Peter Licht

Interferon-γ and tumor necrosis factor-α sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas-mediated signaling during early implantation. Here we show that ESCs are primarily resistant to Fas-mediated apoptosis independently of their state of hormonal differentiation. Pre-treatment of ESCs with interferon (IFN)-γ and tumor necrosis factor (TNF)-α sensitizes them to become apoptotic upon stimulation of Fas by an agonistic anti-Fas antibody. Incubation of ESCs with the early embryonic signal human chorionic gonadotropin (hCG, CGB) does not influence their reaction to Fas stimulation. The sensitizing effect of IFN-γ and TNF-α was accompanied by a significant upregulation of Fas and FLICE-inhibitory protein (FLIP, CFLAR) expression in ESCs. Additionally, we observed an activation of caspase 3, caspase 8 and caspase 9 upon apoptotic Fas triggering. In summary, we demonstrate that IFN-γ and TNF-α sensitize primarily apoptosis-resistant ESCs to Fas-mediated cell death. This might be due to an upregulation of Fas expression, and apoptosis seems to be mediated by active caspase 3, caspase 8 and caspase 9. The observed pro-apoptotic effect of IFN-γ and TNF-α on ESCs could play an important role in the modulation of early implantation.

Human chorionic gonadotropin stimulates matrix metalloproteinases-2 and -9 in cytotrophoblastic cells and decreases tissue inhibitor of metalloproteinases-1, -2, and -3 in decidualized endometrial stromal cells

OBJECTIVE To investigate the influence of hCG on trophoblastic matrix metalloproteinases (MMPs) -2 and -9 as well as endometrial tissue inhibitor of metalloproteinases (TIMPs) -1, -2, and -3. DESIGN In vitro experiment. SETTING Research laboratory at a university medical center. PATIENT(S) Women undergoing legal abortions and premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S) Human first trimester cytotrophoblasts and decidualized endometrial stromal cells were incubated with recombinant hCG. MAIN OUTCOME MEASURE(S) Trophoblastic MMP-2 and -9 were analyzed by enzyme-linked immunosorbent assay (ELISA) and zymography, and endometrial TIMP-1, -2, and -3 were measured by real time reverse transcriptase-polymerase chain reaction and ELISA. RESULT(S) HCG increases the secretion of MMP-2 and -9 in cytotrophoblasts dose dependently. This effect occurs after 4 hours of incubation and becomes less pronounced after 24 hours. In contrast, TIMP-1, -2, and -3 are significantly reduced by hCG in endometrial stromal cells in a time- and dose-dependent manner. CONCLUSION(S) These results suggest a regulatory role of hCG on the MMP/TIMP system at the implantation site. By increasing trophoblastic MMP secretion and reducing endometrial TIMP expression, hCG may be an important tool for the invading embryo to regulate the depth of its implantation.

Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function

OBJECTIVE To determine intrauterine levels of vascular endothelial growth factor (VEGF)-A during the menstrual cycle in the human female and to investigate the impact of decidualization and corpus luteum function. DESIGN Prospective clinical study. SETTING Tertiary university center. PATIENT(S) Fifty-four women with infertility problems. INTERVENTION(S) Intrauterine concentrations of VEGF-A were determined at various time points during the secretory phase using a novel intrauterine microdialysis device. Concomitantly, intrauterine insulin-like growth factor binding protein (IGFBP)-1 levels served as a paracrine parameter for decidualization. Serum progesterone (P) and E(2) levels were determined as markers for corpus luteum function. Intrauterine VEGF levels. RESULT(S) The VEGF levels in utero were clearly cycle dependent with increasing levels during the late secretory and premenstrual phases. There was a significant correlation with the decidualization marker IGFBP-1. In contrast, intrauterine VEGF levels showed a significant negative correlation with serum E(2) and P. CONCLUSION(S) Intrauterine VEGF levels are regulated in a cycle-dependent way. Increasing levels in the late secretory phase are clearly correlated with decidualization. In contrast, decreasing serum levels of steroids produced by the regressing corpus luteum are less likely to be responsible for increasing VEGF levels in the premenstrual phase.

Nonapoptotic effects of tumor necrosis factor–related apoptosis-inducing ligand on interleukin-6, leukemia inhibitory factor, interleukin-8, and monocyte chemoattractant protein 1 vary between undifferentiated and decidualized human endometrial stromal cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is known to exert death-inducing as well as nonapoptotic functions, has been shown to be expressed by the early trophoblast. Here we report that TRAIL has no apoptotic effects on human endometrial stromal cells, but differentially regulates cytokines and chemokines and might therefore play a role in the modulation of the cytokine milieu at the implantation site.

Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells.

Cytotoxic activity of drug conjugates of human chorionic gonadotropin (hCG) and doxorubicin alone was investigated compared to doxorubicin in breast cancer cells with and without expression of hCG receptors. Expression of hCG receptor was determined in MCF-7 and MB231 breast cancer cell line using a multiplex nested rt-PCR approach. The entire sequence of mRNA encoding for hCG receptor was detected in MCF-7 but not in MB231 breast cancer cell line. Cytostatic effect of doxorubicin–hCG conjugates was investigated in these cell lines in comparison to unconjugated doxorubicin. The number of viable cells was determined after 24, 48, 72, 96, and 120 h. To exclude non-specific uptake of the carrier hCG from the culture media, a similar experiment was performed with albumin–doxorubicin conjugates. The number of viable cells decreased in a concentration depending manner after doxorubicin and hCG–doxorubicin conjugate treatment. However, the cytotoxic effect of hCG–doxorubicin conjugate was 10-fold increased compared to unconjugated doxorubin in hCG-receptor positive MCF-7 but not in hCG-receptor negative MB231 cells. Albumin–doxorubicin conjugates showed no increased toxicity compared to doxorubicin. We conclude that the cytotoxic effect of hCG–doxorubicin conjugates is mediated specifically via the hCG receptor. By using hCG conjugates, the development of more selective cytostatics can be achieved.

Inverse regulation of the interferon-γ receptor and its signaling in human endometrial stromal cells during decidualization

OBJECTIVE To investigate whether human endometrial stromal cells (ESCs) express the interferon-gamma-receptor (IFN-gamma R) and whether the process of decidualization or human chorionic gonadotropin (hCG) regulate the IFN-gamma R and its signaling pathway. DESIGN In vitro experiment. SETTING Research laboratory at a medical university center. PATIENT(S) Premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S) Isolation and incubation of ESCs from hysterectomy specimens with 17beta-estradiol, progesterone, recombinant hCG, and IFN-gamma as well as an IFN-gamma R-blocking antibody. MAIN OUTCOME MEASURE(S) We analyzed IFN-gamma R and the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) by flow cytometry. We measured IFN-gamma R and interferon response factor 1 (IRF-1) mRNA using semiquantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). RESULT(S) The IFN-gamma R is up-regulated in human ESCs during decidualization without affecting the phosphorylation of STAT-1. Stimulation of IRF-1 by IFN-gamma is reduced in decidualized ESCs. We found that hCG neither regulates the IFN-gamma R nor its signaling pathway. CONCLUSION(S) These results show an inverse regulation of the IFN-gamma R and its signaling response via STAT-1 and IRF-1 in human ESCs during decidualization. The early embryonic signal hCG has no effect on this process. This mechanism may finely modulate the reactivity of ESCs to IFN-gamma-mediated signals from immune cells at the implantation site.