Pregnancy preparation: redistribution of CCR7-positive cells in the rat uterus.

In brief: Changes in the endometrium prior to implantation may be critical in predicting pregnancy outcomes. This study shows that the endocrine system directs positional changes in CCR7+ cells before implantation, which may be critical for developing maternal tolerance. Abstract: Suppression of the maternal immune system is vital for the implantation of the semi-allogeneic embryo. Although progress in understanding the dialogue between mother and embryo has been made, key interactions between maternal immune cells, hormones, and chemokines remain elusive. Uterine expression of the C-C chemokine receptor type 7 (CCR7) could recruit T regulatory cells and facilitate localized immune suppression. To test this concept, Ccr7 mRNA and protein were assessed in uterine tissue. Ccr7 mRNA expression peaked at day 4 in pregnant rat uteri and then declined at days 5 and 6. CCR7 protein showed similar quantitative changes. To test if female sex steroids affected the spatial distribution of CCR7-expressing cells, uteri from ovariectomized rats, progesterone-pretreated rats (2 mg daily), and progesterone-pretreated rats injected with estradiol (0.2 µg) were analyzed. Progesterone increased CCR7-positive (+) cells in the antimesometrial stroma. Progesterone and estradiol increased CCR7+ cells in the mesometrial stroma. Estradiol increased the density of cluster of differentiation 4 (CD4) positive cells in the mesometrial stromal region over progesterone alone. The density of cells expressing the T regulatory cell marker, forkhead box protein 3 (FOXP3), increased in the antimesometrial stroma in response to progesterone alone. Progesterone and estradiol increased FOXP3+ cells in the antimesometrial region of the stroma. Co-localization of CCR7, CD4, and FOXP3 in the stroma suggests CCR7+ cells are T regulatory cells. Polarization of CCR7+ cells in the endometrial stroma was an intrinsic response regulated by sex steroids and did not require the presence of an embryo.

Gender Bias in Human Systemic Lupus Erythematosus: A Problem of Steroid Receptor Action?

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease resulting from abnormal interactions between T and B cells. The acquisition of SLE is linked to genetic susceptibility, and diverse environmental agents can trigger disease onset in genetically susceptible individuals. However, the strongest risk factor for developing SLE is being female (9:1 female to male ratio). The female sex steroid, estradiol, working through its receptors, contributes to the gender bias in SLE although the mechanisms remain enigmatic. In a small clinical trial, monthly administration of the estrogen receptor (ERα) antagonist, ICI182,780 (fulvestrant), significantly reduced disease indicators in SLE patients. In order to identify changes that could account for improved disease status, the present study utilized fulvestrant (Faslodex) to block ERα action in cultured SLE T cells that were purified from blood samples collected from SLE patients (n = 18, median age 42 years) and healthy control females (n = 25, median age 46 years). The effects of ERα antagonism on estradiol-dependent gene expression and canonical signaling pathways were analyzed. Pathways that were significantly altered by addition of Faslodex included T helper (Th) cell differentiation, steroid receptor signaling [glucocorticoid receptor (GR), ESR1 (ERα)], ubiquitination, and sumoylation pathways. ERα protein expression was significantly lower (p < 0.018) in freshly isolated, resting SLE T cells suggesting ERα turnover is inherently faster in SLE T cells. In contrast, ERα/ERß mRNA and ERß protein levels were not significantly different between SLE and normal control T cell samples. Plasma estradiol levels did not differ (p > 0.05) between SLE patients and controls. A previously undetected interaction between GR and ERα signaling pathways suggests posttranslational modification of steroid receptors in SLE T cells may alter ERα/GR actions and contribute to the strong gender bias of this autoimmune disorder.

The K-INBRE symposium: a 10-institution collaboration to improve undergraduate education.

The Kansas-IDeA Network of Biomedical Research Excellence (K-INBRE) is an infrastructure-building program funded by the National Institute of General Medical Sciences. Undergraduate education, through undergraduate research, is a key component of the program. The K-INBRE network includes 10 higher education institutions in Kansas and northern Oklahoma, with over 1,000 student participants in 16 yr. Since 2003, the K-INBRE has held an annual state-wide research symposium that includes national and regional speakers and provides a forum for undergraduates to give platform and poster presentations. The symposium is well attended by K-INBRE participants and has grown to a size of over 300 participants per year from all 10 K-INBRE schools. Two surveys were distributed to students and mentors to assess the impact of the symposium on student learning. Surveys (153) were distributed to students who participated in K-INBRE from 2013 through 2015 with a 51% response rate. Mentors were surveyed with a response of 111 surveys out of 161. Survey results indicate that students and mentors alike find the symposium to be beneficial and enriching of the student experience. Almost 80% of student respondents indicated that their participation in the symposium fostered appreciation of research. In short, the K-INBRE symposium provides a unique opportunity for students to gain experience in collecting, preparing, and communicating research in a professional environment. The collaborative experience of the annual K-INBRE symposium, the impact it has on student learning, and how it has influenced the research culture at our 10 institutions will be described.

WINGLESS (WNT) signaling is a progesterone target for rat uterine stromal cell proliferation.

Preparation of mammalian uterus for embryo implantation requires a precise sequence of cell proliferation. In rodent uterus, estradiol stimulates proliferation of epithelial cells. Progesterone operates as a molecular switch and redirects proliferation to the stroma by down-regulating glycogen synthase kinase-3ß (GSK-3ß) and stimulating ß-catenin accumulation in the periluminal stromal cells. In this study, the WNT signal involved in the progesterone-dependent proliferative switch was investigated. Transcripts of four candidate Wnt genes were measured in the uteri from ovariectomized (OVX) rats, progesterone-pretreated (3 days of progesterone, 2mg/daily) rats, and progesterone-pretreated rats given a single dose (0.2µg) of estradiol. The spatial distribution of the WNT proteins was determined in the uteri after the same treatments. Wnt5a increased in response to progesterone and the protein emerged in the periluminal stromal cells of progesterone-pretreated rat uteri. To investigate whether WNT5A was required for proliferation, uterine stromal cell lines were stimulated with progesterone (1µM) and fibroblast growth factor (FGF, 50ng/mL). Proliferating stromal cells expressed a two-fold increase in WNT5A protein at 12h post stimulation. Stimulated stromal cells were cultured with actinomycin D (25µg/mL) to inhibit new RNA synthesis. Relative Wnt5a expression increased at 4 and 6 h of culture, suggesting that progesterone plus FGF preferentially increased Wnt5a mRNA stability. Knockdown of Wnt5a in uterine stromal cell lines inhibited stromal cell proliferation and decreased Wnt5a mRNA. The results indicate that progesterone initiates and synchronizes uterine stromal cell proliferation by increasing WNT5A expression and signaling.

Estradiol targets T cell signaling pathways in human systemic lupus.

The major risk factor for developing systemic lupus erythematosus (SLE) is being female. The present study utilized gene profiles of activated T cells from females with SLE and healthy controls to identify signaling pathways uniquely regulated by estradiol that could contribute to SLE pathogenesis. Selected downstream pathway genes (+/- estradiol) were measured by real time polymerase chain amplification. Estradiol uniquely upregulated six pathways in SLE T cells that control T cell function including interferon-alpha signaling. Measurement of interferon-alpha pathway target gene expression revealed significant differences (p= 0.043) in DRIP150 (+/- estradiol) in SLE T cell samples while IFIT1 expression was bimodal and correlated moderately (r= 0.55) with disease activity. The results indicate that estradiol alters signaling pathways in activated SLE T cells that control T cell function. Differential expression of transcriptional coactivators could influence estrogen-dependent gene regulation in T cell signaling and contribute to SLE onset and disease pathogenesis.

Fulvestrant (Faslodex), an estrogen selective receptor downregulator, in therapy of women with systemic lupus erythematosus. clinical, serologic, bone density, and T cell activation marker studies: a double-blind placebo-controlled trial.

OBJECTIVE: Estrogen plays a role in the activation of systemic lupus erythematosus (SLE) and in upregulating intracellular signals by binding to the estrogen receptor(s). Fulvestrant (Faslodex, AstraZeneca Pharmaceuticals, Wilmington, DE, USA), an estrogen selective receptor downregulator, competes for receptor binding in vitro and inhibits estrogen action in target cells. We evaluated the efficacy, side effects, and expression of T cell activation markers, following the administration of fulvestrant or placebo to premenopausal patients with SLE. METHODS: Twenty women with moderate SLE Disease Activity Index (SLEDAI; 7.87 +/- 3.7) were enrolled. They were premenopausal with regular menstrual cycles and not taking exogenous hormones. The study was double-blind and placebo-controlled. Ten patients received 250 mg fulvestrant intramuscularly for 12 months, and 10 received the placebo. All were observed monthly and 3 months after final fulvestrant/placebo injection. Measures studied were monthly SLEDAI scores, routine and serologic markers for lupus, and serum concentrations of estrogen and fulvestrant. Expression of T cell calcineurin and CD154 mRNA in peripheral T cells was measured by polymerase chain reaction. Medications the patients were taking were recorded each visit. Bone density was obtained at baseline and at visit 12. RESULTS: Sixteen patients completed the 15-month study, 8 from each group. SLEDAI improved significantly in the fulvestrant group at both 12 months (p = 0.02) and 15 months (p = 0.002), but serologic markers, routine laboratory tests, and bone density did not. Serum estrogen levels were higher in the fulvestrant group and dropped when fulvestrant was discontinued; these differences were not statistically significant. Medications for therapy of lupus to the fulvestrant group were reduced, whereas the placebo group medications were unchanged or increased. Comparison of relative values at individual timepoints revealed significantly lower median values for the T cell activation markers CD154 (p < 0.001) and calcineurin (p = 0.013) in the fulvestrant arm. CONCLUSION: Blocking estrogen receptors in vivo by an estrogen selective receptor downregulator could be considered as a new and relatively safe therapeutic approach in the management of SLE patients with moderately active disease for the 1-year study period.

Progesterone initiates Wnt-beta-catenin signaling but estradiol is required for nuclear activation and synchronous proliferation of rat uterine stromal cells.

Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-beta. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-beta (0.2 microg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3beta (GSK-3beta) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3beta protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of beta-catenin in these stromal cells. Translocation of beta-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. Beta-catenin binding to TCF/LEF increased (P<0.05) in progesterone-pretreated uteri in response to estradiol. Progesterone stimulated the expression of the Wnt target gene urokinase plasminogen activator receptor (uPA-R) in the periluminal uterine stromal cells. The expression of uPA-R increased in progesterone-pretreated stromal cells in response to estradiol administration. Together, the results indicate that progesterone initiates Wnt signaling in the uterine stroma by down-regulating GSK-3beta. However, nuclear translocation of beta-catenin and sufficient complex formation with TCF/LEF to activate stromal cell cycle entry requires estradiol. Stimulation of a uterine stromal cell line to proliferate and differentiate resulted in beta-catenin accumulation, suggesting that endocrine-dependent Wnt signaling controls proliferation and differentiation (decidualization).

Differential expression of estrogen receptors in women with systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease primarily affecting women. T cell activation markers (calcineurin, CD154) increase in SLE T cells cultured with estradiol 17-beta. Biological effects of estradiol are mediated through 2 receptor proteins, estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta). We compared the amount of estrogen receptor subtypes in T cells and measured the ability of receptor agonist-specific ligands to activate marker gene expression. METHODS: T cells were isolated from 22 female patients with SLE and 17 control women. The amount of ER subtypes was measured by immunoblotting. Some T cells were cultured with ER-alpha or ER-beta-specific agonists. Receptor activation was measured by increased expression of the T cell activation markers CD154 and calcineurin. RESULTS: Although the amount of ER-alpha appeared to be less in SLE T cells than in control T cells, the difference was not statistically significant (p = 0.081). The quantity of ER-beta was similar in SLE and control T cells. The expression of ER-alpha or ER-beta was independent of menstrual cycle phase, age, or SLE disease activity. Calcineurin and CD154 expression increased in SLE T cells cultured in medium containing ER-alpha and ER-beta agonists. CONCLUSION: These data indicate that both ER subtypes activate calcineurin and CD154 in SLE but not in normal T cells. Variation in the amount of ER-alpha in SLE T cells suggests this receptor subtype participates in the sensitivity of SLE T cells to estrogen.

Isolation of hormone responsive uterine stromal cells: an in vitro model for stromal cell proliferation and differentiation.

The female sex hormones estrogen and progesterone stimulate proliferation and differentiation of human and rodent uterine cells. The purpose of this chapter is to provide a method for isolating hormone-responsive rat uterine stromal cell lines that can be used to study steroid control of the cell cycle. Uteri from ovariectomized rats are differentially digested with trypsin to separate epithelial and stromal cells. The stromal cells are cultured in a standard growth medium containing 10% fetal bovine serum. After several passages, the purity of the stromal cell lines is determined using immunocytochemistry. Cell proliferation is studied by culturing the stromal cells in serum-free medium containing sex steroids and other mitogens. Cell cycle progression is assessed by flow cytometry, 3H-thymidine and BrdU incorporation, whereas proliferation is monitored using the MTT assay. Cell cycle regulators are visualized by Northern and Western blotting whereas cyclin-cyclin-dependent kinase activity is monitored using immune complex kinase assays. Uterine stromal cell lines isolated using the methods reported in this chapter provide a suitable model system to investigate the signal transduction events that stimulate hormone-dependent control of the cell cycle.

Transit of rat uterine stromal cells through G1 phase of the cell cycle requires temporal and cell-specific hormone-dependent changes on cell cycle regulators.

Progesterone pretreatment increases the number of synchronously proliferating stromal cells in the ovariectomized rat uterus, but estrogen is necessary to stimulate reentry into the cell cycle. To investigate the mechanisms underlying differential hormone actions, sexually mature ovariectomized rats were injected with progesterone (2 mg) for three consecutive days. Estradiol 17-beta (0.6 microg) was administered to initiate cell proliferation. Uterine samples were collected at timed intervals. Cell entry into DNA replication was monitored by injecting 5-bromo-2'-deoxyuridine (1 mg/100 g body weight) 2 h before necropsy. Demicolchicine (400 microg) was injected 30 min before necropsy to assess transit into M phase. Temporal progress through G1 was determined by spatial changes in cyclin D1/D3 proteins. Total cyclin D1/D3 protein and mRNA was measured by Western and Northern blotting. Estrogen increased the number of 5-bromo-2'-deoxyuridine-positive stromal cells (P < 0.05), compared with the number in rats treated with progesterone alone. An increase (P < 0.05) in the number of M-phase cells occurred at 12 h post estrogen. There was no evidence for epithelial cell proliferation in response to steroid treatments. Cyclin D1/D3 mRNA was expressed in the uteri of ovariectomized and hormone treated rats. The D-type cyclin proteins, however, were not evident in stromal cells without estrogen treatment. Progesterone pretreatment inhibited estrogen-dependent epithelial cell proliferation while redirecting D-type cyclin expression to the uterine stroma. Stromal cell transit through G1 required nongenomic steroid-dependent action on signal transduction pathways that control the nuclear localization and cell type-specific expression of the D-type cyclin proteins.

Increased estrogen-dependent expression of calcineurin in female SLE T cells is regulated by multiple mechanisms.

OBJECTIVE: Calcineurin is a key mediator of T cell activation. Previous studies in our laboratory showed a dose-dependent and hormone-specific increase in calcineurin expression in the T cells from females with systemic lupus erythematosus (SLE). This study investigates whether the estrogen-dependent increase in calcineurin expression is due to stabilization of the messenger RNA (mRNA). METHODS: T cells from female patients with SLE and controls were cultured for 18 hours in a serum-free medium with and without estradiol-17 beta (10(-7) M). Some T cells were activated by further culture on anti-CD3-coated plates. Actinomycin D (25 micrograms/mL) was added to some cultures to inhibit new mRNA synthesis. Calcineurin mRNA stability was assessed by reverse-transcription polymerase chain amplification. RESULTS: Resting SLE (n = 9, P = .59) and normal (n = 5, P = .90) T cells showed no significant differences in mRNA stability in response to estradiol. Calcineurin mRNA was not significantly stabilized in activated SLE (n = 10, P = .12) or activated normal (n = 8, P = .09) T cells in response to estradiol. However, the amount of calcineurin mRNA stabilized in activated normal T cells (n = 8) was significantly greater (P = .02) compared with SLE T cells (n = 10) only after culture in medium without estradiol. CONCLUSIONS: These findings highlight the complex gene regulatory mechanisms underlying the differential action of estrogen on SLE T cells. Furthermore, the data indicate that increased calcineurin expression in SLE T cells is not due solely to estrogen-dependent stabilization of the message, and probably involves additional transcriptional regulatory mechanisms.

Progesterone and the control of uterine cell proliferation and differentiation.

Progesterone is the only steroid hormone that is essential for the establishment and maintenance of pregnancy in all mammalian species that have been studied. Mice lacking the progesterone receptor (PR) by targeted mutagenesis exhibit abnormalities in all aspects of reproduction including sexual behavior, mammary gland development, ovulation, and implantation. Implantation in PR null mice fails, in part, because the uterine stromal cells cannot undergo differentiation (the decidual cell reaction). Uterine stromal cells do not divide without progesterone and proliferation is blocked by progesterone antibodies and PR antagonism. In spite of the preeminence of progesterone in female reproduction, its molecular mechanisms of action on target cell proliferation and differentiation are not well understood. Recent studies suggest that progesterone plays a direct role in regulating cell cycle transit by increasing the expression and activation of cell cycle regulatory complexes. Furthermore, this progesterone-dependent regulation of cell cycle transit may provide a unique window of opportunity for uterine stromal cells to exit the proliferative cycle, and if exposed to appropriate agents, enter into the differentiation pathway.