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Molecular Endocrinology, doi:10.1210/me.2003-0362
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Molecular Endocrinology 18 (9): 2333-2343
Copyright © 2004 by The Endocrine Society

Evidence for the Notch Signaling Pathway on the Role of Estrogen in Angiogenesis

R. Soares, G. Balogh, S. Guo, F. Gärtner, J. Russo and F. Schmitt

Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) (R.S., F.G., F.S.); Medical Faculty (R.S., F.S.), University of Porto; and Molecular Pathology Unit (R.S.), Portuguese Institute of Oncology, 4200 Porto, Portugal; Breast Cancer Research Lab (G.B., S.G., J.R.), Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; and Pathology Department (F.G.), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), 4050 Porto, Portugal

Address all correspondence and requests for reprints to: Raquel Soares, MSc, Ph.D., IPATIMUP, R. Roberto Frias s/n, 4200 Porto, Portugal. E-mail: raquel.soares{at}ipatimup.pt.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have investigated the molecular mechanisms involved in 17ß-estradiol-induced angiogenic pathway. We show here that 17ß-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 17ß-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 17ß-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 17ß-estradiol and angiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANGIOGENESIS, THE FORMATION of new blood vessels from preexisting ones, is essential for the tumors to grow and metastasize. Angiogenesis is a complex process involving endothelial cell proliferation, migration, and anastomosis (1). This process requires the coordinated action of growth factors and their receptors, extracellular proteins, adhesion molecules, and proteolytic enzymes (2). This tightly regulated process is therefore likely to be mediated by specific environment conditions. Increasing evidence indicates that estrogens might be one of the mediators of angiogenesis (3, 4, 5, 6, 7). Estrogens regulate a wide range of cell events by activating their specific receptor family of transcription factors. Estrogen receptors (ERs) are known to regulate a huge number of genes affecting vascular function (6). The presence of ER in endothelial cells and smooth muscle cells that constitute the blood vessel wall (6, 8), and the presence of aromatase, an enzyme involved in estrogen synthesis in endothelial cells (6), also point to the fact that estrogens are implicated in angiogenesis and in blood vessel remodeling. The role of estrogens in mediating vascular protective effects was further highlighted by the development of ER-deficient mice (9). Accordingly, ERß knockout mice developed multiple functional vascular abnormalities (10), emphasizing the relevance of estrogen in the vasculature. In endothelial cells, 17ß-estradiol regulates the expression of several genes implicated in vascular functions (8, 11, 12, 13).

We and others (4, 5, 7, 13, 14) have reported that genes known to play a role in angiogenesis, such as vascular endothelial growth factor (VEGF) and TGF{alpha}, are actually up-regulated by 17ß-estradiol. Although estrogen-induced angiogenic growth factors have been postulated to mediate the effect of this hormone in angiogenesis, the exact mechanism is still unknown (6).

Here we identified genes that were transcriptionally affected by 17ß-estradiol treatment in breast cancer cells (MCF7) and in endothelial cells [human umbilical vein endothelial cells (HUVECs)]. We showed that Notch1, together with Jagged1, are up-regulated by 17ß-estradiol. This increase in receptor and ligand expression resulted in the activation of Notch signaling pathway. Interestingly enough, MCF7 xenografted tumors in nude mice presented higher microvessel density and increased Notch expression when exposed to 17ß-estradiol. Moreover, the role of Notch1 in angiogenesis was highlighted by the finding that Notch1 gene expression was required for tubule-like structure formation in endothelial cells and that Notch1 gene expression clustered with hypoxia-inducible factor (HIF)1{alpha} gene in MCF7 cancer cells by hierarchical analysis. In accordance, the expression of this angiogenic factor was up-regulated whenever MCF7 cells were transfected with a full-length or constitutively active Notch1. Altogether these findings imply Notch signaling pathway in the 17ß-estradiol-mediated angiogenic process both in autocrine and in paracrine pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Treatment with 17ß-Estradiol Up-Regulated Notch1 and Jagged1 Gene Expression
Using the ArrayExplorer software (CLONTECH, Palo Alto, CA), we first analyzed gene expression levels in MCF7 cell line culture treated with 17ß-estradiol for 24 h and compared with untreated MCF7 cells. From an array of 1184 genes, 242 genes were up-regulated more than 2-fold after 17ß-estradiol treatment. We also addressed the gene expression profile of MCF7 cells after incubation with ICI182, 780 (ICI) by cDNA arrays. Because the presence of 17ß-estradiol together with ICI would probably interfere with the pattern of genes expressed upon ICI treatment, MCF7 cells were first kept in medium with 10% fetal bovine serum, containing 17ß-estradiol at nanomolar levels. After washing twice with PBS, cells were incubated in serum-free medium in the presence of 10–9M ICI alone, as described in other studies (15). Knowing that the 17ß-estradiol antagonist ICI blocks ER and leads to its degradation (16), we then identified genes among the 242 that were overexpressed by 17ß-estradiol and abrogated by ICI (Table 1Go). Among the most up-regulated genes, gene 1 and gene 4 were involved in nuclear transport (importin {alpha}3 subunit and nuclear pore complex protein 153) (Table 1Go). The other two were TGFß (gene 2) [previously studied by our group (17)] and Jagged1 (gene 3). Jagged1 is a membrane-bound ligand of Notch family members. Notch is a highly conserved family of transmembrane receptors that plays essential roles in cell fate decisions (18). Notch 1 is activated by a series of proteolytic processing steps, which involve its binding to one of the cell surface ligands (e.g. Jagged1), resulting in cleavage of the intracellular domain, translocation to the nucleus, and transcription of target genes (18).


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  		Table 1. Expressed Genes Up-Regulated by 17ß-Estradiol and Down-Regulated by ICI in MCF7 Cells

 
Our interest in Jagged1 up-regulation increased remarkably when we found out that its receptor, Notch1, was more than 8-fold increased by 17ß-estradiol in these cells compared with vehicle-treated cells, although it was not down-regulated by incubation with ICI (Table 2Go). Thus, our findings that 17ß-estradiol up-regulated both Notch1 and its ligand (Jagged1) suggested that this is an important signaling pathway through which 17ß-estradiol will act.


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  		Table 2. Expression of Notch1 and Jagged1 Genes in MCF7 and HUVEC Cell Lines after Treatment with 17ß-Estradiol or ICI

 
Using the same strategy, we investigated the genes that were up-regulated by 17ß-estradiol treatment in HUVECs. As illustrated in Table 2Go, Notch1 and its ligand Jagged1 presented similar patterns of expression in these cells as well because both were significantly up-regulated by 17ß-estradiol in HUVECs. These results were then confirmed by RT-PCR. The increase in Notch1 expression was confirmed in 17ß-estradiol-treated cell lines, when compared with vehicle-treated cells (Fig. 1AGo). Notch1 mRNA expression was also increased in ICI-treated cultures compared with controls, although to a lesser extent, as observed by cDNA array analyses and RT-PCR (Fig. 1AGo). Up-regulation of Jagged1 by 17ß-estradiol was also found in RT-PCR assay from MCF7 and HUVEC cell cultures, whereas ICI treatment led to Jagged1 gene expression identical with control values by RT-PCR in both cell lines (Fig. 1AGo). To examine whether Notch1 protein expression followed the same pattern, lysates from 17ß-estradiol-treated, ICI-treated or ethanol-treated MCF7 cells were obtained and proteins were separated on a SDS-PAGE. Notch1 protein expression was once more induced by the presence of 17ß-estradiol (Fig. 1BGo). Altogether, these findings indicated that 17ß-estradiol mediates Notch1 and Jagged1 gene expression in the two distinct cell lines.



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  		Fig. 1. Expression of Notch1 and Jagged1 Were Analyzed in MCF7 and HUVEC Cells after 17ß-Estradiol and ICI Treatment

A, RT-PCR analysis for Notch1, Jagged1, and GAPDH were carried out by using RNA derived from either vehicle-treated (control), 17ß-estradiol-treated (17ß-estradiol) or ICI-treated (ICI) MCF7 or HUVEC cell cultures. Both Notch1 and its ligand Jagged1 were up-regulated by 17ß-estradiol in the two cell lines. Treatment with ICI decreased Jagged1 expression in MCF7 and HUVEC cells, confirming cDNA array results. Bars correspond to intensity ratios of Notch1 and Jagged1 gene expression after normalization to GAPDH. Results are representative of three independent experiments. Statistically significant differences in both Notch1 and Jagged1 gene expression were found between 17ß-estradiol-treated and vehicle-treated MCF7 and HUVEC cells (*, P < 0.001). No significant difference in Notch1 expression was observed between 17ß-estradiol-treated and ICI-treated cultures in both cell lines. B, Immunoblotting for Notch1 was performed in MCF7 cells. Notch1 protein was up-regulated in 17ß-estradiol stimulated MCF7 cells and down-regulated by ICI incubation. Control, Proteins from ethanol-treated cells; 17ß-estradiol, proteins from 17ß-estradiol-treated cells; ICI, proteins from ICI-treated cells.

 
The current findings showed that 17ß-estradiol up-regulated Notch1 at 24-h treatment. Thus, we hypothesized that this gene activation could be a secondary event because 17ß-estradiol can regulate immediate early genes in a short period of time (19). We sought, therefore, to analyze whether Notch1 could be an early gene up-regulated by 17ß-estradiol. To address this question, a time-course evaluation of Notch1 expression was performed at short periods of time (1, 3, and 6 h) by RT-PCR in MCF7 cells (Fig. 2Go). A difference in Notch1 expression between MCF7 control cells and 17ß-estradiol-treated MCF7 cells was observed during the whole experiment, indicating that Notch1 expression is up-regulated already in the first hours after 17ß-estradiol exposure. This up-regulation was maintained until 24 h after incubation as observed in Fig 1Go.



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  		Fig. 2. Notch1 Gene Expression Was Evaluated in a Time-Course Assay by RT-PCR in MCF7 Cells

Bars correspond to intensity ratios obtained from Notch1 mRNA levels normalized to GAPDH at different incubation times. Notch1 expression was significantly increased in 17ß-estradiol-treated MCF7 cells during the whole experiment (*, P = 0.01). C, MCF7 cells incubated with vehicle; E, MCF7 cells incubated with 17ß-estradiol.

 
Because the above observations suggested that 17ß-estradiol up-regulated both Notch1 and Jagged1 genes, we next looked for estrogen-responsive elements (EREs) in these genes, using the Swiss-Prot Database. Because the 5'-flanking region of a gene is the most common location of responsive elements for steroid receptors, we searched for EREs in the 3000 bp located in the 5'-flanking region of Notch1 and of Jagged1 genes. Because only a few natural genes contain perfect palindromic EREs (GGTCAnnnTGACC), we looked for 13-bp sequences with strong homology to this consensus ERE. Two imperfect EREs were found in the 5'-flanking region of Notch1 gene: GCTCACCCTGACA (N1a), possessing one base change in each half side relative to the consensus element, and GGGCGGGGGGACC (N1b), with two and one base changes respectively, in each half side relative to the consensus ERE. The same approach identified three imperfect EREs in the 5'-untranslated region of Jagged1 gene: GTTCATACTGAGC (J1a), with one base changed in each half side, and GGGCACATTGAAA (J1b) and GCCCAGGGTGAGC (J1c), presenting one and two base changes in each side, respectively. All of these 13-nucleotide sequences were previously described to be imperfect EREs in other genes (7, 20). We performed luciferase reporter constructs with these putative ERE and measured estrogen responsiveness in transfected MCF7 cells by evaluating expressed luciferase activity. As illustrated in Fig. 3Go, luciferase activity was nearly 3-fold increased by 17ß-estradiol in J1b imperfect ERE but not in cells transfected with any of the other constructs, indicating that Jagged1 gene could be up-regulated by 17ß-estradiol through the activation of this imperfect ERE.



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  		Fig. 3. Role of 17ß-Estradiol on Activation of Imperfect EREs Found in the 5'-Flanking Region of Notch1 and of Jagged1 Was Assessed by Luciferase Assay

MCF7 cells were transfected with luciferase reporter constructs containing N1a, N1b, J1a, J1b, J1c imperfect EREs, and incubated with ethanol (control) or 10–9 M 17ß-estradiol afterward. Reporter gene transactivation was evaluated 2 d after transfection. Luciferase activity induction was measured in 17ß-estradiol-treated transfectants relative to ethanol (control) ones. Incubation with 17ß-estradiol resulted in a statistically significant increase in luciferase activity in J1b-transfected cells (*, P < 0.05). No other significant differences were obtained between 17ß-estradiol-treated vs. vehicle-treated transfected cells. Bars represent mean values of three distinct assays. Error bars represent SD. Experiments were performed in triplicate.

 
Activation of Notch Signaling Pathway by 17ß-Estradiol
To analyze whether or not 17ß-estradiol-induced Notch1/Jagged1 genes resulted in activation of Notch signaling pathway, the receptor activation was assessed with a luciferase reporter assay that responds to the transcriptional activation of RBP-J{kappa}, a downstream component of Notch signaling. Incubation of MCF7 cells with 17ß-estradiol significantly increased Notch reporter expression greater than 2-fold relative to that in ethanol-treated MCF7 cells transfected with the reporter (vehicle), indicating that 17ß-estradiol enhanced Notch signaling (P < 0.05) (Fig. 4AGo). In contrast, ICI treatment did not show any effect on Notch1 activity (Fig. 4AGo).



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  		Fig. 4. Role of 17ß-Estradiol on Notch Signaling Was Assessed by a Luciferase Assay Using Reporter Constructs Containing the RBP-Jk Binding Sites

A, The reporter construct was transfected into a MCF7 cells incubated with ethanol (control), 10–9 M 17ß-estradiol or 10–9 M ICI afterward. Reporter gene transactivation was measured 2 d post transfection. Fold induction of luciferase activity was measured relative to that in MCF7 cells transfected with an empty vector (pGA50–7). *, Differences were statistically significant (P < 0.05). B, Full-length Jagged1 (FLJ1) was transiently expressed in MCF7 cells and then cocultured with MCF7 cells transiently expressing Notch luciferase reporter gene. Cocultures were then incubated with 10–9 M 17ß-estradiol, 10–9 M ICI or ethanol (vehicle). Luciferase activity was measured 1 d after coculturing. Negative controls were performed by transfecting cells with an empty vector (pLNX) incubated with 17ß-estradiol. Bars represent mean values of three distinct assays. Error bars represent SD. Experiments were performed in triplicate. **, Difference between 17ß-estradiol-treated and vehicle-treated samples were statistically significant (P = 0.001).

 
Our former results showed that 17ß-estradiol also up-regulated Jagged1 gene expression. We thus determined whether 17ß-estradiol could promote ligand-induced activity of Notch signaling. Full-length Jagged1 (FLJ1)-expressing MCF7 cells were cocultured with Notch reporter gene-expressing MCF7 cells 24 h after transfections. The expression of Jagged1 (N1/J1-ethanol) led to a 0.3-fold increase in the reporter expression relative to that in control cocultures (N1/–) (Fig. 4BGo). Whenever FLJ1-expressing cells were cocultured with Notch reporter-transfected MCF7 cells in the presence of 17ß-estradiol, a 45-fold increase was observed in the reporter expression. A statistically significant difference between 17ß-estradiol- and vehicle-treated cocultures was obtained (P = 0.001). ICI incubation of these cocultures resulted in low levels of luciferase activity, comparable to controls. These findings showed that 17ß-estradiol promoted ligand-induced Notch signaling.

17ß-Estradiol Promoted Angiogenesis and Induced Notch1 Gene Up-Regulation in MCF7 Tumors
To elucidate the role of 17ß-estradiol in tumor progression, we first analyzed the angiogenic index of MCF7 tumors inoculated in nude mice in an estrogen environment and compared it with placebo-treated (control) mice. Tumor size was monitored weekly until each tumor reached 100 mm3. Tumors in 17ß-estradiol-treated mice proliferated rapidly. In contrast, tumors from placebo-treated mice grew slower, reaching 100 mm3 nearly 4 wk later (Fig. 5AGo), indicative of the 17ß-estradiol role in tumor proliferation. Angiogenesis was evaluated by the immunostaining of tumor vessels using an antibody against CD31 (Fig. 5BGo). Stained vessels were counted in the three most vascularized areas within the tumor. The MCF7 tumors that were exposed to 17ß-estradiol presented a higher microvessel (83.8 ± 7.5) than control tumors, treated with placebo pellet (37.2 ± 9.6). In agreement with previous studies from our group (3, 4, 5), these findings suggested that angiogenesis was induced by the presence of 17ß-estradiol in MCF7 xenografted tumors.



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  		Fig. 5. MCF7 Cells Were Inoculated in the Mammary Fat Pad of 17ß-Estradiol-Treated and Placebo-Treated Nude Mice

A, Tumors grew faster in 17ß-estradiol-treated mice than in placebo-treated mice. B, Mice administered with 17ß-estradiol pellet presented higher number of immunostained microvessels than placebo-treated mice. C, Notch1 expression was analyzed by RT-PCR in MCF7 xenografted tumors. Graph shows increased Notch1 transcript after 17ß-estradiol-treated tumors. Bars refer to Notch1 intensity values normalized to ß-actin. D, Western blotting confirmed the up-regulation of Notch1 gene expression in 17ß-estradiol-treated MCF7 tumors. A representative Western blotting is shown. ß-Actin was used to loading normalization.

 
To elucidate the putative role of Notch1 gene in this cross talk between estrogens and angiogenesis, Notch1 gene expression were next evaluated in the xenografted tumors. As illustrated in Fig. 5Go, C and D, Notch1 transcript and protein were up-regulated in the mice previously treated with 17ß-estradiol. These results confirm the up-regulation of Notch gene expression by 17ß-estradiol in vivo, and suggest a role for this pathway in tumor angiogenesis.

Notch1 Signaling Pathway Activation Resulted in Vessel Assembly
To confirm the role of Notch1 signaling in angiogenesis, we transfected bovine capillary endothelial cells (CRL8659) with full-length Notch1 (FLN1), dominant-negative Notch1 (DNN1), an activated form of Notch1 gene (CAN1), or a full-length Jagged1 insert (FLJ1) and addressed vessel assembly in GFR (growth factor reduced)-Matrigel-coated plates, using a Matrigel (BD Biosciences, San Jose, CA) assay. pSU-ß-Galactosidase revealed a transfection efficiency of more than 47% of CRL8659 endothelial cells. Identical findings were obtained at the three independent transfection experiments. We also analyzed Notch1 protein expression in the transfected cell cultures by Western blotting (Fig. 6AGo), showing that Notch1 protein expression levels were increased in FLN1, DNN1, and CAN1, but not in insert-less plasmid-transfected cells. The presence of a functional Notch1 gene led to cord structure formation on GFR-Matrigel assay (Fig. 6BGo). A statistically significant difference in the number of tubule-like structures was found in cultures of CAN1 (P = 0.001), and in cocultures of FLN1 + FLJ1 transfectants. These structures were significantly enhanced whenever cocultures were incubated with 17ß-estradiol (P < 0.05), indicating the role of 17ß-estradiol in stimulating vessel assembly through Jagged1/Notch1 signaling pathway activation.



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  		Fig. 6. Notch Signaling Pathway Activation in Endothelial Cells Resulted in Tube Formation in In Vitro

A, Western blotting for Notch1 was performed in CRL8659 endothelial cells after transfection with FLN1, DNN1, CAN1, and insert-less plasmid (control), confirming that these cells were efficiently transfected. ßActin was used as loading control. B, Semiquantification of the tube formation index in CRL8659 cells were grown on GFR-Matrigel after transfection with FLN1, DNN1, CAN1, an empty plasmid (Control) or in FLN1-transfectant CRL8659 cells cocultured with FLJ1 CRL8659 cells (FLN1 + FLJ1). CRL8659 cell cocultures were incubated with 17ß-estradiol (FLN1 + FLJ1 + E2). E2, 17ß-Estradiol. Bars correspond to mean of the number of tubule-like structures. Error bars represent SD between different wells. **, Statistically significant difference was found between CAN1-expressing cells and control (P = 0.001). *, FLN1 + FLJ1+ E2 cultures presented a significant increase in tube formation relative to FLN1 + FLJ1 transfected cultures (P < 0.05). Measurements were performed from a representative experiment repeated three times.

 
Genes Clustered with Notch1 in MCF7 Cells
According to our former findings, Notch1 was also present in tumor MCF7 cells. This prompted us to hypothesize that Notch1 gene expression in the tumor cells could also be implicated in angiogenesis in a paracrine manner. Examining the difference in gene expression profile between 17ß-estradiol-treated, ICI-treated and control MCF7 cells by cDNA array, we observed that Notch1 gene clustered with several other genes. Many of these expressed genes encode nuclear repair and cell cycle proteins (p38–2G4, cdkN1A, cdk regulatory subunit, cdc27Hs, DNA glycosylase, GADD153, Rad23A, Rad51C, jun binding protein, HMG17, RbBP4), suggesting an enhanced proliferative activity. Notch1 also associated with several signaling transduction pathways (p21-rac1, tumor necrosis factor receptor-associated protein 1, bone morphogenetic protein receptor-II, hepatocyte-derived growth factor, and {gamma}-interferon) and transcription factors such as HIF1{alpha} and early growth response {alpha} (Table 3Go). These findings point to the fact that Notch1 up-regulation could be accompanied by a very intricate complexity of pathways in 17ß-estradiol-induced cells.


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  		Table 3. Comparative Differential Gene Expression Analysis of 17ß-Estradiol or ICI-Treated MCF7 Cells Versus Controls

 
One of the genes found to be clustered in MCF7 cells was HIF1{alpha}, a transcription factor up-regulated during hypoxia that stimulates transcription of several angiogenic genes. To address the question whether Notch1 expression resulted in HIF1{alpha} gene up-regulation, we transiently transfected MCF7 cells with DNN1, FLN1, or CAN1 constructs and analyzed the expression of this gene by Western blotting. Consistent with clustering data, HIF1{alpha} gene was overexpressed in cultures with functioning (FLN1) or activated (CAN1) Notch1 gene, whereas a decrease in HIF1{alpha} expression was found in the dominant-negative form (Fig. 7Go). The above observations indicate that HIF1{alpha} could be a putative target gene of activated Notch1 pathway, emphasizing therefore that Notch1 may mediate angiogenesis in a paracrine way as well, through the up-regulation of HIF1{alpha} in tumor cells.



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  		Fig. 7. Notch1 Expression Induces Up-Regulation of HIF1{alpha}

Western blotting analysis was conducted in MCF7 cells transfected with DNN1, FLN1, or CAN1 plasmids to evaluate expression of HIF1{alpha}. Proteins were extracted from MCF7 cells transfected with each plasmid and separated in a 10% PCR. The presence of functional Notch1 up-regulated HIF1{alpha} protein. Insert-less MCF7 transfectants were used as negative controls. ß-Actin was used for loading control. Immunoblots are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although an increasing amount of growth factors have been associated with 17ß-estradiol-induced angiogenic phenotype, little is known regarding the mechanisms involved. Here, we demonstrated that 17ß-estradiol induced up-regulation of Notch1 gene in MCF7 and in HUVEC cells, using cDNA array analyses and RT-PCR. Consistently, Notch1 protein followed the same increased expression pattern found in RNA in MCF7 cells. The fact that Notch1 ligand (Jagged1) was also up-regulated by 17ß-estradiol incubation provided evidence that Notch1 signaling is a relevant pathway promoted by 17ß-estradiol in these two cell types. Estrogen-induced Notch1 up-regulation was already found at 1 h time point after 17ß-estradiol incubation. The finding that Notch1 is an early response gene upon 17ß-estradiol exposure in MCF7 cells further emphasizes the relevance of this gene in estrogenic response. In agreement with these data, we found that an imperfect ERE in the 5' flanking region of Jagged1 was able to activate Jagged1 gene transcription in the presence of 17ß-estradiol, emphasizing that this gene can be up-regulated by 17ß-estradiol. Inversely, no up-regulation of Notch1 gene transcription by 17ß-estradiol was found through the two imperfect ERE tested, implying that 17ß-estradiol-induced Notch1 up-regulation probably occurs in an ERE-independent manner. These findings are in accordance with the fact that ICI was able to down-regulate Jagged1 but not Notch1 gene expression as shown in our previous experiment, suggesting that 17ß-estradiol could activate distinct regulatory mechanisms in the two genes. Estrogens are known to confer a complex regulation of target genes transcription. Hyder and collaborators (7) identified several EREs in VEGF flanking regions, which behaved differently in the presence of the pure estrogen antagonist, ICI 182,780.

We also found that up-regulation of these genes by 17ß-estradiol actually resulted in activation of the Notch1 signaling cascade, as evidenced by the luciferase reporter gene activity (Fig. 4Go). Quite remarkably, ICI did not appreciably change Notch1 signaling activity, despite its effect on Notch1 transcript up-regulation. These findings, together with the fact that Jagged1 expression strongly potentiated 17ß-estradiol-induced Notch1 signaling activity, led to the assumption that 17ß-estradiol promoted activation of the Notch1 downstream signaling in a ligand-dependent manner.

Altogether, the current data indicate that Notch1 signaling might be a relevant pathway in both cell types. Previous studies have reported that aberrant Notch signaling is implicated in many cancers (21, 22, 23, 24). One of the first evidences came from a subset of T-cell acute lymphoblastic leukemia characterized by chromosomal translocations that comprise Notch1 locus (21). Overexpression of Notch pathway components has also been found in several other cancers, such as renal cell carcinoma (22), head and neck squamous cell carcinoma (23), neuroblastoma (24), and endometrial cancer (25). Moreover, Notch nuclear localization was detected in cervical carcinomas (26) by immunohistochemical techniques, showing its nuclear activity. Murine mammary tumorigenesis showed that Notch1 signaling might play a role in human breast cancer. Expression of Notch1 and Notch4 was found in ductal carcinoma in situ of the breast (27). However, the mechanism by which Notch signaling contributes to tumor progression remains unknown. Unlike DNA repair genes or cell cycle machinery, aberrant Notch signaling does not obviously cause unregulated cell proliferation or genetic instability. In Drosophila and vertebrate embryogenesis, only a few cells are able to differentiate (27). This is accomplished by Notch1 activity, which prevents neighboring cells to adopt this undifferentiated fate. In agreement, most of the reports on Notch signaling in tumors implicate this pathway in triggering an undifferentiated tumor stage with increased proliferative activity (27). In our clustering data, expression of several genes involved in cell cycle and proliferation was associated with Notch expression, implying a proliferative role for this gene in tumor cells. Therefore, evaluation of the role of these clustered genes in future studies is essential to further determine the complex role of Notch1 signaling during tumorigenesis.

The scope of this study was focused, however, in the role of Notch1 in the cross talk between estrogens and angiogenesis. Dysfunction of Jagged/Notch1 signaling pathway has been associated with human pathologies involving cardiovascular abnormalities (28). Notch1 mutant embryos displayed severe defects in angiogenic remodeling (29). Another important evidence that Notch signaling is involved in vascular disorders was elucidated by the finding of Notch3 mutants in CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a disorder characterized by arteriopathy of cerebral arterioles (30). Recently, it was reported that Notch pathway regulates cell-cell or cell-matrix interaction, contributing, hence, to cell migration in situations of tissue remodeling (31). Notch1- and Jagged1-expressing cells or a constitutive activated form of Notch1 resulted in differentiation of endothelial cells into vessel-like structures in the present study. These observations were in total agreement with the ones from Uyttendaele et al. (32), where activated Jagged1 or Notch4 genes promoted vessel assembly. More importantly, tubule-like structures were further enhanced whenever Notch1/Jagged1 expressing cells were incubated with 17ß-estradiol (Fig. 6BGo), indicating that Notch1 pathway might be implicated in 17ß- estradiol regulation of angiogenesis. The present study also addressed the role of Notch1 signaling in tumorigenesis by evaluating its role in breast tumor cells. Up-regulation of Notch1 gene was found in MCF7 murine xenografts with higher microvessel counts, suggesting a role of Notch signaling in promoting tumor angiogenesis.

An interesting observation was that Notch1 gene clustered with HIF1{alpha}. Notch1 signaling pathway must obviously act in concert with other signaling pathways implicated in angiogenesis. HIF1{alpha} is a transcription factor stabilized during hypoxia that activates several angiogenic genes that act in a paracrine manner in endothelium. In a recent report, Jögi et al. (33), demonstrated that hypoxia induced complex changes in gene expression pattern of neuroblastomas, namely increasing Notch1 expression. Accordingly, the present study showed that the expression of either an active form (CAN1) or a full-length (FLN1) form of Notch1 resulted in the up-regulation of HIF1{alpha} protein expression. The induction of HIF1{alpha} transcript by FLN1-transfected MCF7 cells is remarkable, given that FLN1 is probably inactive in the absence of the ligand. We know, however, that MCF7 cells express Jagged1. Therefore, the baseline levels of Jagged1 could trigger Notch1 downstream effects. In addition, Notch transformation can be dependent on gene transcription activation through the ankyrin repeats of Notch1 protein instead. This ankyrin-dependent activation has been observed in a few cells, including human breast cancer cells (34), and is known to require the interaction of Deltex, a cytoplasmic protein, although no ligand binding was required (34). According to this, futures studies addressing whether HIF1{alpha} gene transcription by Notch1 depends on the presence of Jagged1 ligand or not are required.

In conclusion, this study identified factors activated by 17ß-estradiol, which might play important roles in tumor progression, namely in the cross talk between tumor cell growth and angiogenesis. In vivo studies aiming at blocking Notch1 signaling are required to address the relevance of this pathway in 17ß-estradiol-induced tumor progression. Nevertheless, our findings provide evidence for the first time that Jagged1/Notch1 signaling pathway can be mediated by 17ß-estradiol. The current study focused on the involvement of Notch1 signaling in tumor angiogenesis, demonstrating that the 17ß-estradiol-induced Notch1 signaling activation associated with angiogenic factors. Because the presence of 17ß-estradiol promoted increased microvessel density in vivo, the current study also suggested that Notch1 can be implicated in driving tumors toward an angiogenic phenotype. Understanding how Notch1 signaling modulates these processes may provide new therapeutic strategies for human cancers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and In vitro Studies
The hormone-dependent human breast cancer cell line MCF7 (ATCC, Barcelona, Spain) was maintained in Eagle MEM (Invitrogen Life Technologies, Paisley, Scotland, UK). HUVECs, harvested in 0.1% collagen-coated flasks were cultured in F12 K medium and supplemented with endothelial cell growth supplement (Sigma-Aldrich Quimica, Portugal). Bovine adrenal medulla capillary endothelial cells (CRL8659, ATCC) were grown in RPMI medium (Invitrogen Life Technologies). Every cell culture media were supplemented with 10% inactivated fetal bovine serum (Invitrogen Life Technologies) and 1% penicillin/streptomicin (Invitrogen Life Technologies).

Before treatments cultures were serum-starved for 16 h. Cell lines were then treated with 10–9 M 17ß-estradiol (Sigma-Aldrich Quimica) or 10–9 M ICI 182, 780 for 24 h as described for each experiment. Concentrations of 17ß-estradiol and ICI were established from previous viability studies, from a set of five different concentrations, ranging from 10–11 M to 10–7 M, and assessed by the trypan blue exclusion assay (35). Controls were obtained using cells incubated with ethanol (vehicle) for the same period of time.

In vivo Studies
The animal experimentation was conducted according to the accepted standards of humane animal care, namely the National Institutes of Health (NIH) animal guidelines. Ten female nude mice [N: NIH (s) II strain], 4–6 wk old, were housed in a pathogen-free environment under control conditions of light and humidity. Five mice were administered (sc) with a 17ß-estradiol pellet and the other five with a control (placebo) pellet 24 h before cell inoculation. Mice were then inoculated with 5 x 107 MCF7 cells in the mammary fat pad. Tumor growth was monitored weekly, and tumors were removed whenever tumor size reached 100 mm3. Xenografted tumors were then divided in two fragments: one was formalin fixed and paraffin embedded for immunohistochemistry analysis, and the other was frozen in liquid nitrogen and used for RNA and protein isolation.

Immunohistochemistry Analysis
Immunohistochemistry was performed in formalin fixed, paraffin-embedded xenografted MCF7 tumors. Avidin-biotin-peroxidase complex method was used for CD31 (Novocastra, Newcastle upon Tyne, UK) immunostaining. Briefly, sections were cut from paraffin blocks at 4 µm slide thickness, dewaxed, and hydrated. CD31 immunostaining was preceded by pepsin digestion at room temperature for 30 min. Negative control for immunostaining was carried out by omission of the primary antibody. As positive controls, sections from breast cancer known to express CD31 were used. Any positive single cell or cluster of cells stained clearly separated from adjacent clusters and background, with or without lumen, was considered an individual vessel. Microvessels were counted in the three most vascularized areas in a x200 field (0.74 mm2) by four observers simultaneously (1).

cDNA Array Analyses
Total RNA obtained from MCF7 and HUVEC cell cultures after 24 h incubation with either ethanol (vehicle), 10–9 M 17ß-estradiol, or 10–9 M ICI was isolated using the TriPure method (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. The Atlas Human Cancer 1.2 Arrays (CLONTECH) were assayed as previously described (35, 36). Arrays from ethanol-treated (control), 17ß-estradiol-treated and ICI-treated cells were compared. Mean values of intensity for each spot detected from multiple arrays were generated by the computer software. Comparative analysis was performed with ImaGene 4.2 image analysis software from Biodiscovery, Inc. (El Segundo, CA). Comparative differential gene expression analysis of 17ß-estradiol or ICI-treated cells vs. control was performed using GeneSight 5.0 software (Biodiscovery) and the specific statistical study in base log2. We considered the gene expression up and down-modulated more and less than 2-fold with P < 0.0005. The differentially expressed genes demonstrate a broad range of functional activity.

RT-PCR Analyses
RT-PCR was performed to confirm the results obtained by cDNA array hybridization in MCF7 and HUVEC cultures using the OneStep RT-PCR QIAGEN (Valencia, CA) kit. For each sample, 1.0 µg of RNA was reverse transcribed in a reaction volume of 25 µl in the presence of 10 mM deoxynucleotide triphosphate and 2 µl RT-PCR enzyme mix. Gene-specific primers for Notch1 (forward, gcc gac aaa aca ccc aaa c; reverse, aga act aca agc cct cag ac) or jagged1 (forward, ata cgg gat gat ggg aac c; reverse, cca agc cac agt taa gac ag), coamplified with a set of primers for the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used in RT-PCR. Quantifications were done in triplicate. The PCR products were separated on ethidium bromide-stained 2% agarose gel. The intensity of the fluorescence was automatically measured and integrated using genescan software (Image Master, Pharmacia, Lisbon, Portugal).

Luciferase Reporter Assay
Transient transfections were performed in MCF7 cells by Lipofectamine (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). To assess Notch signaling, MCF7 cells (1 x 105 cells), cultured 1 d earlier in 24-well culture plates, were transfected in triplicate with 0.4 µg of luciferase vector (pGA981-6) (37, 38). Cells were then incubated with 10–9 M 17ß-estradiol, 10–9 M ICI, or vehicle (ethanol). To determine 17ß-estradiol effects on ligand-induced Notch signaling, coculture assays were performed using MCF7 cell line. MCF7 cells (3 x 106) seeded 1 d earlier in 10-cm culture plates, were transfected with 8 µg pGA981–6 or with either 20 µg of full-length Jagged1 (FLJ1) or 20 µg of the empty vector (pLNCX). One day after transfection, pGA981-6-transfected MCF7 cells were cocultured in triplicate with FLJ1 or pLNCX transfected cells on 24-well plates for 24 h and incubated with 10–9 M 17ß-estradiol, 10–9 M ICI or vehicle (ethanol). An insert-lacking luciferase vector plasmid (pGA50-7) was used as negative control for luciferase assays. A similar approach was used to test the function of imperfect EREs identified in the 5'-flanking region of Notch1 (N1a and N1b) and Jagged1 (J1a, J1b, and J1c). MCF7 cells (1 x 105) seeded 1 d earlier in 24-well plates were transfected with luciferase reporter constructs containing each of these putative EREs or with an insert-less luciferase reporter plasmid. Transfected cells were incubated either with vehicle or 10–9M 17ß-estradiol 24 h after transfection. Experiments were performed in triplicate and repeated three times. Luciferase activity was determined 2 d post transfection using an enhanced luciferase assay kit (BD Biosciences) in a scintillation counter (Amersham Biosciences).

Transfection Studies
For transient transfection, MCF7 cells (1 x 105) or CRL8659 cells (1 x 105) were cultured on 24-well plates 1 d before transfection. For each well, 2.5 µg of each plasmid DNA (FLN1, DNN1, CAN1, insert-less plasmid-control, and FLJ1) were transfected using Lipofectamine reagent (Amersham Biosciences). Experiments were done in triplicate. To establish transfection efficiency, 20 ng per well of pSU-ß-Galactosidase control vector (VWR International, Lisbon, Portugal) was included with each transient transfection and measured on cell lysates by colorimetric analysis.

Matrigel Assay and Semiquantification Analysis
One day after transfection, bovine endothelial CRL8659 cells (1 x 105) were cultured on growth factor reduced-Matrigel-coated plates, and a Matrigel assay was performed as previously described (35, 39). The plates were photographed on an inverted phase-contrast microscope 24 h after incubations (Nikon, Kingston upon Thames, UK). Experiments were performed in triplicate.

A semiquantitative measurement of cord formation in GFR-Matrigel cultured CRL8659 cells was developed (tube formation index). Comparisons among CRL8659 cell cultures plated simultaneously with different transfectants were performed. Micrographs of four fields representative of each culture were obtained in a low magnification (x40), comprising 25% of the total surface area of the well and were representative of the conditions throughout the well. The numbers of cord-like structures were then measured in each field by two observers simultaneously. Each cord portion between the ramifications was considered one cord unit. Mean values were obtained by evaluating the four fields of each of the three wells under the same treatment.

Western Blotting Analysis
Two days after transfection, MCF7 cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100). Lysates were centrifuged at 14,000 rpm for 5 min, and protein content was quantified by spectrophotometry using BSA as a standard. Equal amounts of protein were subjected to 10% SDS-PAGE. The same procedure was used to evaluate Notch1 expression in 17ß-estradiol-treated, ICI-treated or ethanol-treated (vehicle) MCF7 cells as well as in MCF7 tumors by Western blotting. CRL8659 bovine endothelial cells were also tested for Notch1 expression 24 h after transfection with FLN1, DNN1, CAN1, and insert-less plasmid. Antibodies against Notch, HIF1{alpha}, and ß-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Bands were visualized by chemiluminescence (Amersham Biosciences, ECL kit).

Statistical Analysis
All experiments were performed in triplicate with reproducible results; quantifications are expressed as mean ± SD. Samples were evaluated by the ANOVA test.


   ACKNOWLEDGMENTS
 
The authors are grateful to Lothar Strobl (Cologne University, Cologne, Germany) for generously providing Notch reporter constructs and Sergio Dias (CIPM, Lisbon, Portugal) for the Notch1 and Jagged1 plasmids. We would also like to thank George Lam and Carla Costa (Cornell University, New York, NY) for providing the HUVEC cell line, Mrs. Conceição Magalhães for her helpful assistance in the animal house, and Astra Zeneca for the ICI 182, 780 estrogen antagonist.


   FOOTNOTES
 
This work was supported by grants from Liga Portuguesa Contra o Cancro (Portugal) and Department of Defense Breast Cancer Research Program Grant DAMD-17-00-1-0249.

Abbreviations: CAN1, Activated form of Notch1 gene; DNN1, dominant-negative Notch1; ER, estrogen receptor; ERE, estrogen-responsive element; FLJ1, full-length Jagged1 insert; FLN1, full-length Notch1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFR, Matrigel growth factor reduced-Matrigel; HIF, hypoxia-inducible factor; HUVEC, human umbilical vein endothelial cells; ICI, ICI182, 780; VEGF, vascular endothelial growth factor.

Received for publication September 17, 2003. Accepted for publication June 3, 2004.


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 DISCUSSION
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