorescence and immunofluorescence were used to determine subcellular localization of TLR3, TLR4 and TLR9 in both uterine tissue and in polarized primary UECs. Finally, polarized primary UECs were stimulated with ligands for TLR3, TLR4, TLR9 and NOD2 to determine their functional innate immune response. Uterine epithelial cells ( and ) were shown to express TLR1-7, TLR9, NOD1, NOD2, NLRP3, NLRP6, NLRX1, RIG1, MDA5 and LGP2. Subcellular localization of and polarized primary UECs exhibited TLR3 and TLR9 localized to the apical cell surface whereas TLR4 was localized to the intracellular space. Polarized primary UECs stimulated with TLR3, TLR4 and TLR9 ligands showed induced secretion of IL-6, IL-13 and IL-10, respectively indicating that these receptors were functional. These results indicate that pig uterine epithelial cells are functional innate immune cells that may act as sentinels to protect against uterine infection.