Regulation of aromatase P450 expression in endometriotic and endometrial stromal cells by CCAAT/enhancer binding proteins (C/EBPs): decreased C/EBPbeta in endometriosis is associated with overexpression of aromatase

In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.     

Induction of CCAAT/enhancer binding protein-delta by cytokinins, but not by retinoic acid, during granulocytic differentiation of human myeloid leukaemia cells

Cytokinins, purine derivatives that act as hormones to control many processes in plants, are very effective at inducing the granulocytic differentiation of human myeloid leukaemia cells. Isopentenyladenine (IPA), a potent cytokinin, significantly induced the expression of CCAAT/enhancer-binding protein (C/EBP)delta, but not C/EBP alpha protein, whereas all-trans retinoic acid, a well-known inducer of granulocytic differentiation, induced C/EBP alpha but not C/EBP delta protein. Antisense oligonucleotide for C/EBP delta, but not C/EBP alpha or C/EBP beta, effectively suppressed IPA-induced differentiation, suggesting that the expression of C/EBP delta protein is necessary for cytokinin-induced differentiation. Although C/EBP alpha is known to be crucial for granulocytic differentiation, the function of C/EBP delta has not been well documented in the regulation of haematopoiesis. The role of C/EBP delta in the granulocytic differentiation of myeloid leukaemia cells is discussed.     

Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.     

Induction of HLA-DR expression in endometrial epithelial cells by endometrial T-cells: potential regulatory role of endometrial T-cells in vivo.

Endometrial epithelial cells express HLA-DR molecules of the major histocompatibility complex in vivo adjacent to aggregates of T-cells in the endometrial stroma. To test whether HLA-DR expression on endometrial epithelium is mediated by T-cells, endometrial T-cells were isolated from human endometrium by the sheep red blood cell rosetting technique. In contrast to the resting T-cells from peripheral blood and similar to the peripheral blood T-cells activated with Concanavalin-A, endometrial T-cells formed colonies in vitro. Direct addition of the endometrial T-cells to epithelial cell cultures derived from autologous glands induced both morphological changes as well as HLA-DR molecules in the epithelial cells. The intensity of the immunostained HLA-DR molecules in the epithelial cells as well as the percentages of the HLA-DR-positive epithelial cells correlated with the number of T-cells added to the epithelial cell cultures. T-Cells were bound to the HLA-DR-positive epithelial cells as single cells or aggregates, and they were not found bound to the HLA-DR-negative epithelial cells. The supernatant of the endometrial T-cells induced expression of HLA-DR molecules and morphological changes in cultures of HLA-DR-negative epithelial cells. This expression could be inhibited by a neutralizing antiserum to interferon-gamma. These findings are consistent with the hypothesis that endometrial T-cells are activated and suggest that the expression of HLA-DR molecules in glandular epithelium in vivo is mediated by the interferon-gamma secreted by the endometrial T-cells.     

Inhibition of proliferation and differentiation by RU 486 in human endometrial stromal and epithelial cells in vitro.

The effects of progesterone and RU 486 on cellular proliferation and differentiation in long term cultures of mixed human endometrial cells were studied. The endometrial tissue was obtained from women with normal menstrual cycles who were undergoing hysterectomy for benign growths. Estradiol supplemented cultures were treated with progesterone and/or RU 486 for 27 days. Cell number was measured by crystal violet assay, and prolactin secretion was used as a marker of differentiation. Progesterone doubled the rate of proliferation, but the addition of RU 486 reduced it to baseline again. The gestagen increased prolactin secretion up to 30 times, while the addition of RU 486 suppressed it to baseline levels. When administered to cells that were pretreated with progesterone for 15 days RU 486 abolished the progesterone effects. RU 486 alone was without any effect. Our results indicate that (1) in vitro progesterone is essential for the initiation and maintenance of proliferation and differentiation of endometrial cells and (2) RU 486 acts as a pure progesterone antagonist in our culture model.     

Hepatic expression of CCAAT/enhancer binding protein α: hormonal and metabolic regulation in rats

Summary There is a significant body of evidence which suggests that the α-isoform of the CCAAT/enhancer binding protein (C/EBPα) plays a central regulatory role in energy metabolism in the liver. However, there is little information available regarding regulation of its expression in this tissue. In this study, we examined the effect of hormones and diabetes on its expression in rat H4IIE hepatoma cells and in rat liver. Treatment of H4IIE cells with dexamethasone led to a threefold increase in C/EBPα mRNA within 4 h. Insulin treatment produced a bi-phasic response, initially reducing mRNA levels up to the 4 h time point, but after 8 h a twofold increase in C/EBPα mRNA was observed. Treatment with 8-chlorophenylthio-cAMP produced a twofold induction of C/EBPα mRNA after 8 h. Western analysis indicated that the changes in mRNA in response to hormonal treatment generally resulted in corresponding alterations in C/EBPα protein levels. Finally, we observed an inhibition of C/EBPα gene expression in streptozotocin-diabetic rat liver, reflected by a decrease in both mRNA and protein levels that were partially reversed by insulin treatment. These results indicate that the expression of C/EBPα in liver is under complex control by both hormonal and metabolic signals, which is consistent with its role as a trans -regulator of genes which play a role in energy metabolism. [Diabetologia (1997) 40: 1117–1124] Received: 21 January 1997 and in revised form: 28 April 1997     

Facilitative glucose transporter type 1 is differentially regulated by progesterone and estrogen in murine and human endometrial stromal cells

Embryo implantation is a highly synchronized event between an activated blastocyst and a receptive endometrium. The success of this process relies on the dynamic interplay of estrogen (E(2)) and progesterone (P(4)), however, the details of this interaction are not entirely clear. Recent data implicate E(2) and P(4) in the regulation of glucose utilization by affecting facilitative glucose transporter (GLUT) expression. In this study we examine GLUT1 expression in murine and human endometrial stromal cells (ESCs) using a primary culture system. We show that expression of GLUT1 is increased during ESC decidualization in vitro. P(4) up-regulates, whereas E(2) down-regulates, GLUT1 expression. In addition, P(4) increases and E(2) decreases glucose uptake in ESCs, suggesting that GLUT1 may be a major player in glucose utilization in these cells. Moreover, GLUT1 expression is increased in human ESCs when decidualized in vitro with P(4) and dibutyryl cAMP, suggesting a similar role for P(4) in human endometrium. In conclusion, an imbalance between P(4) and E(2) seen in patients with polycystic ovary syndrome, luteal phase defect, and recurrent pregnancy loss may have a critical impact on glucose utilization in the endometrial stroma, and, thus, may be responsible for endometrial dysfunction and failure of embryo implantation in these patient populations.     

Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.

Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.     

Dedifferentiation of adult monkey Sertoli cells through activation of extracellularly regulated kinase 1/2 induced by heat treatment

Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.     

Induction of cyclooxygenase-2 in monocyte/macrophage by mucins secreted from colon cancer cells

Up-regulation of cyclooxygenase-2 (COX-2) and overproduction of prostaglandins have been implicated in the initiation and/or progression of colon cancer. However, it is uncertain in which cells and how COX-2 is induced initially in the tumor microenvironment. We found that a conditioned medium of the colon cancer cell line, LS 180, contained a factor to induce COX-2 in human peripheral blood mononuclear cells. This factor was purified biochemically and revealed to be mucins. A small amount of mucins (approximately 100 ng of protein per ml) could elevate prostaglandin E2 production by monocytes. The mucins induced COX-2 mRNA and protein levels of monocytes in a dose- and time-dependent manner, indicating a COX-2-mediated pathway. We also have examined immunohistochemically the localization of COX-2 protein and mucins in human colorectal cancer tissues. It is noteworthy that COX-2-expressing macrophages were located around the region in which mucins were detectable, suggesting that COX-2 also was induced by mucins in vivo. These results suggest that mucins produced by colon cancer cells play a critical role in the initial induction of COX-2 in the tumor microenvironment.