Progesterone modulates extracellular heat-shock proteins and interlukin-1ß in human choriodecidual after Escherichia coli infection.

INTRODUCTION: Chorioamnionitis is an adverse condition in human pregnancy caused by many bacterial pathogens including Escherichia coli (E. coli); which has been associated with higher risk of preterm birth. We recently reported that human maternal decidua (MDec) tissue responds to E. coli infection by secreting extracellular heat-shock proteins (eHsp)-60, -70 and interlukin-1ß (IL-1ß). Previous studies have shown that progesterone (P4) regulates the immune response, but it is unknown whether P4 inhibits the secretion of eHsp. The aim of this investigation was to determine the role of P4 on the secretion of eHsp-27, -60, -70 and IL-1ß in MDec after 3, 6, and 24 h of E. coli infection. METHODS: Nine human feto-maternal interface (HFMi) tissues were included and mounted in the Transwell culture system. Only the maternal decidua (MDec) was stimulated for 3, 6 and 24 h with E. coli alone or in combination with progesterone and RU486. After each treatment, the HFMi tissue was recovered to determine histological changes and the culture medium recovered to evaluate the levels of eHsp-27, -60, -70 and IL-1ß by ELISA and mRNA expression by RT-PCR. RESULTS: No structural changes were observed in the HFMi tissue treated with P4 and RU486. However, stimulation with E. coli produces diffuse inflammation and ischemic necrosis. E. coli induced infection decreases, in time- and dose-dependent manner, eHsp-27 and increases eHsp-60, eHsp-70 and IL-1ß levels. In contrast, incubation of HFMi tissue with E. coli + P4 reversed eHsp and IL-1ß secretion levels relative to E. coli stimulation group but not relative to the control group. The same profile was observed on the expression of eHsp-27 and eHsp-60. DISCUSSION: we found that progesterone modulates the anti-inflammatory (eHsp-27) and pro-inflammatory (eHsp-60 and eHsp-70) levels of eHsp induced by E. coli infection in human choriodecidual tissue. eHsp-60 and eHsp-70 levels were not completely reversed; maintaining the secretion of IL-1ß, which has been associated with adverse events during pregnancy.

Ribosomal S6 kinase 4 (RSK4) tumor suppressor gene promoter methylation status in ovarian cancer.

BACKGROUND: Previously, we reported lower RSK4 mRNA and protein levels in malignant ovarian tumors compared to normal and benign ovarian tissues. Also, we observed a significant inverse correlation between the advanced ovarian cancer stages and RSK4 mRNA levels. We did not investigate the mechanisms involved in RSK4-reduced expression in ovarian cancer. Thus, this study investigates whether RSK4 promoter methylation in ovarian cancer tissues is responsible for its low expression. Additionally, the reactivation of RSK4 expression and its effect was studied in ovarian cancer cell lines. METHODS AND RESULTS: RSK4 promoter methylation percentage in malignant and benign ovarian tumors and normal ovary tissues was determined by combined bisulfite restriction analysis. The reactivation of RSK4 expression by decitabine treatment was studied in OVCAR3, SKOV3, TOV-112D, and TOV-21G cells by Western blotting. Cell proliferation was determined by XTT. A significantly high methylation percentage of the RSK4 promoter was observed among malignant and benign ovarian tumors but not in normal ovarian tissue. RSK4 promoter methylation was not associated with age, histological subtype, or stages of ovarian cancer. RSK4 promoter methylation correlates weakly but not significantly with RSK4 protein expression. No correlation was shown between RSK4 methylation and RSK4 mRNA expression. Decitabine induces RSK4 reactivation in all cell lines. However, cell proliferation was reduced only in TOV-112D cells. CONCLUSION: These data indicate that although RSK4 promoter methylation is increased in malignant ovarian tumors, this mechanism is unlikely to regulate its expression in ovarian cancer. RSK4 reactivation reduced cell proliferation only in the endometroid histological subtype.

Cadmium exposure reduces invasion of the human trophoblast-derived HTR-8/SVneo cells by inhibiting cell adhesion and matrix metalloproteinase-9 secretion.

Preeclampsia and intrauterine growth restriction, multisystemic disorders characterized by a shallow trophoblast invasion, have been associated with maternal cadmium (Cd) exposure. The molecular mechanisms of this association remain unknown. Cell adhesion and matrix metalloproteinase production are essential for an adequate trophoblast invasion. Thus, the aim of this study was to determine the effect of Cd exposure on invasion, adhesion, and matrix metalloproteinase-9 (MMP-9) production in the trophoblast-derived HTR-8/SVneo cell line. Cultured HTR-8/SVneo trophoblast cells were incubated with different concentrations of CdCl2 for 6 h. Cell invasion was determined by the transwell assay, while cell adhesion was examined on collagen type I. MMP-9 release and activity were measured by ELISA and zymography, respectively. MMP-9 mRNA expression was detected by reverse-transcription polymerase chain reaction, while intracellular MMP-9 protein was assessed by Western blotting. Cd exposure significantly decreased the invasion and adhesion of HTR-8/SVneo cells. Also, MMP-9 levels and activity in the culture medium were significantly reduced after Cd incubation. In contrast, MMP-9 mRNA expression and intracellular protein levels were significantly increased. These data indicate that Cd reduces trophoblast cells invasiveness by inhibiting cell adhesion and MMP-9 secretion.

Progranulin expression induced by follicle-stimulating hormone in ovarian cancer cell lines depends on the histological subtype.

Epithelial ovarian cancer (EOC) is a heterogeneous disease that can be categorized into four major histological subtypes. Its etiology remains poorly understood due mainly to this heterogeneity. Follicle-stimulating hormone (FSH) has been implicated as a risk factor in EOC and has been suggested that may influence the development of specific subtypes. In addition, FSH regulates different aspects of ovarian cancer tumorigenesis. FSH downstream target genes in EOC have not been fully identified. Progranulin (PGRN) overexpression is associated with cell proliferation, invasion, chemoresistance, and shortened overall survival in ovarian cancer. Recently, we demonstrated that PGRN expression is regulated through the PI3K signaling pathway in clear cell ovarian carcinoma (CCOC) cells. In contrast, we also demonstrated that PGRN synthesis in serous ovarian cancer (SOC) cells is regulated via PKC but not by the PI3K signaling pathway. Several studies have demonstrated that FSH induces PKC and PI3K activation. Thus, this study was to investigate the effect of FSH on PGRN production in the CCOC cell line TOV-21G as compared to the SOC cell lines SKOV3 and OVCAR3. Cultured TOV-21G, SKOV3, and OVCAR3 cells were incubated with different concentrations of FSH for 48 h. PGRN mRNA and protein expression were assessed by RT-PCR and Western blotting, while PGRN secretion was measured by ELISA. PGRN mRNA and protein expression, as well as PGRN secretion, significantly increased after FSH stimulation in TOV-21G but not in SKOV3 and OVCAR3 cells. These data indicate that FSH induces PGRN expression and secretion only in CCOC cells. Establishing specific features for CCOC could reveal potential diagnostic and therapeutic targets.

Inhibition of PI3K/AKT/mTOR and MAPK signaling pathways decreases progranulin expression in ovarian clear cell carcinoma (OCCC) cell line: a potential biomarker for therapy response to signaling pathway inhibitors.

Patients with advanced stage ovarian clear cell carcinoma (OCCC) have a poor prognosis due to resistance to conventional platinum chemotherapy. Recent studies have demonstrated that PI3K/AKT/mTOR and ERK1/2 signaling pathways are involved in this chemoresistance. Progranulin (PGRN) overexpression contributes to cisplatin resistance of epithelial ovarian cancer cell lines. Also, PGRN expression is regulated by AKT/mTOR and ERK1/2 signaling pathways in different cell types. Thus, the present study was designed to identify if PGRN expression is regulated by AKT, mTOR, and ERK1/2 signaling pathways in the OCCC cell line TOV-21G. Cultured TOV-21G cells were incubated with different concentrations of pharmacological cell signaling inhibitors. PGRN expression and phosphorylation of ERK1/2, AKT, and mTOR were assessed by Western blotting. Inhibition of AKT, mTOR, and ERK1/2 significantly reduced PGRN expression. Cell viability was not affected, while cell proliferation significantly decreased with all inhibitors used in this study. These observations demonstrated that inhibition of PI3K/AKT/mTOR and ERK1/2 signaling pathways reduces PGRN expression in TOV-21G cells. Thus, PGRN could be considered as a candidate for explaining the high resistance to platinum-based treatment and a potential biomarker for therapy response to cell signaling inhibitors in patients with OCCC.

Cadmium exposure induces interleukin-6 production via ROS-dependent activation of the ERK1/2 but independent of JNK signaling pathway in human placental JEG-3 trophoblast cells.

Maternal exposure to cadmium (Cd) has been associated with preeclampsia (PE), which is a multisystemic disorder characterized by endothelial dysfunction. Elevated interleukin (IL)-6 expression is linked to PE and has been suggested to contribute to maternal endothelial dysfunction. Cd induces IL-6 production in various cell types through different signaling pathways. Thus, this study was designed to investigate the effect of Cd on IL-6 production and the underlying mechanisms in a trophoblast-derived cell line. Cultured JEG-3 trophoblast cells were exposed to non-toxic concentrations of CdCl2 in the presence or absence of various MAPK inhibitors or N-Acetyl-L-cysteine (NAC). IL-6 was measured by ELISA. Phosphorylation of ERK1/2, JNK, and c-Jun was assessed by Western blotting. Cd exposure induced IL-6 production and increased ERK1/2, JNK, and c-Jun phosphorylation. NAC and the inhibition of ERK1/2 significantly reduced Cd-induced IL-6 production. These data indicate that Cd induces IL-6 production in trophoblast cells through a ROS-dependent activation of ERK1/2.

Frequency of the T307A, N680S, and -29G>A single-nucleotide polymorphisms in the follicle-stimulating hormone receptor in Mexican subjects of Hispanic ancestry.

BACKGROUND: FSHR SNPs may influence the ovarian sensitivity to endogenous and exogenous FSH stimulation. Given the paucity of data on the FSHR c.919A > G, c.2039A > G and - 29G > A SNPs in Hispanic population, we here analyzed their frequency distribution in Mexican mestizo women. METHODS: Samples from 224 Mexican mestizo women enrolled in an IVF program as well as a genotype database from 8182 Mexican mestizo subjects, were analyzed for FSHR SNPs at positions c.919, c.2039 and - 29G > A. Association between the genetic variants and reproductive outcomes was assessed. RESULTS: The c.919 and c.2039 SNPs were in strong linkage disequilibrium and their corresponding genotype frequencies in the IVF group were: AA 46.8%, AG 44.2%, and GG 8.9%, and AA 41.9%, AG 48.2% and GG 9.8%, respectively. For the -29G > A SNP, genotype frequencies were 27% (GG), 50% (GA) and 23% (AA). In normal oocyte donors with the c.2039 GG genotype, the number of oocytes recovered after ovarian stimulation (COS) were significantly (p < 0.01) lower than in those bearing other genotypes in this or the -29G > A SNP. Analysis of the large scale database revealed that both allelic and genotype frequencies for the three SNPs were very similar to those detected in the IVF cohort (p ≥ 0.38) and that female carriers of the c.2039 G allele tended to present lower number of pregnancies than women bearing the AA genotype; this trend was stronger when women with more Native American ancestry was separately analyzed (OR = 2.0, C.I. 95% 1.03-3.90, p = 0.04). There were no differences or trends in the number of pregnancies among the different genotypes of the -29G > A SNP. CONCLUSIONS: The frequency of the GG/GG combination genotype for the c.919 and c.2039 SNPs in Mexican hispanics is among the lowest reported. The GG genotype is associated with decreased number of oocytes recovered in response to COS as well as to lower pregnancy rates in Hispanic women from the general population. The absence of any effect of the -29AA genotype on the response to COS, indicates that there is no need to perform this particular genotype testing in Hispanic women with the purpose of providing an individually-tailored COS protocol.

Methods to Study the Role of Progranulin in Preimplantation Mouse Embryo Development.

Progranulin is a 67-88 kDa glycoprotein, also known as acrogranin, PC-cell-derived growth factor, granulin-epithelin precursor, and proepithelin. This protein is present in a variety of mouse, rat, and human tissues. Progranulin, which is a growth factor, mediates cell cycle progression and cell migration in normal and pathological conditions. In several types of cancers, progranulin expression is upregulated, whereas function-interfering mutations in the granulin gene in humans have been linked to a subset of heritable cases of frontotemporal lobar degeneration. Also, progranulin has important effects on mouse preimplantation embryo development in vitro, including regulation of the appearance of the epithelium in the developing mouse blastocyst and growth of trophectoderm. Furthermore, progranulin promotes mouse blastocyst hatching, adhesion, and outgrowth in vitro. In this chapter, we describe some of the techniques that may be useful in the study of progranulin in embryo development.

The follicle-stimulating hormone receptor Asn680Ser polymorphism is associated with preterm birth in Hispanic women.

OBJECTIVE: Recently, a study based on the analysis of accelerated evolution of related genes at birth identified the follicle-stimulating hormone receptor (FSHR) as a possible candidate for the development of preterm delivery. Additionally, FSHR expression has been described in extragonadal tissue including the placenta. Therefore, the aim of the present study was to determine the association between the N680S polymorphism of the follicle-stimulating hormone receptor and preterm birth in a population of Hispanic women. METHODS: Placenta samples were obtained from 64 women who had preterm births and 54 control cases. DNA was extracted and genotyped for the N680S FSHR gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The χ2 test and t-test were used to calculate statistical significance. RESULTS: Statistically significant differences in genotype frequencies for the N680S polymorphism were observed between preterm and term groups (p = .04). Based on the Akaike information criterion values, the dominant model showed that the NN genotype had a significantly increased risk of preterm birth compared with the SS + NS genotype (OR 2.52, 95% CI 1.20-5.33, p = .02). CONCLUSIONS: The results herein suggest that the FSHR polymorphism N680S is significantly associated with preterm birth in the Hispanic population.

Progranulin and its biological effects in cancer.

Cancer cells have defects in regulatory mechanisms that usually control cell proliferation and homeostasis. Different cancer cells share crucial alterations in cell physiology, which lead to malignant growth. Tumorigenesis or tumor growth requires a series of events that include constant cell proliferation, promotion of metastasis and invasion, stimulation of angiogenesis, evasion of tumor suppressor factors, and avoidance of cell death pathways. All these events in tumor progression may be regulated by growth factors produced by normal or malignant cells. The growth factor progranulin has significant biological effects in different types of cancer. This protein is a regulator of tumorigenesis because it stimulates cell proliferation, migration, invasion, angiogenesis, malignant transformation, resistance to anticancer drugs, and immune evasion. This review focuses on the biological effects of progranulin in several cancer models and provides evidence that this growth factor should be considered as a potential biomarker and target in cancer treatment.

The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells.

Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy.

Synergistic effect of DDT and its metabolites in lipopolysaccharide-mediated TNF-α production is inhibited by progesterone in peripheral blood mononuclear cells.

Increased TNF-α levels have been associated with adverse pregnancy outcomes. Lipopolysaccharide (LPS), 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) induce TNF-α release in peripheral blood mononuclear cells (PBMC). Conversely, progesterone (P4) inhibits TNF-α secretion. Pregnant women in malaria endemic areas may be co-exposure to these compounds. Thus, this study was to investigate the synergistic effect of LPS and these pesticides in PBMC and to assess P4 influence on this synergy. Cultured PBMC were exposed to each pesticide in the presence of LPS, P4, or their combination. TNF-α was measured by ELISA. All pesticides enhanced TNF-α synthesis in PBMC. Co-exposure with LPS synergizes TNF-α production, which is blocked by progesterone. These results indicate that these organochlorines act synergistically with LPS to induce TNF-α secretion in PBMC. This effect is blocked by P4.

Ribosomal S6 kinase 4 (RSK4) expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines.

Survival rate in ovarian cancer depends on the stage of the disease. RSK4, which has been considered as a tumor suppressor factor, controls cells invasion due to its antiinvasive and antimetastatic properties. Modulation of RSK4 expression could be an important event to increase the survival rate in ovarian cancer patients. Thus, the goal of the present study was to establish the differences in RSK4 expression among normal, benign and malignant ovarian tissues and to determine whether antineoplastic drugs regulate its expression in SKOV3 and TOV-112D cells. RSK4 levels in 30 malignant ovarian tumors, 64 benign tumors and 36 normal ovary tissues were determined by reverse transcription polymerase chain reaction and Western blot. Modulation of RSK4 expression by two antineoplastic drugs (cisplatin and vorinostat) was also studied in the SKOV3 and TOV-112D ovarian cancer cell lines using the same techniques. RSK4 mRNA and protein levels were decreased in malignant ovarian tumors as compared to benign tumors and normal tissue. These low-RSK4 levels were significantly associated with advanced stages of ovarian cancer. RSK4 expression was increased after incubation of SKOV3 and TOV-112D cell lines with cisplatin and vorinostat for 24 h. The combination of these antineoplastic drugs did not produce a synergistic or additive effect. These results suggest that RSK4 is expressed at low levels in malignant ovarian tumors, which correlates with advanced stages of the disease. Additionally, RSK4 expression is regulated by cisplatin and vorinostat in two ovarian cancer cell lines.

[Clinical implications of basic research in preeclampsia: immunological tolerance]. / Implicaciones clínicas de la investigación básica de la preeclampsia: tolerancia inmunológica.

Preeclampsia is one of the main causes of maternal and perinatal mortality in the world; however, the pathophysiologic pathways haven't been clearly elucidated. It is thought to result from a breakdown of maternal tolerance to paternal antigens in placenta that start an immune response against the trophoblast inducing a defective placentation and a hipoxic/isquemic environment which in turn triggers a systemic inflamatory response. This review gives an overview of the mechanims involved in maternal tolerance, how these are disrupted in preeclampsia, and how they contribute to the inflamatory response.

[Characterization of hemodynamic ex vivo model to study endothelial activation by TNF-α in prefunded human veins]. / Caracterización de un modelo hemodinámico ex vivo para el estudio de la activación endotelial por el factor de necrosis tumoral α (TNF-α) en venas humanas perfundidas.

Inflammation is recognized as part of the etiology of numerous diseases. The interaction among cells of the immunological system with local cells and molecules, such as cytokines and chemokines, allows cellular activation and response amplification. The importance of several physicochemical factors like frictional force, vascular flow, shear stress, and pressure is now recognized because they are known to modulate genetic expression and endothelial activation; however, there are very few studies that recreate such cellular microenvironments. Hence, it is of paramount importance to develop new models that will mimic physiological conditions. Our aim was to improve a human vein ex vivo model that would allow endothelial activation in flow conditions, to study the molecular components during adhesion, taking into consideration physicochemical parameters such as flow and shear stress. Endothelial umbilical human vein was used and activated with TNF-a in order to determine U937 monocytic cells adhesion, as well as cytokines secretion and ICAM-1 expression. This model will allow leukocyte adhesion studies, using different inflammatory stimulus, along with the signaling pathways involved in several pathologies.

Adrenal androgen excess and body mass index in polycystic ovary syndrome.

CONTEXT: Adrenal hyperandrogenism affects approximately 25% of polycystic ovary syndrome (PCOS) patients but its relation to obesity is not totally understood. OBJECTIVE: This study aimed to assess dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) levels in relation to body mass index (BMI) in PCOS. DESIGN AND SETTING: This was a prospective observational study at an institutional practice at an obstetrics/gynecology hospital. PARTICIPANTS: The study included 136 PCOS patients, 20-35 years old, and 42 age-matched control women. The participants were classified with the BMI cutoff value of 27 kg/m(2) as follows: 1) high-BMI PCOS patients; 2) low-BMI PCOS patients; 3) high-BMI control women; and 4) low-BMI control women. The data were reanalyzed with the BMI cutoff value of 30 kg/m(2) to corroborate the findings in obese and nonobese patients. MAIN OUTCOME MEASURE(S): Blood samples were taken and LH, FSH, insulin, T, androstenedione (A4), DHEA, DHEAS, and glucose levels were determined. Homeostatic model assessment was calculated. Pelvic and abdominal ultrasound for ovarian morphology and adipose tissue, respectively, were performed. RESULTS: Obese PCOS patients presented significantly more insulin resistance than nonobese PCOS patients. The LH levels and LH/FSH ratio were significantly higher in low-BMI than in high-BMI PCOS patients. The A4 and DHEAS levels were significantly higher in nonobese than in obese PCOS patients. A significant correlation between LH and A4 in nonobese PCOS patients was observed. The frequency of hyperandrogenism by increased A4, and DHEA along with DHEAS was significantly higher in low-BMI PCOS patients compared with high-BMI PCOS patients. Some findings observed with the BMI cutoff value of 27 kg/m(2) changed with the cutoff value of 30 kg/m(2). CONCLUSIONS: Low BMI more so than high BMI is associated with increased LH, high A4, DHEA, and DHEAS levels in PCOS patients. The BMI cutoff value of 27 kg/m(2) classified better than 30 kg/m(2) for hormonal and metabolic characteristics.

Matrix metalloproteinase-8 promoter gene polymorphisms in Mexican women with ovarian cancer.

Increased levels of matrix metalloproteinase-8 (MMP-8) have been associated with tumor grade and stage in ovarian cancer. Also, it has been reported that higher concentrations of this enzyme in fluid from malignant ovarian cysts compared with benign ovarian cysts. However, no genetic analysis has been conducted yet to assess the contribution of MMP-8 polymorphisms in ovarian cancer. Thus, this study was performed to investigate the frequencies of MMP-8 genotypes in Mexican women with ovarian cancer. MMP-8 promoter genotypes were examined in 35 malignant ovarian tumors, 51 benign tumors, and 37 normal ovary tissues. Two single nucleotide polymorphisms were selected and characterized using polymerase chain reaction-restriction fragment length polymorphism analysis. The chi-square test was used to calculate statistical significance. Haplotype analysis was performed using the SNPstats web tool. Of the two polymorphisms, only the MMP-8 -799 T/T genotype was significantly associated with an increased risk of ovarian cancer (OR 3.78, 95 % CI 1.18-12.13). The Kaplan-Meier analysis for this polymorphism showed that patients with the T/T genetic variant had a tendency toward significant worse overall survival compared with patients with the C/C + C/T genotypes. Haplotype analysis revealed no significant differences in haplotype distribution between benign ovarian tumors, malignant ovarian cancer, and controls. This study suggests that MMP-8 promoter gene polymorphism -799 T/T is significantly associated with an increased risk of ovarian cancer in Mexican women.

Reduced frequency of a CD14+ CD16+ monocyte subset with high Toll-like receptor 4 expression in cord blood compared to adult blood contributes to lipopolysaccharide hyporesponsiveness in newborns.

The human innate immune response to pathogens is not fully effective and mature until well into childhood, as exemplified by various responses to Toll-like receptor (TLR) agonists in newborns compared to adults. To better understand the mechanistic basis for this age-related difference in innate immunity, we compared tumor necrosis factor alpha (TNF-α) production by monocytes from cord blood (CB) and adult blood (AB) in response to LAM (lipoarabinomannan from Mycobacterium tuberculosis, a TLR2 ligand) and LPS (lipopolysaccharide from Escherichia coli, a TLR4 ligand). LPS or LAM-induced TNF-α production was 5 to 18 times higher in AB than in CB monocytes, whereas interleukin-1α (IL-1α) stimulated similar levels of TNF-α in both groups, suggesting that decreased responses to LPS or LAM in CB are unlikely to be due to differences in the MyD88-dependent signaling pathway. This impaired signaling was attributable, in part, to lower functional TLR4 expression, especially on CD14(+) CD16(+) monocytes, which are the primary cell subset for LPS-induced TNF-α production. Importantly, the frequency of CD14(+) CD16(+) monocytes in CB was 2.5-fold lower than in AB (P < 0.01). CB from Kenyan newborns sensitized to parasite antigens in utero had more CD14(+) CD16(+) monocytes (P = 0.02) and produced higher levels of TNF-α in response to LPS (P = 0.004) than CB from unsensitized Kenyan or North American newborns. Thus, a reduced CD14(+) CD16(+) activated/differentiated monocyte subset and a correspondingly lower level of functional TLR4 on monocytes contributes to the relatively low TNF-α response to LPS observed in immunologically naive newborns compared to the response in adults.

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

Differential effect of DDT, DDE, and DDD on COX-2 expression in the human trophoblast derived HTR-8/SVneo cells.

The purpose of this study was to investigate the effect of 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) isomers on COX-2 expression in a human trophoblast-derived cell line. Cultured HTR-8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX-2 mRNA and protein expression were assessed by RT-PCR, Western blotting, and ELISA. Prostaglandin E2 production was also measured by ELISA. Both COX-2 mRNA and protein were detected under control (unexposed) conditions in the HTR-8/SVneo cell line. COX-2 protein expression and prostaglandin E2 production but not COX-2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX-2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX-2 by these organochlorines pesticides appears to be at the translational level.