hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma.

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immuno-conjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF. METHODS: A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3+ levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h (51)Cr assays were also conducted in the presence of low doses of IL-2 (i.e., 50-100 IU ml(-1)). RESULTS: Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; P<0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC (mean killing+/-s.d., 65.6+/-3.7%, range 57.5-77.0%, P<0.001), although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC. CONCLUSION: hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities.

Cell type-specific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection.

Placental infection is associated with adverse fetal outcomes. Toll-like receptors (TLRs) are critical regulators of the innate immune response based on their ability to recognize and respond to pathogen-associated molecular patterns expressed by microbes. To date, cell-type specific expression and regulation of TLR function in human term placenta remains largely unelucidated. The goal of the current study was to examine the in vivo and in vitro patterns of TLR expression and function in major cell types of term placenta. Immunohistochemical analysis of terminal and stem villi localized TLR-2, which recognizes peptidoglycan (PG) from Gram-positive bacteria, to endothelial cells and macrophages, and to a lesser extent to syncytiotrophoblast (SCTs) and fibroblasts (FIBs). Staining for TLR-4, the receptor for Gram-negative bacterial lipopolysaccharide (LPS), was most prominent in SCTs and endothelial cells. Results from Western blotting, conventional, and quantitative PCR (qRTPCR) analyses using protein and mRNA isolated from cultures of SCTs and myofibroblasts (mFIBs) revealed that SCTs expressed TLR-2 and TLR-4, whereas mFIBs expressed only TLR-4. In addition, qRTPCR showed that LPS treatment increased TLR-2 expression in SCTs, indicating that infection with Gram-negative bacteria may enhance innate immune responses in placenta toward a broad range of microorganisms. In addition, treatment with LPS increased IL-8 levels in both SCTs and mFIBs, whereas PG treatment only stimulated IL-8 levels in SCTs. Our results indicate that there exist cell type-specific patterns of TLR function in placenta which likely regulate innate immune response at the maternal-fetal interface.

Long-term progestin-only contraceptives result in reduced endometrial blood flow and oxidative stress.

CONTEXT: Because of their safety and efficacy, long-term progestin-only contraceptives (LTPOCs) are well-suited for women with restricted access to health care. However, abnormal uterine bleeding (AUB) causes half of all users to discontinue therapy within 12 months. Endometria of LTPOC-treated patients display aberrant angiogenesis with abnormally enlarged, thin-walled, fragile blood vessels, inflammation, and focal hemorrhage. In this study, similar effects were observed with a new third-generation implantable LTPOC. OBJECTIVE: We hypothesized that LTPOC reduces uterine and endometrial blood flow, leading to hypoxia/reperfusion, which triggers the generation of reactive oxygen species. The latter induce aberrant angiogenesis, causing AUB. DESIGN: Endometrial perfusion was measured by laser-Doppler fluxmetry in women requesting LTPOCs. Endometrial biopsies were obtained for in vivo and in vitro experiments. SETTING: The study was conducted in the Yale University School of Medicine and Family-Planning Center in Western Australia. PATIENTS: Seven women 18 yr or older requesting implantable LTPOCs were recruited in Western Australia. INTERVENTION: Women received etonorgestrel implants. MAIN OUTCOME: LTPOC treatment resulted in reduced endometrial perfusion and increased endometrial oxidative damage. CONCLUSIONS: We propose that LTPOCs result in hypoxia reperfusion, which leads to aberrant angiogenesis resulting in AUB.

Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells.

OBJECTIVE: Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity. METHODS: Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture. RESULTS: Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells. CONCLUSIONS: Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Increased microsomal oxidation of alcohols after pyrazole treatment and its similarities to the induction by ethanol consumption.

Microsomes isolated from rats treated for 3 days with 200 mg/kg body wt. per day of pyrazole, a potent inhibitor of alcohol dehydrogenase, catalyzed the oxidation of ethanol and 2-butanol at rates 2-3-fold higher than saline controls. This increase was blocked by carbon monoxide, and was not associated with an increase in the oxidation of aminopyrine or in the content of cytochrome P-450, suggesting the possibility of an induction of an alcohol-preferring cytochrome P-450 by pyrazole. Microsomes from the pyrazole-treated rats displayed a stereochemical preference for the oxidation of the (+)-2-butanol isomer over the (-)-2-butanol isomer, which was blocked by carbon monoxide, and also displayed a type-2 binding spectrum with dimethylsulfoxide or 2-butanol. No such spectrum was found with the saline controls. These properties are similar to those which are observed with microsomes from chronic ethanol-fed rats. These similarities suggest the possibility that pyrazole treatment may induce a cytochrome P-450 isozyme with properties similar to the ethanol-inducible cytochrome P-450.

Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells.

Vascular endothelial cells play a critical role in the maintenance of endometrial homeostasis. Indeed many pathological conditions causing abnormal endometrial bleeding including progestin only contraception, hormone replacement therapy, endometrial polyps, myomas, hyperplasia and cancer are associated with aberrant angiogenesis. Critical to the process of angiogenesis is the breakdown of the surrounding tissues by matrix metalloproteases (MMPs). In addition to the cells surrounding the endometrial endothelial cells, the endothelial cells themselves produce their own panel of MMPs. We now characterize the specific MMPs that are expressed by endothelial cells derived from human endometrium. These include MMP-1, MMP-2 and MMP-10 but not MMP-3. In addition, in order to successfully carry out consistent, homogeneous and sufficient numbers of studies we investigated the in vitro expression of the MMPs with both freshly isolated, early passaged endometrial endothelial cells (HEECs) as well as with newly telomerase immortalized HEECs (T-HEECs). The latter were karyotypically normal and expressed classic endothelial cell endpoints such as tubulogenesis on matrigel and expression of the endothelial cell markers CD-31 (PECAM), von Willebrand's factor, and the Tie-2 receptors. The levels of MMP expression as well as that of the metalloprotease inhibitors TIMP-1 and TIMP-2 were similar in parent and immortalized endothelial cells.

Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3.

Prior studies indicate that tissue factor (TF), the primary cellular initiator of hemostasis, is persistently up-regulated in human endometrial stromal cells (HESCs) undergoing progestin-induced decidualization in vivo and in vitro. The mechanism underlying progestin enhancement of TF mRNA and protein levels in these cells involves transcriptional activation of the TF gene. Transient transfections of HESCs with the truncated TF promoters driving the luciferase reporter gene have demonstrated that the region spanning -111 to +14 bp retained differential progestin-enhancing effects. We now demonstrate that RU486 displays inhibitory effects on the progestin-induced TF promoter activity, confirming the involvement of the progesterone receptor. Since the TF minimal promoter (pTF 111 spanning -111 to +14 bp) contains three overlapping Sp1 and three Egr-1 sites, the present study determined whether Sp1 and/or Egr-1 were required for progestin-regulated TF expression. The results indicate that the three Sp1 sites are primarily responsible for both the constitutive and progestational activity of the pTF 111 promoter, whereas the Egr-1 sites have only a minor involvement in both activities. Overexpression of the Sp1 protein resulted in greater than a 6-fold induction in TF promoter activity. In contrast, no enhancement was observed when the Sp3 protein was overexpressed. The concomitant overexpression of Sp1 and Sp3 demonstrated that Sp3 completely blocked the induction of TF promoter activity by Sp1. Moreover, the addition of 10 nM mithramycin, a concentration that inhibits Sp1 binding to target DNA, blocked the progestational induction of TF mRNA expression. Immunohistochemical studies demonstrated increased Sp1 levels in perivascular stromal cells in secretory phase compared with proliferative phase endometria. In contrast, Sp3 expression was greatly decreased in stromal cells of secretory, compared with proliferative phase tissues. The levels of Egr-1 were low in both proliferative and secretory endometria. Immunocytochemistry of E2 vs. E2 + medroxyprogesterone acetate-treated HESCs demonstrated a dramatic reduction in Sp3 expression after progestin treatment, and Northern blots demonstrated progestational increases in Sp1 and reduction in Sp3 mRNA expression compared with controls. Taken together, our results demonstrate that progestin enhancement of TF gene expression in HESCs is mediated principally by Sp1. We propose that progestins regulate HESC TF gene expression in vivo by altering the ratio of Sp1 to Sp3 nuclear factors.

Glucocorticoid suppression of human placental fibronectin expression: implications in uterine-placental adherence.

Increased levels of glucocorticoids are associated with human parturition whether occurring before or at term. In the present study we examined the effects of glucocorticoids on placental fibronectin (FN) expression in cytotrophoblasts, isolated from human term placentas, to provide a potential mechanism through which glucocorticoids may influence uterine-placental adherence near parturition. Based on immunoassays, relative to controls, media levels of placental FNs bearing an oncofetal epitope (onfFN) were inhibited 65-80% by treatment with 10(-7) M dexamethasone (DEX) during experiments in which cumulative levels and daily release of onfFN were measured. DEX treatment increased human CG production by cytotrophoblasts approximately 3-fold without affecting the levels of total protein, suggesting that DEX treatment did not reduce placental function. DEX and cortisol inhibited onfFN expression with an EC50 of 2 and 16 nM, respectively. Other steroids were not effective in down-regulating onfFN expression, indicating that this was a glucocorticoid-specific response. In immunoprecipitation studies, treatment of cytotrophoblasts with 10(-7) M DEX for 3 days inhibited both release of labeled FN to the media and its incorporation into cell-associated material by approximately 80%. Results from Northern blotting indicated that DEX treatment suppressed levels of FN messenger RNA approximately 90% relative to controls. Levels of labeled laminin in media were inhibited approximately 80% by a 3-day treatment with 10(-7) M DEX, suggesting that glucocorticoids may coordinately suppress the synthesis of multiple extracellular matrix proteins in cytotrophoblasts. In our model, we propose that glucocorticoids may suppress placental extracellular matrix protein synthesis, which could lead to decreased uterine-placental adherence and be associated with parturition.

Matrix metalloproteinase and matrix metalloproteinase inhibitor expression in endometrial stromal cells during progestin-initiated decidualization and menstruation-related progestin withdrawal.

Estradiol (E) primes human endometrial stromal cells (HESCs) for the decidualizing effects of progesterone in vivo and in vitro. Matrix metalloproteinase (MMP) expression was evaluated in confluent HESCs incubated in control medium, and in medium supplemented with either E, or the synthetic progestin medroxyprogesterone acetate (P), or E + P. Measurements with a specific ELISA indicated that basal pro-MMP-1 output was unaffected by E, whereas E + P, which induces the expression of several decidualization-related markers, produced a time-dependent inhibition in HESC-secreted levels of pro-MMP-1. Consistent with progestin inhibition of MMP-1 protein expression in the HESCs, P but not E, reduced steady state levels of MMP-1 messenger RNA (mRNA) as determined by Northern analysis. By contrast, mRNA levels for MMP-2 and the MMP inhibitor TIMP-1 were not altered by either P or E. Steroid withdrawal studies indicated that after MMP-1 expression was suppressed by incubation of the HESCs with E + P, 4 days of exposure to the antiprogestin RU 486 (mifepristone) significantly up-regulated MMP-1 levels in the conditioned medium by severalfold compared with cultures maintained in E + P. The change to steroid-free control medium required a more prolonged period of withdrawal to attain up regulatory effects that were comparable with those evoked by RU 486. The ELISA measurements were validated by immunoblot analysis with a specific MMP-1 antibody, which showed corresponding changes in a band at the expected mobility of about 50 kDa. Moreover, Northern analysis revealed parallel changes in MMP-1 mRNA levels, whereas neither MMP-2 nor TIMP-1 mRNA levels were modulated by adding or withdrawing steroids. The contrast between regulated MMP-1 expression and constitutive MMP-2 expression observed in the cultured HESCs is consistent with the demonstrated presence on the MMP-1 promoter of regulatory elements such as AP-1 and PEA-3 that are absent from the MMP-2 promoter. Extrapolation of these in vitro changes in HESCs to in vivo endometrial events suggests that: 1) inhibition of MMP-1 expression by E and progesterone would stabilize the perivascular endometrial ECM to prevent local hemorrhage during endovascular invasion by the implanting trophoblast; 2) enhanced expression of MMP-1 evoked by steroid withdrawal would mediate endometrial ECM degradation leading to sloughing of the functional layer during menstruation.

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell plasminogen activator and plasminogen activator inhibitor expression.

The abortifacient and menstrual effects of the potent antiprogestin, RU 486, are associated with both endometrial hemorrhage and extracellular matrix (ECM) degradation. Such processes reflect reduced perivascular decidual cell hemostatic and increased ECM-degrading protease activity. Therefore, we assessed the effects of RU 486 administration on the expression of immunoreactive (ir) endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activator and their activities as well as levels of ir type 1 plasminogen activator inhibitor (PAI-1) using a well characterized in vitro model of decidualization. Thus, confluent stromal cell cultures were exposed to vehicle control, 10(-8) mol/L estradiol (E2), 10(-7)-10(-8) mol/L medroxyprogesterone acetate (MPA), E2 plus MPA, or 10(-6)-10(-7) mol/L RU 486 alone or in combination with MPA or E2 plus MPA for 3-4 days. Compared to the vehicle control, E2 and RU 486 used alone had no effect on levels of ir PAI-1, uPA, or tPA or on PA activity in the conditioned medium. In contrast, MPA and E2 plus MPA decreased ir uPA and tPA levels and their corresponding activities, whereas MPA increased, and E2 plus MPA further increased ir PAI-1 release. These effects of progestin were blocked by a log higher concentration of RU 486. Similar results were obtained for steady state PAI-1 messenger ribonucleic acid levels. To determine if RU 486 reversed progestin-inhibited stromal cell uPA and tPA release and progestin-enhanced PAI-1 expression, confluent cultures were exposed to 10(-8) mol/L E2 plus 10(-7) mol/L MPA for 10 days, washed, and reexposed to E2 plus MPA, steroid-free medium, or RU 486 for 3-5 or 9-11 days. Compared with cultures maintained in E2 plus MPA for 3-5 days, withdrawal to a steroid-free medium failed to affect stromal cell ir PAI-1, uPA, or tPA levels. In contrast, exposure to RU 486 for 3-5 days increased ir uPA and tPA levels 5- to 8-fold (P < 0.02) while reducing PAI-1 levels by 85% (P < 0.04). By 9-11 days of treatment, steroid withdrawal and RU 486 exerted similar effects on ir PAI-1, tPA, and uPA levels. Comparable results were obtained for PAI-1, uPA, and tPA steady state messenger ribonucleic acid levels. These findings indicate that RU 486 blocks and reverses progestin-inhibited PA expression, suggesting a mechanism for RU 486-induced endometrial hemorrhage and ECM dissolution.

Effects of thrombin on steroid-modulated cultured endometrial stromal cell fibrinolytic potential.

By virtue of their unique chronic expression of tissue factor, the primary initiator of hemostasis, decidualized endometrial stromal cells are capable of significant thrombin generation after vascular disruption. In addition to its potent procoagulant effects, thrombin modifies endothelial and glomerular cell fibrinolytic activity. Therefore, we evaluated whether thrombin affected the expression of endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their primary inhibitor, type 1 plasminogen activator inhibitor (PAI-1), and whether ovarian steroids modulated putative thrombin effects. Confluent stromal cell cultures were incubated in a defined medium containing vehicle control, 10(-8) mol/L estradiol (E2), 10(-7) mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA for 4 days. The medium was then collected and exchanged for medium containing the corresponding steroids with or without thrombin and the specific thrombin inhibitor, D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, for an additional 24 h. The conditioned medium was then collected and analyzed for immunoreactive (ir) uPA, tPA, and PAI-1 by enzyme-linked immunosorbent assay and for PA activity by chromogenic assay, whereas Northern analysis of the cells was employed to evaluate the expression of thrombin receptor, uPA, tPA, and PAI-1 messenger ribonucleic acid (mRNA) species. The latter studies revealed that confluent cultures incubated in defined medium expressed the 3.45-kilobase thrombin receptor message. Steady state levels of thrombin receptor mRNA were unaffected by exogenous steroids. Thrombin added in the absence of exogenous steroids elevated concentrations of ir tPA, uPA, and PAI-1 compared with control cultures. Conversely, in the absence of added thrombin, MPA added alone or together with E2 inhibited levels of ir tPA and uPA while stimulating PAI-1 levels despite the lack of a response to E2 alone. Interestingly, thrombin counteracted this progestin inhibition of tPA and uPA expression and augmented the progestin-enhanced expression of PAI-1. Northern analysis revealed that steady state levels of tPA and uPA mRNA were also enhanced by thrombin in both control and steroid-containing cultures. Net PA activity reflects the balance between PA and PAI-1. In the absence of thrombin, there is virtually no detectable tPA activity and minimal uPA activity in progestin-exposed cultures. However, thrombin elicited significant increases in tPA and uPA activity in control and E2-treated cultures. Despite the molar excess of PAI-1 in MPA-treated and E2- plus MPA-treated cultures, thrombin reversed progestin inhibition of PA activity. Predictably, the addition of D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, blocked the effects of thrombin on PAI-1, tPA, and uPA protein and mRNA expression and PA activity. In summary, thrombin enhances endometrial stromal cell fibrinolytic and extracellular matrix-degrading protease activity in vitro. Such processes occurring in vivo would probably play a role in menstruation and abnormal uterine bleeding.

Transcriptional regulation of the tissue factor gene by progestins in human endometrial stromal cells.

Decidualization of estradiol (E2)-primed human endometrial cells (HESCs) by progesterone is associated with elevated levels of tissue factor (TF), the primary initiator of hemostasis. Similarly, in cultured human HESCs, the synthetic progestin, medroxyprogesterone acetate (MPA), enhances TF protein and messenger ribonucleic acid (mRNA) levels. Although ineffective alone, E2 potentiates this progestin enhancement of TF expression by HESCs. The current study examines mechanisms underlying MPA enhancement of TF mRNA expression in HESCs. In the presence of the transcription-blocking agent dichlororibofuranosylbenzimidazole, no significant differences were noted in the half-lives of TF mRNA isolated from HESCs treated with E2 alone or with E2 plus MPA. This indicates that MPA-enhanced TF mRNA levels do not reflect changes in the stability of the TF message. To test the effect of progestin on TF promoter activity and to ascertain the mechanism of promoter regulation, primary or first passaged HESCs were transfected with TF promoter constructs spanning the regions -2106 to +121 (TFp(-2106)), -278 to +121 (TFp(-278)), and -111 to +14 (TFp(-111)) bp upstream of the transcription start site. MPA was found to enhance TF transcription by 20-fold in HESCs transfected with TFp(-2106) after correcting for transfection efficiencies with a beta-galactosidase reporter plasmid. Interestingly, levels of E2- plus MPA-stimulated transcription were significantly increased using TFp(-278) compared to TFp(-2106), suggesting that the region between -2106 and -278 bp may contain an inhibitory element. In addition, rates of MPA-stimulated transcription using TFp(-111) were significantly reduced compared to values obtained using TFp(-2106) and were even further reduced compared to values obtained using TFp(-278). This suggests that regulatory elements in the -111 bp region of the TF promoter are necessary for progestin-mediated regulation of the TF gene in HESCs, but are not sufficient to account for maximal rates of TF gene transcription. Our results also demonstrated that induction of steady state TF mRNA by MPA was abolished by treating cells with E2 plus MPA in conjunction with the protein synthesis inhibitor cycloheximide. In light of the absence of a complete progesterone or estrogen response element in the published 5'-sequence of the TF promoter, our results suggest that progestin-enhanced transcription of TF mRNA in stromal cells may be mediated by an uncharacterized protein intermediate(s).

Biological mechanisms underlying RU 486 clinical effects: inhibition of endometrial stromal cell tissue factor content.

Despite the pronounced hemorrhagic effects of RU 486 administration on luteal phase and early gestational endometrium, no information is available on the effect of RU 486 on endometrial hemostatic potential. The expression of endometrial stromal cell tissue factor (TF), the primary initiator of hemostasis, has been shown to be progestationally regulated in vivo and in vitro. To evaluate the effects of RU 486 on progestin-enhanced TF expression, confluent stromal cell cultures derived from proliferative phase endometria were exposed to vehicle control, 10(-8) mol/L estradiol (E2), 10(-6) mol/L dexamethasone, 10(-7) mol/L medroxyprogesterone acetate (MPA), E2 plus MPA, E2 plus 10(-6) mol/L progesterone (P), or 10(-6) mol/L RU 486 alone or with E2 plus MPA or E2 plus P for 3-4 days. Compared to the vehicle control, E2, dexamethasone, and RU 486 alone had no effect on the content of immunoreactive and functionally active TF protein, whereas MPA increased and the combination of E2 and MPA further increased TF protein content. Similarly, E2 and P enhanced the stromal cell TF content. These progestin effects were blocked by RU 486. Similar results were obtained for steady state TF messenger ribonucleic acid (mRNA) levels. Possible RU 486-mediated reversal of progestin-enhanced stromal cell TF expression was assessed by incubating confluent cultures in E2 plus MPA for 3-10 days to enhance TF content, then washing the cultures and reexposing them to either E2 plus MPA or to RU 486 alone or with E2 plus MPA for 3, 4, or 7 days. Exposure to RU 486 alone or with E2 plus MPA greatly reduced levels of stromal cell TF protein and mRNA expression compared to those in cultures maintained in E2 plus MPA. These findings demonstrate that RU 486 not only blocks but also reverses in vitro progestin-enhanced stromal cell TF protein and mRNA expression, suggesting an additional mechanism for RU 486-induced menses and early abortion.

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell stromelysin-1 and prolactin expression.

During in vitro decidualization of human endometrial stromal cells (HESCs), medroxyprogesterone acetate (MPA) inhibits expression of the potent extracellular matrix (ECM)-degrading protease stromelysin-1 (MMP-3), but enhances PRL expression. Consistent with its priming role in vivo, estradiol (E2) augments these effects. In the current study, immunoblot analysis revealed that coincubation with 10(-6) M RU 486 blocked the inhibition in HESC-secreted MMP-3 levels (50,000 mol wt) evoked by 10(-8) M E2 + 10(-7) M MPA. Although MPA can act as a glucocorticoid, the HESCs were refractory to 10(-7) M dexamethasone added alone or with E2. Because E2 elevates progesterone but not glucocorticoid receptor levels, MPA and RU 486 control MMP-3 expression as a progestin and antiprogestin, respectively. To study RU 486 involvement in steroid withdrawal leading to menstruation, HESCs were decidualized during 10 days incubation with E2 + MPA, and parallel cultures were kept in E2 + MPA or withdrawn to either control or RU 486-containing medium. Compared with E2 + MPA-suppressed HESCs, increases in levels of secreted MMP-3 (2.0-fold), and its 2.1-kilobase messenger RNA (10-fold) were observed in HESCs after 4 days of withdrawal to control medium, with much greater increases seen in RU 486-containing medium (10-fold protein, 100-fold messenger RNA). Previously, we showed that RU 486 up-regulated E2 + MPA-inhibited plasminogen activator expression in the cultured HESCs. Extrapolation of these in vitro observations to endometrial events following RU 486 administration suggests that coordinate enhancement of MMP-3 and plasminogen activator expression promotes proteolysis of the stromal/decidual ECM, which leads to endometrial sloughing. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy menstrual bleeding stemming from RU 486 administration. However, in contrast to the marked RU 486-initiated reversal of MMP-3 expression, RU 486 did not significantly reverse E2 + MPA-enhanced PRL secretion by the cultured HESCs. Interestingly, decidual PRL, unlike decidual MMP-3, does not appear to play a role in menstruation. Interleukin-1 beta counteracted E2 + MPA-mediated inhibition of secreted MMP-3 levels, implying that leukocyte/trophoblast-derived cytokines can modulate steroid-regulated MMP-3 expression by stromal/decidual cells during menstruation and pregnancy.

Progestin-epidermal growth factor regulation of tissue factor expression during decidualization of human endometrial stromal cells.

Perivascular decidualized human endometrial stromal cells (HESCs) are ideally positioned to prevent peri-implantational hemorrhage during endovascular trophoblast invasion by expressing tissue factor (TF), the primary cellular mediator of hemostasis. Earlier in vivo and in vitro studies have demonstrated enhanced TF expression in estradiol (E2)-primed HESCs during progestin-induced decidualization. However, the absence of estrogen or progesterone response elements from the TF gene promoter suggests that paracrine factor(s) may mediate these effects. We now demonstrate that significant elevation of TF messenger RNA and protein levels in the cultured HESCs require incubation with both epidermal growth factor (EGF) and the progestin medroxyprogesterone acetate (MPA) added, with or without E2. By contrast, no effects were elicited by adding EGF with E2, or by the separate additions of EGF, MPA, or E2 plus MPA. Our finding, that transforming growth factor-alpha, but not transforming growth factor-beta or interleukin 1-beta mimics these EGF effects, indicates that progestin-enhanced TF expression in cultured HESCs requires activation of the EGF receptor (EGFR). Western blot analysis indicated that MPA increased EGFR levels 2-to 3-fold in cultured HESCs. The current results suggest that the progestin up-regulation of TF levels in decidualized HESCs is mediated by enhanced EGFR expression.

Effects of norplant on endometrial tissue factor expression and blood vessel structure.

Abnormal uterine bleeding after Norplant administration is primarily responsible for the high discontinuation rate of this safe and effective long-acting implantable progestin-only contraceptive agent. Although tissue factor (TF) is the primary initiator of hemostasis, previous studies indicated that Norplant-associated bleeding persists despite relatively high TF levels in the stromal compartment. Recently, we determined that progestin-enhanced TF expression during decidualization of human endometrial stromal cells involves both the epidermal growth factor receptor and progesterone receptor (PR]. The current study evaluated TF levels in endometrial bleeding (BL) and nonbleeding (NBL) sites obtained by camera-guided hysteroscopy during Norplant contraception. After 1 yr of therapy, immunohistochemical TF levels were unexpectedly higher at BL than at NBL sites. Use of immunohistochemistry and Western blotting indicated that both sites displayed elevated epidermal growth factor receptor levels and that the BL sites exhibited high levels of the PR, as well as the PR(A) and the PR(B) isoforms. Microscopic examination of 1-yr biopsies revealed that significantly larger numbers of enlarged, distended vessels were present in BL, compared with NBL sites. Elevated TF levels and abnormally enlarged blood vessels in the BL sites are consistent with the recently discovered angiogenic role of TF. By promoting aberrant angiogenesis, chronic endometrial overexpression of TF could produce fragile vessels, which are at increased risk to bleed. Analysis of endometrial BL and NBL sites, during Norplant contraception, offers the potential of elucidating local mechanisms that control enhanced TF expression, leading to abnormal angiogenesis at specific endometrial sites.

Glucocorticoid effects in the human placenta: evidence that dexamethasone-mediated inhibition of fibronectin expression in cytotrophoblasts involves a protein intermediate.

Oncofetal fibronectin is an extracellular matrix protein that is suggested to play an important role in regulating adherence at uterine-placental interfaces. The purpose of the present study was to elucidate a mechanism through which glucocorticoids (GCs) inhibit the synthesis of FN in human placenta as part of their matrix-suppressive action near parturition. We observed that treatment of cytotrophoblasts isolated from human term placentas for 48 h with 10(-7) mol/L dexamethasone (DEX) down-regulated levels of FN expression to 13-19% of control levels in immunoprecipitation, Northern blotting, and enzyme-linked immunosorbent assay experiments. Conversely, GC treatment increased FN expression in placental fibroblasts to 164-310% of control levels in Northern blotting and enzyme-linked immunosorbent assay procedures, suggesting that GC-mediated suppression of FN expression is specific to cytotrophoblasts. Results indicated that the DEX-mediated suppression of FN expression in cytotrophoblasts was not mediated through changes in the stability of FN messenger ribonucleic acid (mRNA). Run-on transcription assays using isolated nuclei suggested that GC treatment did not markedly affect transcription of the FN gene in cytotrophoblasts. To test whether the GC-mediated suppression of FN expression was mediated through a protein intermediate, levels of FN mRNA were examined by Northern blotting in cells treated for 48 h with and without 10(-7) mol/L DEX and cycloheximide (CHX; 125 ng/mL). We observed that CHX treatment increased FN expression in DEX-treated cells to 91% of control values. We noted that whereas the presence of 100-300 ng/mL CHX reversed the DEX-mediated inhibition of FN mRNA expression in cytotrophoblasts, it did not alter the overall rates of protein synthesis in DEX-treated and control cells. These data suggest that suppression of FN mRNA expression by GC in cytotrophoblasts requires de novo protein synthesis and is mediated through a short lived intermediate, the synthesis of which is inhibited at low concentrations of CHX. Thus, GC-induced protein intermediates may influence uterine-placental adherence by modulating levels of oncofetal FN at sites of uterine-placental contact.

Chronic antagonism of nuclear factor-kappaB activity in cytotrophoblasts by dexamethasone: a potential mechanism for antiinflammatory action of glucocorticoids in human placenta.

Circulating glucocorticoids are present in increasing quantities as human gestation progresses, peaking during labor whether it occurs before or at term. Although the precise role of glucocorticoids in pregnancy is not well defined, it is clear that glucocorticoids suppress inflammation in many cell types by antagonizing the acute stimulatory actions of members of the Rel/nuclear factor-kappaB (NF-kappaB) family on cytokine gene expression. In the present study we tested the hypothesis that during pregnancy, glucocorticoids chronically suppress inflammation in the human placenta. Cytotrophoblasts obtained from human term placentas were maintained for 48 h in culture medium supplemented with 10% charcoal-stripped calf serum with and without 100 nmol/L dexamethasone (DEX). Enzyme-linked immunosorbent assay studies revealed that cytotrophoblasts constitutively express interleukin-8 (IL-8), a known mediator of placental inflammation, between 24-96 h of culture. A 48-h treatment of cytotrophoblasts with 100 nmol/L DEX significantly reduced the production of IL-8 to 24+/-1% of control levels (P < 0.01). DEX and cortisol mediated a dose-dependent inhibition of IL-8 expression, with ED50 values of 5 and 50 nmol/L, respectively. DEX treatment also significantly reduced levels of IL-6 and tumor necrosis factor-alpha in culture medium, suggesting that glucocorticoids coordinately reduce cytokine levels in cytotrophoblasts. As cytokine expression is regulated by NF-kappaB and activator protein-1 (AP-1) transcription factors, electrophoretic mobility shift assays (n = 4) were used to determine whether DEX treatment altered the binding of nuclear proteins from cytotrophoblasts to labeled oligonucleotides corresponding to the kappaB and AP-1 response elements. We observed that a 48-h treatment of cytotrophoblasts with 100 nmol/L DEX markedly reduced binding of nuclear extracts from cytotrophoblasts to the kappaB response element. DEX treatment promoted a relatively smaller reduction of binding to the AP-1 response element. Northern blotting experiments revealed that DEX treatment did not alter the level of IkappaB, p50, or p65 messenger ribonucleic acid, suggesting that the antiinflammatory action of glucocorticoid in cytotrophoblasts did not directly involve alterations in the level of NF-kappaB proteins. Our results demonstrate a novel chronic suppressive action of glucocorticoid on cytokine production and nuclear binding of NF-kappaB and AP-1 proteins in cytotrophoblasts, providing a potential mechanism through which glucocorticoids may suppress inflammation at maternal-fetal interfaces across gestation.

Steroid-modulated stromal cell tissue factor expression: a model for the regulation of endometrial hemostasis and menstruation.

This study examined steroidal regulation of tissue factor expression by cultured endometrial stromal cells. Confluent stromal cell cultures derived from cycling human endometria were exposed to vehicle control, 10(-8) mol/L estradiol (E2), 10(-8)-10(-6) mol/L medroxyprogesterone acetate (MPA), or both E2 and MPA for 2-24 days in serum-containing medium. The progestin enhanced immunoreactive and functionally active stromal cell tissue factor content, achieving peak effects by 8-12 days of culture. Although E2 alone was ineffective, it augmented MPA-enhanced tissue factor content by 8 days of culture, with continued increases beyond 20 days. Dose-dependent effects on tissue factor protein content were observed between 10(-8)-10(-6) mol/L MPA added alone or together with E2. The content of tissue factor mRNA was also increased by MPA and synergistically increased by E2 plus MPA. Similar steroidal effects on stromal cell tissue factor protein and mRNA content were observed using a defined medium. After optimal induction of tissue factor expression by E2 plus MPA, removal of these steroids reduced levels of stromal cell tissue factor mRNA and protein, with virtually complete reversal by day 7 of withdrawal. These time-course and dose-response relationships establish in vitro conditions with which to dissect factors controlling endometrial hemostasis, whereas the observed effects of steroid withdrawal establish a novel model for the study of mechanisms regulating normal and abnormal uterine bleeding.

Progestational regulation of human endometrial stromal cell tissue factor expression during decidualization.

Decidualized endometrial stromal cells from mid- to late secretory phase and decidual cells from gestational human endometrium displayed prominent immunohistochemical staining for tissue factor, a primary initiator of hemostasis. Consistent with the regulation by progesterone of the decidualization process in vivo, medroxy-progesterone acetate elevated the tissue factor content of primary stromal cell cultures 8-fold over basal values. This increase paralleled the release of immunoreactive PRL, a marker of decidualization. The induced, as well as basal, tissue factor displayed full functional activity and the expected electrophoretic mobility (46 kilodaltons). Moreover, Northern analysis of RNA from cultured stromal cells indicated that medroxyprogesterone acetate increased tissue factor mRNA levels approximately 10-fold relative to control levels. In contrast, cultured stromal cell tissue factor protein content and mRNA levels were unaffected by exogenous estradiol. These findings indicate that enhancement of endometrial stromal cell tissue factor content is associated with progesterone induction of the decidualization process. In humans, trophoblastic invasion of the endometrial vasculature during blastocyst implantation risks hemorrhage. Therefore, increases in perivascular decidual cell tissue factor expression could serve to promote periimplantational endometrial hemostasis.