Evidence for the notch signaling pathway on the role of estrogen in angiogenesis

We have investigated the molecular mechanisms involved in 17 beta-estradiol-induced angiogenic pathway. We show here that 17 beta-estradiol promoted a 6-fold increase in Jagged1 expression and an 8-fold increase in Notch1 expression by cDNA arrays in breast cancer MCF7 cells. Interestingly, Jagged1 was abrogated by incubation with the estrogen antagonist, ICI182,780. A similar up-regulation of both Notch1 receptor and Jagged1 ligand was found in endothelial cells. Additionally, imperfect estrogen-responsive elements were found in the 5' untranslated region of Notch1 and Jagged1 genes. Treatment with 17 beta-estradiol also led to an activation of Notch signaling in MCF7 cells expressing Notch1 reporter gene or by promoting Jagged1-induced Notch signaling in coculture assays. Inoculation of MCF7 cells in 17 beta-estradiol-treated nude mice resulted in up-regulation of Notch1 expression as well as increased number of tumor microvessels in comparison to placebo-treated mice. Notch1-expressing endothelial cell cultures formed cord-like structures on Matrigel in contrast to cells expressing a dominant-negative form of Notch1, emphasizing the relevance of Notch1 pathway in vessel assembly. Finally, Notch1-expressing MCF7 cells up-regulated hypoxia-inducible factor 1 alpha gene, a well-known angiogenic factor that clustered with Notch1 gene. This study implicates Notch signaling in the cross talk between 17 beta-estradiol and angiogenesis.     

Differing transcriptional responses to pulsed or continuous estradiol exposure in human umbilical vein endothelial cells

This study used human umbilical vein endothelial cells (HUVECs) that were treated with 17beta-estradiol for 5 days as 1h pulse or 24h continuous treatment at concentrations such that the 24h exposure (concentration x time) was identical in both conditions. Cell proliferation was studied and gene expression profiling was carried out using the Affymetrix GeneChip microarray analysis. Changes in morphology and apoptosis in HUVECs were examined with electron microscopy. Time-course studies of expression of genes vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed by quantitative PCR. We observed that cell proliferation was significantly decreased over days 3-5 with pulsed estradiol treatment relative to constant exposure. Microarray results showed that after 5 days, 801 genes differed (P<0.05) between continuous versus pulsed estradiol treatment. Functional analysis showed a significant number of genes to be associated with apoptosis and cell cycle pathways. We did not find any evidence of apoptosis from flow cytometry or electron microscopy examination. Our study highlights a large number of significantly different molecular responses to estradiol depending upon the mode of administration of estradiol. Significant changes were observed in genes involved in apoptosis and proliferation including VEGF, IGF receptors, and tumor protein p53.     

Estradiol increases the secretion of hepatic lipase by rat hepatocyte cultures.

Hepatic lipase (EC 3.1.1.3) is synthesized and secreted by parenchymal hepatocytes and binds to endothelial cells of liver sinusoids. The present study shows that the activity of hepatic lipase secreted by hepatocyte cultures from male rats in increased approx. 6-fold after 10 h culture with 10 microM 17 beta-estradiol. The stimulatory effect of 17 beta-estradiol is biphasic and declines at higher concentrations. In hepatocytes from male rats: progesterone, unlike 17 beta-estradiol, had only a small stimulatory effect when present as the sole hormone and a small inhibitory effect in the presence of 17 beta-estradiol, while testosterone and dexamethasone had no effect. Hepatocyte cultures from female rats had a higher basal rate of hepatic lipase secretion than cells from male rats and showed a smaller stimulation by 17 beta-estradiol. These results suggest that 17 beta-estradiol might regulate the secretion of hepatic lipase by hepatocytes, and presumably the activity of the enzyme at either the endothelial surface of the liver sinusoids or at extrahepatic sites.     

On the role of 17 alpha-estradiol and 17 beta-estradiol in the proliferation of MCF7 and T47D-A11 human breast tumor cells

A comparative study of the proliferative effect of 17 beta-estradiol and 17 alpha-estradiol on human estrogen-sensitive cell lines was performed. When using charcoal-dextran stripped human female sera-supplemented media the administration of the hormones, 17 alpha-estradiol at 3 X 10(-10)M, and 17 beta-estradiol at 3 X 10(-11)M, resulted in a ten-fold increase in cell yield when compared with non-estrogen supplemented controls after cells were grown for periods between 10 to 14 days. No significant metabolization of 17 alpha-estradiol into 17 beta-estradiol occurred as measured by the E2 levels in the supernatants of the cell culture flasks. Increased concentrations of 17 beta-estradiol and 17 alpha-estradiol added to the media bathing C7MCF7-173 cells resulted in a triggering of a partially successful shut-off effect; this phenomenon was not observed with T47D-All cells. These results are compatible with predictions stemming from the indirect and direct negative working hypothesis for the regulation of cell proliferation.     

Endothelial monocyte activating polypeptide-II induced gene expression changes in endothelial cells

In the current study we used microarray (MA) analysis to examine gene expression changes in human umbilical vein endothelial cells (HUVEC) exposed to the tumor-derived cytokine, endothelial monocyte-activating polypeptide-II (EMAP-II). HUVEC treated with EMAP-II for 0.5, 1, 2, 4 and 8 h, were analyzed using 10K cDNA arrays. Our results demonstrated that changes in gene expression of <0.5 and >2 fold were seen for 69 genes and the majority of gene changes occurred early. Validation of MA analysis for 10 genes by real time RT-PCR, demonstrated the gene changes to be consistent and specific to HUVEC when compared to human fibroblasts treated with EMAP-II. Among these genes, downregulated in ovarian cancer 1 (DOC1) gene was studied further because of its possible role in EMAP-II induced cytoskeletal remodeling. DOC1 expression was silenced using small interfering RNA. SiRNA to DOC1 completely abolished EMAP-II stimulated gene expression of DOC1. Silencing of DOC1 gene expression reversed the modulatory effect of EMAP-II on 4 other genes, suggesting that DOC1 might play a role in mediating some of the effects of EMAP-II on endothelial cells.     

Protective effects of 17beta-estradiol and trivalent chromium on interleukin-6 secretion, oxidative stress, and adhesion of monocytes: relevance to heart disease in postmenopausal women

Postmenopausal diabetic women are at greater risk for heart disease compared with men of similar age and with other risk factors. We examined the hypothesis that 17beta-estradiol and trivalent chromium inhibit secretion of the pro-inflammatory cytokine interleukin (IL)-6 and oxidative stress in monocytes exposed to high glucose (HG). U937 human monocytes were cultured with HG (30 mM) with and without 17beta-estradiol (0-1000 nM) and chromium chloride (Cr(3+), 0-10 muM) at 37 degrees C for 24 h. Results show that 17beta-estradiol inhibits IL-6 and adhesion to endothelial cells (p <. 05) by HG-treated monocytes. Treatment with 17beta-estradiol+Cr(3+) required a significantly lower dose of estradiol-17beta compared with 17beta-estradiol alone for IL-6 inhibition. 17beta-Estradiol+Cr(3+) also inhibited lipid peroxidation and the adhesivity to human endothelial cells in HG-treated monocytes. Thus, 17beta-estradiol+Cr(3+) inhibits oxidative stress, IL-6 secretion, and monocytic adhesion to endothelial cells, risk factors in the development of heart disease. The female body requires E but studies on some patients indicate side effects with increased amounts of 17beta-estradiol-supplementation. The potential benefit of a lower estrogen dose in combination with chromium is novel and needs to be explored in postmenopausal diabetic women.     

Synergistic regulation of endothelial tight junctions by antioxidant (Se) and polyunsaturated lipid (GLA) via Claudin-5 modulation

Tight junctions (TJs) in endothelial cells act as cell-cell adhesion structures, governing paracellular permeability (PCP). Disruption can lead to leaky vascular bed and potentially to oedema and swelling of tissues, the aetiology of mastalgia. These changes may also cause vascular spread of cancer cells. This study aimed to determine whether the function of TJs in endothelial cells can be strengthened by gamma linolenic acid (GLA), selenium (Se) and iodine (I) in the presence of 17beta-estradiol (17beta-estradiol), which causes leakage of endothelial cells by disruption of TJs in endothelium. GLA, I, and Se individually increased transendothelial resistance. The combination of all three agents also had a significant effect on TER. Addition of GLA/Se/I reduced PCP of the endothelial cells. Treatment with GLA/Se/I reversed the effect of 17beta-estradiol in reducing TER and increasing PCP. Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h there was increasing relocation to endothelial cell-cell junctions of the TJ proteins Claudin-5, Occludin, and ZO-1. Interestingly, this relocation was particularly evident with treatments containing I when probing with Claudin-5 and those containing Se for Occludin. There was a small increase in overall protein levels when examined by Western blotting after treatment with GLA/Se/I when probed with Claudin-5 and Occludin. We report that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells, by way of regulating the distribution of Claudin-5, Occludin, and ZO-1. Interestingly, this combination was also able to completely reverse the effect of 17beta-estradiol in these cells.     

Sex steroid hormone metabolism takes place in human ocular cells

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.     

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

Comparative analysis of vascular endothelial cell activation by TNF-α and LPS in humans and baboons

As an Old World nonhuman primate, baboons have been extensively used for research on dyslipidemia and atherogenesis. With increasing knowledge about the endothelium's role in the initiation and progression of atherosclerosis, the value of the baboon model can be increased by developing it for research on the role of dysfunctional endothelium in atherogenesis. Toward that goal, we have established and validated methods of isolating and culturing baboon femoral artery endothelial cells (BFAECs) and compared baboon endothelial cellular characteristics with those of humans. Our results indicated that baboon and human endothelial cells share similar growth and culture behaviors. As was the case for human endothelial cells, BFAECs responded to tumor necrosis factor (TNF)-α stimulation with increased expression of adhesion molecules (maximum increase for intracellular adhesion molecule (ICAM): 1.76±0.26-fold; vascular cell adhesion molecule (VCAM): 1.65±0.25-fold; E-selectin: 2.86±0.57-fold). However, BFAECs were hyporesponsive to lipopolysaccharide (LPS) (range, 0.25–20 μg/mL) in adhesion molecule expression, whereas 1 μg/mL LPS induced 2.14- to 3.71-fold increases in human endothelial cells. The differential responses to LPS were not related to TLR-2 and toll-like receptor (TLR)-4 expression on the cell surface. And baboon microvascular endothelial cells had similar features as BFAECs. We observed constitutive expression of interleukin (IL)-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)-1 in both human and baboon endothelial cells, and these cytokines were further induced by TNF-α and LPS. We also demonstrated that the responses to TNF-α or LPS varied among baboons maintained under the same dietary and environmental conditions, suggesting that response may be controlled by genetic factors.     

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.     

Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells

Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

Estrogen induces phospholipase A2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells

The principal secreted estrogen, 17beta-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17beta-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2-3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A(2) (PLA(2)) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA(2)alpha to perinuclear membranes occurred 1-2 min after 17beta-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA(2)alpha at serine residue 505. The phosphorylation and translocation of cPLA(2) were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA(2) could be stimulated by membrane impermeable 17beta-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA(2) and the eicosanoid biosynthetic pathway are involved in the 17beta-estradiol induced rapid calcium responses of breast cancer cells.     

Effects of endocrine disruptors on genes associated with 17beta-estradiol metabolism and excretion

In order to provide a global analysis of the effects of endocrine disruptors on the hormone cellular bioavailability, we combined 17beta-estradiol (E2) cellular flow studies with real-time PCR and Western blot expression measurements of genes involved in the hormone metabolism and excretion. Three endocrine disruptors commonly found in food were chosen for this study, which was conducted in the estrogen receptor (ER) negative hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol (RES). We showed that 24h after a single dose treatment with genistein, resveratrol or bisphenol A, the expression of ATP-binding cassette transporters (the multidrug resistance or MDR, and the multidrug resistance associated proteins or MRP) uridine diphosphate-glucuronosyltransferases (UGT) and/or sulfotransferases (ST) involved in 17beta-estradiol elimination process were significantly modulated and that 17beta-estradiol cellular flow was modified. Resveratrol induced MDR1 and MRP3 expressions, bisphenol A induced MRP2 and MRP3 expressions, and both enhanced 17beta-estradiol efflux. Genistein, on the other hand, inhibited ST1E1 and UGT1A1 expressions, and led to 17beta-estradiol cellular retention. Thus, we demonstrate that bisphenol A, genistein and resveratrol modulate 17beta-estradiol cellular bioavailability in HepG2 and that these modulations most probably involve regulations of 17beta-estradiol phase II and III metabolism proteins. Up to now, the estrogenicity of environmental estrogenic pollutants has been based on the property of these compounds to bind to ERs. Our results obtained with ER negative cells provide strong evidence for the existence of ER-independent pathways leading to endocrine disruption.     

Significantly increased cortisol secretion in normal adrenocortical cells transfected with K-ras mutants derived from human functional adrenocortical tumors

Our previous study showed that the mutation hotspots of the K-ras proto-oncogene in human functional adrenocortical tumors are in codons 15, 16, 18, and 31, thus differing from the sites in other tumors. In addition, analyzing the K-Ras protein by a recombinant DNA technique showed that the activity of endogenic GTPase and the GTPase-activating protein (GAP)-binding ability were significantly decreased in patients with these tumors. The aim of this study was to understand whether those K-ras mutants, which were found only in human adrenocortical tumors, play an important role in these tumors. Thus, the mutant K-ras cDNA was constructed with mammalian expression vectors and transfected into normal adrenocortical cells. The amount of cortisol secreted by the transfected cells was 20 to 30 times that of normal cells. Furthermore, Northern blot analysis revealed that the expression of the three steroidogenesis-related genes P450(scc) (cholesterol side-chain cleavage enzyme), P450(C17) (17alpha-hydroxylase/17, 20-lyase), and P450(C21) (steroid 21-hydroxylase) gene increased in the transfected cells. The K-ras oncogene significantly increases cortisol secretion by normal adrenocortical cells.     

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.