Joon Song

Interaction of the Estrogen Receptors with the Fas Ligand Promoter in Human Monocytes

The predominance of autoimmune diseases among women suggests that estrogen may modulate immune function. Monocytes and macrophages are important in initiating, maintaining, and resolving inflammatory responses through cell-signaling molecules, which control immune cell survival. One important mechanism of cell survival is mediated by the Fas/Fas ligand (FasL) system. In this study, the link between estrogen, monocytes/macrophages, and the Fas/FasL system was investigated. Estrogen treatment increased FasL expression in monocytes through the binding of the estrogen receptors (ER) to the estrogen recognizing elements and AP-1 motifs present at the FasL promoter. Furthermore, estrogen induced apoptosis in monocytes expressing ERβ, but not in monocyte-differentiated macrophages expressing ERα. The expression of either ERα or ERβ and their response to estrogen in monocytes was found to be dependent on the their stage of cell differentiation. Previously, we have shown that estrogen replacement therapy in postmenopausal women decreased the number of circulating monocytes. In this study, we have characterized the molecular mechanism by which estrogen regulates monocytes homeostasis. These findings indicate that estrogen may regulate immune cell survival through the Fas/FasL system. There is biological relevance to these findings in view of studies showing that accumulation of activated monocytes is involved in the pathogenesis of conditions such as vasculititis, arteriosclerosis, and rheumatoid arthritis.

Roles of Fas and Fas ligand during mammary gland remodeling

Mammary involution is associated with degeneration of the alveolar structure and programmed cell death of mammary epithelial cells. In this study, we evaluated the expression of Fas and Fas ligand (FasL) in the mammary gland tissue and their possible role in the induction of apoptosis of mammary cells. FasL-positive cells were observed in normal mammary epithelium from pregnant and lactating mice, but not in nonpregnant/virgin mouse mammary tissue. Fas expression was observed in epithelial and stromal cells in nonpregnant mice but was absent during pregnancy. At day 1 after weaning, high levels of both Fas and FasL proteins and caspase 3 were observed and coincided with the appearance of apoptotic cells in ducts and glands. During the same period, no apoptotic cells were found in the Fas-deficient (MRL/lpr) and FasL-deficient (C3H/gld) mice. Increase in Fas and FasL protein was demonstrated in human (MCF10A) and mouse (HC-11) mammary epithelial cells after incubation in hormone-deprived media, before apoptosis was detected. These results suggest that the Fas-FasL interaction plays an important role in the normal remodeling of mammary tissue. Furthermore, this autocrine induction of apoptosis may prevent accumulation of cells with mutations and subsequent neoplastic development. Failure of the Fas/FasL signal could contribute to tumor development.

Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen.

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.

The role of the Fas/Fas ligand system in estrogen-induced thymic alteration

PROBLEM: Estrogen induces atrophy in the thymus by an unknown mechanism. Since the Fas/FasL system is one of the main pathways in T cell apoptosis, we tested the hypothesis that estrogen‐induced thymic atrophy is mediated by the Fas/FasL system.
 METHODS OF STUDY: In vivo experiments were done using ovariectomized female rats treated with estrogen or saline. In vitro experiments were performed using isolated thymocytes. Estrogen receptor (ER) α and β expression was characterized using flow cytometry, RT‐PCR and immunofluorescence. Fas and FasL mRNA and protein expression was evaluated using RT‐PCR and Western blot analysis respectively.
 RESULTS: ERα and ERβ are present in thymocytes and stromal cells. ER expression is mainly localized in the Double Positive CD4+CD8+ thymocytes. Estrogen treatment decreases thymus size and increase FasL expression.
 CONCLUSION: CD4+CD8+ thymocytes and thymic stroma cells express ERα and ERβ. In vivo and in vitro we showed that estrogen treatment increases FasL expression while decreasing thymus cell number. These findings support the hypothesis that estrogen‐induced thymic atrophy occurs as a result of apoptosis and is mediated by estrogen‐induced FasL expression.

Mechanisms involved in the evolution of progestin resistance in human endometrial hyperplasia—precursor of endometrial cancer

BACKGROUND Successful treatment of endometrial hyperplasia with progestins is commonly accompanied by the finding of an inactive or suppressed endometrium after therapy. However, approximately 30% of the endometrial hyperplasia cases do not respond to progestins and hyperplastic glands persist. The Fas/FasL system is known to play a role in tissue remodeling as a result of changes in menstrual hormone levels. The aims of this study are to examine Fas/FasL expression in endometrial hyperplasia of pre- and postprogestin treatment samples and to study the Fas/FasL regulation in vitro with Ishikawa cells after progestin stimulation. DESIGN Pre- and posttreatment paraffin-embedded endometrial hyperplasia tissue samples from 26 women were examined by immunohistochemistry for changes in Fas/FasL expression related to the administration of progestins. Among 26 patients, 18 were successfully treated with progestins and 8 failed treatment. Fas/ FasL positivity was defined by the presence of 10% or more immunoreactive epithelial cells in each specimen. In positive cases, a percentage or an immunoscore of immunoreactive cells was given by counting 500 cells. Cell viability was evaluated by the MTT assay. The in vitro effects of progesterone on Fas/FasL expression and apoptosis in Ishikawa cells were examined by using Western blot and TUNEL assays, respectively. RESULTS Fas immunoreactivity was present in 4/26 (15%) preprogestin cases with an average of 16% of the epithelial cells expressing Fas. FasL was expressed in 21/26 (80%) pretreatment cases with an average of 42% of the hyperplastic glandular cells being positive. In postprogestin cases, an increase of Fas expression (14/18, 77%) with an average of 47% stained cells was seen in responders (P < 0.001), while FasL was found in 16/18 (89%) responders with an average of 65% of cells positive (P = 0.587). In nonresponders, no significant changes in Fas/FasL expression were detected compared to pretreatment samples. With in vitro Ishikawa cells, a slight increase (10-20%) of Fas and FasL protein expression was detected after 24 h of progesterone treatment, but a more significant increase (220-343%) of both Fas and FasL expression was found after 48 h of withdrawing progesterone, which parallels apoptotic activity. CONCLUSIONS The Fas/FasL system may be involved in the development of endometrial hyperplasia. Part of the molecular mechanisms of progestin therapy for endometrial hyperplasia is through upregulation of Fas/FasL expression. Dysregulation of Fas/FasL expression in hyperplastic endometrium may be part of the molecular mechanisms for nonresponders to progestin treatment. Intermittent, rather than continuous, progestin treatment may be more effective clinically for the treatment of endometrial hyperplasia.

17α-Methyl testosterone is a competitive inhibitor of aromatase activity in Jar choriocarcinoma cells and macrophage-like THP-1 cells in culture ☆

17alpha-methyl testosterone is a synthetic androgen with affinity for the androgen receptor. 17alpha-methyl testosterone is used widely as a component of hormone replacement therapy. Previous reports have indicated that contrary to testosterone, 17alpha-methyl testosterone is not aromatized. However, 17alpha-methyl testosterone still could affect local estrogen formation by regulating aromatase expression or by inhibiting aromatase action. Both possibilities have important clinical implications. To evaluate the effect of 17alpha-methyl testosterone on the expression and activity of aromatase, we tested the choriocarcinoma Jar cell line, a cell line that express high levels of P450 aromatase, and the macrophage-like THP-1 cells, which express aromatase only after undergoing differentiation. We found that in both cell lines, 17alpha-methyl testosterone inhibits aromatase activity in a dose-related manner. The curve of inhibition parallels that of letrozole and gives complete inhibition at 10(-4) M 17alpha-methyl testosterone, determined by the tritium release assay. 17alpha-methyl testosterone does not have detectable effects on aromatase RNA and protein expression by Jar cells. Undifferentiated THP-1 cells had no aromatase activity and showed no effect of 17alpha-methyl testosterone, but differentiated THP-1 (macrophage-like) cells had a similar inhibition of aromatase activity by 17alpha-methyl testosterone to that seen in Jar cells. The Lineweaver-Burke plot shows 17alpha-methyl testosterone to be a competitive aromatase inhibitor. Our results show for the first time that 17alpha-methyl testosterone acts as an aromatase inhibitor. These findings are relevant for understanding the effects of 17alpha-methyl testosterone as a component of hormone replacement therapy. 17alpha-methyl testosterone may, as a functional androgen and orally active steroidal inhibitor of endogenous estrogen production, also offer special possibilities for the prevention/treatment of hormone-sensitive cancers.

Chromosome 17p13.2 transfer reverts transformation phenotypes and fas-mediated apoptosis in breast epithelial cells

Transformation of the human breast epithelial cells (HBEC) MCF‐10F with the carcinogen benz(a)pyrene (BP) into BP1‐E cells resulted in the loss of the chromosome 17 p13.2 locus (D17S796 marker) and formation of colonies in agar‐methocel (colony efficiency (CE)), loss of ductulogenic capacity in collagen matrix, and resistance to anti‐Fas monoclonal antibody (Mab)‐induced apoptosis. For testing the role of that specific region of chromosome 17 in the expression of transformation phenotypes, we transferred chromosome 17 from mouse fibroblast donors to BP1‐E cells. Chromosome 11 was used as negative control. After G418 selection, nine clones each were randomly selected from BP1‐E‐11neo and BP1‐E‐17neo hybrids, respectively, and tested for the presence of the donor chromosomes by fluorescent in situ hybridization and polymerase chain reaction‐based restriction fragment length polymorphism (PCR‐RFLP) analyses. Sensitivity to Fas Mab–induced apoptosis and evaluation of transformation phenotype expression were tested in MCF‐10F, BP1‐E, and nine BP1‐E‐11neo and BP1‐E‐17neo clones each. Six BP1‐E‐17neo clones exhibited a reversion of transformation phenotypes and a dose dependent sensitivity to Fas Mab‐induced apoptosis, behaving similarly to MCF‐10F cells. All BP1‐E‐11neo, and three BP1‐E‐17neo cell clones, like BP1‐E cells, retained a high CE, loss of ductulogenic capacity, and were resistant to all Fas Mab doses tested. Genomic analysis revealed that those six BP1‐E‐17neo clones that were Fas‐sensitive and reverted their transformed phenotypes had retained the 17p13.2 (D17S796 marker) region, whereas it was absent in all resistant clones, indicating that the expression of transformation phenotypes and the sensitivity of the cells to Fas‐mediated apoptosis were under the control of genes located in this region.

SEX HORMONES, APOPTOSIS AND THE FAS/FAS LIGAND SYSTEM IN NORMAL ENDOMETRIAL TISSUE REMODELING

INTRODUCTION. Tissue remodeling is an important physiological mechanism for maintaining adequate cellular balance in differentiated tissues showing high turnover, and for cancer prevention. The human endometrium is continuously undergoing remodeling mediated by survival and differentiation factors such as estrogen and progesterone. Absence of survival factors at the end of each differentiation cycle results in cellular death of the endometrium, via activation of apoptotic genes such as the Fas/Fas Ligand (FasL) system. In contrast to past studies, and consistent with the above information, new data indicate that menstruation results from activation of apoptotic genes. We hypothesized that remodeling of endometrial tissue is mediated by activation of the Fas/FasL system as result of hormonal changes in the endometrium’s microenvironment. METHOD. We performed in vitro studies using primary cultures of human endometrial cells and the estrogen and progesterone receptor (ER and PR)-positive Ishikawa endometrial cancer cell line. Cells were incubated with physiologic concentrations of estradiol, progesterone or a combination of both (10 -8 M). The endometrial cells were then incubated in a hormone-depleted milieu and evaluated for apoptosis (MTT assay), Fas and FasL mRNA (RT-PCR) and protein (Western blot) expressions. Sensitivity to Fas-mediated apoptosis was evaluated using an anti-Fas mAb to trigger the Fas signal and MTT assay. RESULTS. Estrogen and/or progesterone-withdrawal induced apoptosis of endometrial cells (48% and 43% respectively, p<0.05). At the same time, hormonal withdrawal increased Fas and FasL expression as determined by RT-PCR and Western blot analysis. Treatment of the endometrial cells with an anti-Fas antibody further increases apoptosis after hormonal removal (69%), suggesting that the Fas pathway is functional in these cells. Furthermore, application of anti-FasL antibody prior to hormonal withdrawal prevents cellular death. DISCUSSION. Apoptosis in reproductive tissues is a physiological mechanism that eliminates differentiated, senescent, or dysfunctional cells. The cyclic changes of estrogen and progesterone promote a clean up of the endometrium preventing the accumulation of differentiated/mutated cells. In the present study we show that sexhormone withdrawal represents the signal for apoptosis, in part via the Fas/FasL system. These results underscore the importance of sex-hormone cyclicity for providing a protective effect against neoplastic transformation in the endometrium. It