help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1394
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kazi, A. A.
Right arrow Articles by Koos, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kazi, A. A.
Right arrow Articles by Koos, R. D.
Endocrinology Vol. 148, No. 5 2363-2374
Copyright © 2007 by The Endocrine Society

Estrogen-Induced Activation of Hypoxia-Inducible Factor-1{alpha}, Vascular Endothelial Growth Factor Expression, and Edema in the Uterus Are Mediated by the Phosphatidylinositol 3-Kinase/Akt Pathway

Armina A. Kazi and Robert D. Koos

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Robert D. Koos, Ph.D., Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201-1559. E-mail: rkoos{at}umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) plays an essential role in normal uterine physiology and function as well as endometrial cancer and other uterine disorders. Recently we showed that estrogen regulation of VEGF expression in the rat uterus involves rapid recruitment of both estrogen receptor (ER)-{alpha} and hypoxia-inducible factor (HIF)-1{alpha} to the VEGF promoter. Estrogen is known to stimulate both the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways, which have been linked to the activation of both of these transcription factors. Therefore, the involvement of these pathways in estrogen-induced VEGF expression was investigated. Inhibitors of the MAPK (U0126) or PI3K pathways (wortmannin or LY294002) were administered ip to immature female rats 1 h before 17ß-estradiol (E2) treatment. E2 activation of both pathways occurred and was completely inhibited by the appropriate antagonist. Only PI3K inhibitors, however, blocked E2 stimulation of VEGF mRNA expression and E2-induced uterine edema. In vivo chromatin immunoprecipitation analysis showed that this was associated with a failure of both HIF-1{alpha} and ER{alpha} to bind to the VEGF promoter. To determine whether inhibiting the PI3K pathway affected ER{alpha} induction of other estrogen target genes, the expression of creatine kinase B and progesterone receptor A/B was also examined. The expression of each was also inhibited by wortmannin, as was ER{alpha} binding to the creatine kinase B promoter. In conclusion, although estrogen activates both the MAPK and PI3K pathways in the rat uterus, activation of HIF-1{alpha} and ER{alpha}, and therefore regulation of VEGF gene expression is dependent only on the PI3K/Akt pathway. Furthermore, activation of the PI3K pathway appears to be a common requirement for the expression of estrogen-induced genes. These findings not only shed light on estrogen action in normal target tissues but also have important implications for cancer biology because excessive PI3K, HIF-1{alpha}, and VEGF activity are common in estrogen-dependent tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASCULAR ENDOTHELIAL GROWTH factor (VEGF) plays a central role in the induction of increased microvascular permeability and angiogenesis (1), processes that are absolutely essential for both normal development and reproduction but that also contribute to the progression of a wide range of pathological conditions, most notably the growth and spread of tumors. In the uterus, VEGF expression, and the edema that follows, is essential for the cyclic growth of the endometrium and for implantation (2, 3, 4). Estrogen [17ß-estradiol (E2)] rapidly induces a robust increase in VEGF expression in the rodent uterus (5, 6), primarily in endometrial epithelial cells (7, 8, 9). Recently we showed that this involves the recruitment of both estrogen receptor (ER)-{alpha} and hypoxia-inducible factor 1 (HIF-1) to the VEGF promoter (10). HIF-1 is a heterodimeric transcription factor consisting of an inducible {alpha}-subunit and a constitutively expressed ß-subunit, also known as aryl hydrocarbon receptor nuclear translocator, which is best known for regulating the expression of VEGF and a wide range of other genes in response to hypoxia (11). HIF-1 has recently been shown to also mediate the induction of VEGF expression in a variety of cell types by a number of other hormones, growth factors, and cytokines under nonhypoxic conditions (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Basal levels of HIF-1{alpha} are present in normoxic tissues (23), including the uterus (10). This raises the question of how estrogen induces the rapid activation of this basal pool of HIF-1{alpha} and its recruitment to the VEGF promoter in endometrial epithelial cells.

HIF-1{alpha} activation by nonhypoxic stimuli has been linked primarily to the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and less frequently to the MAPK pathway (24, 25, 26, 27). A rapidly growing body of evidence indicates that estrogen activates these same two cytoplasmic signaling pathways in target cells (28, 29). In endothelial cells, for example, ER{alpha} has been reported to interact directly with the p85{alpha} regulatory subunit of PI3K (30). This appears to involve a small subset of membrane-associated ERs (31) and the formation of a complex that also includes c-Src kinase (32). This interaction leads to activation of Akt, increased endothelial nitric oxide synthase (eNOS) expression, and synthesis of the vasodilator nitric oxide (NO). Estrogen also induces eNOS expression in the rat uterus (33) and in cultured human endometrial epithelial cells (34). The coinduction of nitric oxide synthase and VEGF by estrogen provides the two factors necessary for maximum exudation of fluid from the endometrial microvasculature: 1) a vasodilator, NO, that increases flow through microvessels and 2) a permeabilizing agent, VEGF, that makes those vessels highly porous (2, 4, 35, 36). Estrogen also induces the activity of PI3K/Akt in both primary rodent neurons (37, 38) and ER-positive human breast cancer cells (39, 40, 41, 42, 43, 44). As in endothelial cells, this appears to involve direct ER{alpha} binding to the p85{alpha} PI3K subunit (41) and c-Src (39), perhaps via the adaptor protein p130Cas (45). Estrogen activation of PI3K/Akt is also observed in the rat and mouse endometrium, specifically in the epithelial cells, in vivo (46, 47, 48, 49). Phospho-Akt levels are also greatest in human endometrial glandular epithelial cells during the late proliferative phase, when estrogen is highest (50).

Despite the growing evidence that estrogen activates PI3K/Akt and other cytoplasmic signaling pathways in target cells, the functional significance of this relative to estrogen’s better-known nuclear ER{alpha}-mediated effects is not well understood. In addition to the induction of eNOS discussed above, in vitro studies link PI3K/Akt activation to estrogen stimulation of cell proliferation. For example, inhibiting either PI3K or Akt activity, or inhibiting p130Cas expression with small interfering RNA, blocks estrogen-induced cyclin D1 expression in breast cancer cells (39, 45). Even more significantly, activation of PI3K/Akt was recently linked to estrogen-induced endometrial epithelial cell proliferation in the mouse uterus (49). Estrogen activation of the PI3K/Akt pathway has also been shown to protect cultured neurons from glutamate-induced apoptosis (37) and irradiation-induced apoptosis of ER{alpha}-expressing breast cancer cells (43).

Here we demonstrate for the first time that PI3K/Akt also mediates estrogen induction of VEGF expression in the rat uterus and the subsequent increase in microvascular permeability that is the sine qua non of cyclic endometrial growth and implantation. Furthermore, we demonstrate that PI3K/Akt activation of VEGF expression is mediated through the recruitment of both HIF-1{alpha} and ER{alpha} to the VEGF gene promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council) and approved by the Institutional Animal Care and use Committee, University of Maryland School of Medicine. Immature (21 d old), female Sprague Dawley rats (Charles River, Wilmington, MA) were injected ip with 0.2 ml dimethyl sulfoxide (DMSO) vehicle, 100 µg/g body weight (BW) U0126 (MAPK kinase 1/2 inhibitor; LC Labs, Woburn, MA), 1.4 µg/g BW wortmannin (PI3K inhibitor; Biosource, Camarillo, CA), or 20 µg/g BW LY294002 (PI3K inhibitor, LC Labs) 1 h before being injected with either 0.2 ml ethanol/PBS vehicle (1:500; controls) or E2, 0.05 µg/g BW in 0.2 ml. The dosages of U0126 (51, 52), wortmannin (53, 54, 55), and LY294002 (56, 57, 58, 59) were based on previous studies in mice, which showed them to be both tolerable and effective. Animals treated with E2 were killed 1, 4, or 5 h later by cervical dislocation. The reproductive tract was then exposed through a midline incision and the uterus and ovaries were excised together and placed on a moistened paper towel on top of a frozen gel pack. The ovaries and oviducts, fat, and mesometrial membranes were quickly trimmed away. Uteri collected from vehicle-treated control animals and the 4- or 5-h E2-treated animals were also quickly weighed. Uterine tissue was either stored in RNAlater (QIAGEN, Valencia, CA) for RNA extraction, flash frozen in liquid nitrogen for whole-cell protein extraction, or immediately fixed for chromatin immunoprecipitation (ChIP) analysis. In the latter case, the horns of each uterus were cut open longitudinally using fine scissors, immersed in 10 ml of 2% formaldehyde in DMEM-F12 medium in a 15-ml polypropylene tube, and rocked for 15 min at room temperature. Fixation was stopped by adding 1.5 ml of 1 M glycine and rocking for an additional 5 min.

Western blot analysis
Rat uterine tissue (half of one horn) was homogenized on ice in 200 µl radioimmunoprecipitation buffer [50 mM Tris-HCl (pH 7.4); 1% Nonidet P-40; 0.25% deoxycholic acid; 1 mM EDTA; protease inhibitors (Complete mini-EDTA-free protease inhibitor cocktail, 1 tablet/10 ml; no. 1 836 170; Roche, Indianapolis, IN); and phosphatase inhibitors (cocktails I and II, 1:100 each; Sigma, St. Louis, MO)] using a Tissue-Tearor rotor-stator homogenizer (Biospec Products, Bartlesville, OK) at power setting 1.5. The homogenate was then centrifuged at 3000 rpm for 30 min at 4 C, and the supernatant was collected, aliquoted, and stored at –80 C. The protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). To 12- to 20-µg protein samples (depending on which protein was being analyzed), 15 µl of either reducing [0.125 M Tris (pH 6.8); 4% sodium dodecyl sulfate (SDS); 20% glycerol; 10% ß-mercaptoethanol; 0.004% bromophenol blue] or nonreducing (Quality Biologicals, Gaithersburg, MD) 2x SDS loading buffer was then added before incubation at 100 C for 5 min. Protein samples were then loaded onto NuPAGE 4–12% Bis Tris gels (Invitrogen, San Diego, CA) and transferred overnight onto polyvinylidene fluoride membranes. Membranes were then blocked with 5% nonfat dry milk/1x Tris-buffered saline [TBS (pH 7.5)] at 37 C for 1 h before incubation for 2 h at room temperature with a mouse monoclonal antibody to HIF-1{alpha} (1:250; BD Biosciences, San Jose, CA), rabbit polyclonal antibody to ER{alpha} (1:300, Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antibody to ER{alpha} (1:50, Labvision, Fremont, CA), or one of the following rabbit polyclonal or monoclonal antibodies (1:1000; Cell Signaling, Danvers, MA): phospho-ERK1/2Thr202/Tyr204 (no. 9101S), ERK1/2 (no. 9102), phospho-AktSer473 (no. 4058), phospho-AktThr308 (no. 4056), and Akt (no. 9272). After four 5-min washes in 1x TBS with 0.1% Tween 20, the membranes were incubated at room temperature for 1 h with either goat antimouse IgG horseradish peroxidase-conjugated antibody (1:10,000; Jackson Immunoresearch Laboratories, West Grove, PA) or goat antirabbit IgG horseradish peroxidase-conjugated antibody (1:5000, no. sc-2054; Santa Cruz Biotechnology). The membranes were then washed 4 x 5 min in 1x TBS with 0.1% Tween 20 at room temperature. Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, MA) was used for visualization of protein bands, according to the manufacturer’s instructions.

RNA extraction and reverse transcription (RT)
Uterine tissue (half of one horn) was homogenized in buffer RLT (QIAGEN RNeasy minikit) using a mini-Beadbeater and 1.0 mm zirconia/silica beads (Biospec Products). The beads were removed and the samples further homogenized/sheared by centrifugation through QIAshredder spin columns (QIAGEN). RNA was extracted and purified from homogenates using the RNeasy minikit (QIAGEN). Total RNA concentration and purity were calculated based on 260/280 nm absorbance. RNA was diluted with water to 0.08 µg/µl and reverse transcribed. The RT reaction mixture consisted of 6 µl diluted RNA (0.48 µg), 4 µl 5 x reverse transcriptase buffer, 4 µl deoxynucleotide triphosphate mix (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 2 µl 0.1M dithiothreitol, 2 µl of 1 mg/ml BSA, 1 µl of 0.5 µg/µl random primers, and 1 µl (200 U) of Moloney murine leukemia virus reverse transcriptase (all from Invitrogen). This mixture was incubated for 1 h at 37 C.

PCR
PCR analysis of VEGF, creatine kinase B (CKB), and progesterone receptor-A/B (PR-A/B) mRNA expression was done by both conventional and real-time PCR. For conventional PCR, each 30 µl reaction consisted of 3 µl RT reaction mixture, 3 µl 10x buffer (QIAGEN), 2.4 µl deoxynucleotide triphosphate mix (as for RT, above), 3 µl 5 µM primer mix, 0.15 µl Taq DNA polymerase (QIAGEN), and 18.45 µl molecular grade water. For amplification of 18S rRNA, the RT reaction mixture was first diluted 1:1000. The optimal number of PCR cycles (the number of cycles yielding detectable product but still within the linear range of amplification) was first determined for VEGF mRNA (24 cycles), CKB mRNA (22 cycles), PR-A/B (22 cycles), and 18S rRNA (16 cycles). An annealing temperature of 60 C was used in all cases. The following primers were used for both conventional and real-time PCR analysis: rat VEGF +12 to +644, forward, 5'-GCTCTCTTGGGTGCACTGGA-3' and reverse, 5'-CACCGCCTTGGCTTGTCACA-3' (GenBank no. AF215725); rat CKB +1443 to +1806, forward, 5'-CATCCAGACTGGCGTAGACA-3' and reverse, 5'-AGGTTGTCTGGGTTGAGGTC-3' (GenBank no. M18668); rat PR-A/B +1910 to +2359, forward, 5'-TTATGAGAGCCCTCGATGGT-3' and reverse, 5'-GCGAGTAGAATGACAACTC-3' (GenBank no. L16922); human 18S rRNA +364 to +647, forward, 5'-CAACTTTCGATGGTAGTCGC-3' and reverse, 5'-CGCTATTGGAGCTGGAATTAC-3' (GenBank no. X01117).

The VEGF, CKB, and PR-A/B primer pairs spanned intron-exon borders. The PCR products were run on 8% polyacrylamide gels and visualized after staining with ethidium bromide.

Real-time PCR analysis was done using a DNA Opticon system (MJ Research, Boston, MA). Each 30-µl reaction mixture included 3 µl RT reaction mixture, 15 µl 2x DyNAmo SYBR green qPCR mix (New England Biolabs, Ipswich, MA), 1.2 µl primer mix (5 µM each primer), and 10.8 µl water. Each sample was assayed in duplicate. A standard curve was generated by serially diluting an RT reaction mixture from a 1-h E2-treated uterus. The yield of product for each unknown sample was calculated by applying its threshold cycle value (the cycle at which the sample’s fluorescence exceeds background noise and begins to increase linearly) to the standard curve using the Opticon Monitor analysis software (version 2.1; MJ Research). Values were normalized to corresponding 18S rRNA real-time RT-PCR values and expressed as the fold increase relative to 0 h.

In vivo ChIP
After fixation, uterine tissue (one or two horns) was rinsed in 1 ml cold Dulbecco’s PBS containing protease and phosphatase inhibitors (as described previously) to remove residual formaldehyde and glycine. ChIP was performed on each individual uterus (the n value for each group is shown in the figure captions). Each horn(s) was then transferred to a clean 1.5 ml microcentrifuge tube, frozen by immersion in liquid nitrogen, and stored at –80 C. To continue, 200 µl of radioimmunoprecipitation buffer was added to each tube and homogenization carried out as described previously for Western blots. Homogenates were centrifuged at 12,000 rpm for 5 min at 4 C and the supernatants removed and discarded. Pellets were resuspended in 600 µl nuclear lysis buffer [50 mM Tris-HCl (pH 8.1); 5 mM EDTA; 1% SDS; and protease inhibitors (as above)] and incubated on ice for 15 min. Samples were sonicated on ice for 10 x 10 sec cycles, with 20 sec pauses between each cycle, using a Microson Ultrasonic cell disruptor (Misonix, Farmingdale, NY) at power level 2.5. After sonication, the samples were centrifuged at 14,000 x g for 10 min at 4 C. The supernatants were then collected, divided into 100-µl aliquots, and stored at –80 C.

Sonicated sample aliquots were thawed on ice and diluted 1:10 with dilution buffer [20 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease inhibitors (as above)] before being immunocleared in a solution containing 45 µl of a 50% slurry of Protein A or G Sepharose 4 Fast Flow (Amersham, Piscataway, NJ) in Tris/EDTA (TE) buffer (pH 8), 2 µg salmon sperm DNA (Invitrogen), and 20 µl of either normal mouse or rabbit serum (Sigma) for 2 h at 4 C. Supernatants were collected and incubated overnight at 4 C with 2.5 µg of a mouse monoclonal antibody to HIF-1{alpha} (BD Biosciences) or 2 µg of a mouse monoclonal antibody to ER{alpha} (Labvision), or 5 µg of a rabbit polyclonal antibody to specificity protein 1 (Sp1) (Santa Cruz Biotechnology). For the nonspecific antibody control, an equal volume of normal mouse or rabbit serum was substituted for the specific antibody. Protein A or G Sepharose beads (45 µl of a 50% slurry in TE buffer) and salmon sperm DNA (2 µg) were then added and incubated for 1 h at 4 C. The beads were then washed sequentially with 1 ml each of Tris/NaCl/EDTA (TSE) I, TSE II, and buffer III, and then 2 x 1 ml of TE buffer (pH 8.0); each wash was for 10 min at 4 C (10). The protein-DNA complexes were then eluted by twice incubating beads in 100 µl of elution buffer (1% SDS, 0.1 M NaHCO3) for 10 min at room temperature with vigorous mixing. To separate immunoprecipitated protein and DNA, the pooled eluates were incubated at 65 C overnight. The DNA was purified using the Qiaquick PCR purification kit (QIAGEN) and eluted in 50 µl of elution buffer [10 mM Tris-HCl (pH 8.5); supplied in kit].

The yield of target region DNA in each sample after ChIP was analyzed by both conventional (30 cycles, 60 C annealing temperature) and real-time PCR, as described previously. In both cases, 3 µl of each 50-µl sample were amplified. For real-time PCR, standard curves were generated by serially diluting an input chromatin sample. The following primers, which encompass regions on the VEGF or CKB promoter, were used: rat VEGF –944 to –611 [hypoxia response element (HRE)-containing region], forward, 5'-TCTGCCAGACTCCACAGTG-3' and reverse, 5'-TGCGTGTTTCTAACACCCAC-3' (GenBank no. U22373); rat VEGF –173 to +114 (proximal Sp1/ER{alpha} binding region), forward, 5'-CAGGCTATGGACCCTGGTAA-3' and reverse, 5'-ATAGTCTGCCTTGTCGCTGC-3' (GenBank no. U22373); rat CKB –678 to 32 –319, forward, 5'-GGAAAGAACCTGGGGATTTG-3' and reverse, 5'-GTTAGCACTTGAGGTTCCTG-3' (GenBank no. M18668).

As negative controls, ER{alpha} immunoprecipitation samples were amplified using primers for the HRE containing region of the promoter and HIF-1{alpha} immunoprecipitation samples were amplified with primers to the proximal GC-rich region. As an additional control, primers to a region spanning a portion of the 3'-untranslated region (+2537 to +2799) were used. As a negative control for the ChIP analysis of the CKB promoter, primers encompassing a portion of the CKB coding region (+502 to +865) were used.

Statistical analysis
Statistical analyses were done using factorial ANOVA and appropriate post hoc tests except for the uterine weight data, which was analyzed using the Kruskall-Wallis test (StatView version 4.5; Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen activates both the MAPK and PI3K pathways in the rat uterus
Several recent studies (60, 61, 62, 63, 64) demonstrated that estrogen can rapidly activate the MAPK pathway in a wide range of cell types in vitro. Because the MAPK pathway has also been linked in some cases to activation of HIF-1{alpha} and VEGF expression (25, 26), we first determined whether estrogen induces phosphorylation of the MAPKs ERK1 and ERK2 in the rat uterus. As seen in Fig. 1Go, Western blot analysis showed that there was a 7-fold increase in phosphorylated ERKs 1 h after E2 treatment, whereas total ERK levels were unchanged. This effect of E2 was completely blocked by the MEK inhibitor U0126.


Figure 1
View larger version (39K):
[in this window]
[in a new window]

  		FIG. 1. Effect of E2 and U0126 on ERK1/2 phosphorylation in the rat uterus. Immature female rats were treated (ip) with either DMSO vehicle (–) or U0126 (100 µg/g BW) for 1 h before treatment (ip or sc) with either EtOH/PBS vehicle (0 h) or E2 (1 h; 0.05 µg/g BW). Whole-cell protein was extracted from uteri and phosphorylated (p-ERK1/2) and total ERK1/2 were analyzed by Western blot analysis. A, Representative gel. B, Densitometry. Densitometry results are expressed as fold increase in p-ERK1/2, compared with uteri not exposed to either U0126 or E2 (DMSO vehicle-0 h E2) after normalization to total ERK1/2 (means ± SEM, n = 5 uteri per group). a, P < 0.05 vs. DMSO vehicle-0 h E2, U0126–0 h E2, and U0126–1 h E2.

 
Estrogen has also been shown to activate PI3K both in a variety of cells in vitro (30, 31, 32, 37, 38, 39, 40, 41, 42, 43, 44) and endometrial epithelial cells in vivo (46, 47, 48, 49). The PI3K/Akt pathway is also widely linked to activation of HIF-1{alpha} (25, 26). As shown in Fig. 2Go, both phospho-AktSer473 and phospho-AktThr308 were detected in the uterus before treatment, and E2 induced a 75% increase (P < 0.05) in the level of phosphorylation at both sites. Pretreatment of animals with the PI3K inhibitor wortmannin resulted in a complete loss of both basal and E2-induced phosphor-Akt, without affecting total Akt. A second inhibitor, LY294002, also blocked the E2-induced increase in Akt phosphorylation, again without affecting total Akt, although at the dose used it did not reduce basal Akt phosphorylation.


Figure 2
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 2. Effect of E2 and wortmannin or LY294002 on Akt phosphorylation in the rat uterus. Immature female rats were treated (ip) with either DMSO vehicle (–) or PI3K inhibitor [A and B, wortmannin (1.4 µg/g BW; Wort); or C and D, LY294002 (20 µg/g BW; LY)] for 1 h before treatment with either EtOH/PBS vehicle (0 h) or E2 (1 h; 0.05 µg/g BW). Whole-cell protein was extracted from uteri, and phosphorylated Akt (p-AktSer473 and p-AktThr308) and total Akt were analyzed by Western blot analysis. A and C, Representative gels. B and D, Densitometry. Densitometry results are expressed as fold increase in p-AktSer473 and p-AktThr308, compared with uteri not exposed to either PI3K inhibitor (Wort or LY) or E2 (DMSO vehicle-0 h E2) after normalization to total Akt (means ± SEM, n = 4–5 uteri/group). a, P < 0.05 vs. DMSO vehicle-0 h E2, Wort- or LY-0 h E2, and Wort- or LY-1 h E2.

 
Inhibiting the PI3K/Akt pathway, but not the MAPK pathway, inhibits estrogen-induced VEGF expression
As expected, E2 induced marked increases, ranging from 3- to 20-fold in different experiments, in VEGF mRNA levels within 1 h of treatment (Figs. 3Go and 4Go). As shown in Fig. 3Go, inhibiting the MAPK pathway with U0126 had no effect on this induction. Thus, although E2 activates the MAPK pathway in the rat uterus, E2-induced VEGF expression is not MAPK dependent. In contrast to this, the PI3K inhibitors wortmannin and LY294002 both completely blocked the E2-induced increase in VEGF mRNA at 1 h (Fig. 4Go). These results are consistent with other reports demonstrating an essential role for the PI3K/Akt pathway in both hypoxic and nonhypoxic VEGF induction (25, 26).


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 3. Effect of E2 and U0126 on VEGF mRNA expression in the rat uterus. Immature female rats were treated as described in Fig. 1Go. Total RNA was extracted from uteri and VEGF mRNA analyzed using conventional (representative gel, A) and real-time RT-PCR (B). The VEGF primers encompass the alternative splice sites and generate products for both the VEGF164 and VEGF120 isoforms. Real-time RT-PCR results are expressed as fold increase in VEGF mRNA levels, compared with uteri not exposed to either U0126 or E2 (DMSO vehicle-0 h E2) after normalization to 18S rRNA (means ± SEM, n = 5 uteri/group). a, P < 0.05 vs. DMSO vehicle-0 h E2 and U0126-0 h E2.

 

Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 4. Effect of E2 and wortmannin (Wort) or LY294002 (LY) on VEGF mRNA expression in the rat uterus. Animals were treated as described in Fig. 2Go and VEGF mRNA analyzed using conventional (A and C, representative gel) and real-time RT-PCR (B and D). Real-time RT-PCR results are expressed as the fold increase in VEGF mRNA levels, compared with uteri not exposed to either PI3K inhibitor or E2 (DMSO vehicle-0 h E2) after normalization to 18S rRNA (means ± SEM, n = 4–5 uteri/group). a, P < 0.05 vs. DMSO vehicle-0 h E2 and Wort-1 h E2; b, P < 0.01 vs. Wort-0 h E2; c, P < 0.0001 vs. DMSO vehicle-0 h E2, LY-0 h E2, and LY-1 h E2.

 
Inhibiting the PI3K/Akt pathway blocks estrogen-induced uterine edema
As we have previously demonstrated, blocking VEGF action using a neutralizing antibody prevents estrogen-induced uterine edema (2). Blocking VEGF expression by inhibiting PI3K therefore should also block edema. To confirm this, groups of rats were killed 4 or 5 h after treatment with E2 alone or E2 plus wortmannin and their uteri weighed. Edema normally peaks approximately 4–6 h after E2 treatment. As expected, uteri from E2-treated rats were approximately 30% heavier than those from vehicle-treated controls at 4 or 5 h (P < 0.01). This increase in wet weight was completely blocked by wortmannin (Fig. 5Go).


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 5. Effect of E2 and wortmannin (Wort) on uterine weight. Immature female rats were weighed and treated either with DMSO vehicle (–) or wortmannin (Wort, 1.4 µg/g BW) for 1 h before treatment with either EtOH/PBS vehicle (0 h) or E2 (0.05 µg/g BW) for either 4 h or 5 h. Uteri were weighed and percent increase in wet weight (normalized to BW) vs. untreated controls (DMSO-0 h E2) were calculated (n = 7–11 uteri per group). Because weights within groups were not different at the two time points, the data were combined. a, P < 0.01 vs. DMSO-0 h E2; b, P < 0.05 vs. Wort-0 h E2 and Wort-E2.

 
Taken together, these results indicate that E2 rapidly activates the PI3K/Akt pathway in the endometrium, and that this leads to increased VEGF gene expression and, consequently, edema, the hallmark of E2’s uterotrophic effects.

Inhibiting PI3K activity blocks recruitment of HIF-1{alpha} to the VEGF gene promoter
We have recently shown that estrogen-induced VEGF expression in the uterus correlates closely with the recruitment of HIF-1 to the VEGF promoter (10). We hypothesized therefore that the decrease in estrogen-induced VEGF expression caused by PI3K inhibitors was due to inhibition of HIF-1{alpha} activation and recruitment. To test this, we used in vivo ChIP to look at HIF-1{alpha} binding to the HRE on the VEGF promoter in rat uteri 1 h after E2 treatment with or without pretreatment with wortmannin or LY294002. Consistent with our previous results, there was a 2.5-fold increase (P < 0.01) in the yield of PCR product for the HRE-containing region of the promoter after immunoprecipitation of HIF-1{alpha}, indicating increased binding of HIF-1{alpha} with the HRE 1 h after E2 treatment (Fig. 6Go, B–D); no product was obtained when normal serum was substituted for the HIF-1{alpha} antibody or when PCR was carried out using primer pairs encompassing either the GC-rich region of the promoter (–173 to +114; Fig. 7BGo) or a downstream segment of the 3' untranslated region of the gene (+2537 to +2799; Fig. 6EGo). This E2-induced recruitment of HIF-1{alpha} to the VEGF promoter was completely prevented by either wortmannin or LY294002. Thus, the attenuation of E2’s uterotrophic effects by wortmannin correlates with a lack of E2-induced HIF-1{alpha} recruitment to the VEGF promoter. Western blot analysis of HIF-1{alpha} protein levels in the uteri at 1 h showed no difference in any of the groups. Thus, the inhibitory effect of wortmannin on HIF-1{alpha} recruitment to the VEGF promoter is not due to a loss of HIF-1{alpha}. This is consistent with our earlier observation that the HIF-1{alpha} recruited to the VEGF promoter 1 h after E2 treatment originates from the basal pool normally present in endometrial cells, not an increase in the level of HIF-1{alpha}.


Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 6. Effect of E2 and wortmannin (Wort) or LY294002 (LY) on recruitment of HIF-1{alpha} to the VEGF promoter in the rat uterus. Immature female rats were treated as described in Fig. 2Go. ChIP analysis was carried out on each individual uterus. Immunoprecipitation was carried out using antibodies to either HIF-1{alpha} or ER{alpha} or an equivalent volume of normal mouse serum (NS). Primers for the –944 to –611 region of the rat VEGF promoter (A), which contains the HRE to which HIF-1 binds, were used for PCR. AP1, activator protein 1. B and D, Representative ChIP gels from experiments with Wort and LY294002 (LY; n = 2 uteri), respectively. C, ChIP real-time PCR results from the Wort experiment; results are expressed as the fold increase, compared with uteri not exposed to either Wort or E2 (DMSO vehicle-0 h E2; means ± SEM, n = 9 uteri/group). a, P < 0.001 vs. DMSO-0 h E2 and Wort-1 h E2; b, P < 0.05 vs. Wort-0 h E2. E, PCR results using primers targeting a downstream region of the VEGF gene (+2537 to +2799); the expected product was obtained only in the input samples. F, Western blot of HIF-1{alpha} at 0 and 1 h after treatment with E2 in the presence or absence of Wort; 20 µg of protein was loaded in each well.

 

Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 7. Effect of E2 and wortmannin (Wort) on recruitment of ER{alpha} to the VEGF promoter in the uterus. Immature female rats were treated (ip) with either DMSO vehicle (–) or Wort (1.4 µg/g BW) for 1 h before treatment with either EtOH/PBS vehicle (0 h) or E2 (1 h; 0.05 µg/g BW). Immunoprecipitation was carried out using antibodies to either ER{alpha} or HIF-1{alpha} or an equivalent volume of normal mouse serum (NS). Primers for the –173 to +114 region of the rat VEGF promoter (A), which contains at least three Sp1 sites [and an activator protein (AP)-2 site] were used for PCR. B, Representative ChIP gels. C, ChIP real time PCR results expressed as the fold increase, compared with uteri not exposed to either Wort or E2 (DMSO vehicle-0 h E2; means ± SEM, n = 4 uteri/group). a, P < 0.05 vs. DMSO-0 h E2, Wort-0 h E2, and Wort-1 h E2. D, PCR results using primers targeting a downstream region of the VEGF gene (+2537 to +2799); the expected product was obtained only in the input samples. E, Western blot of ER{alpha} at 0 and 2 h after treatment with E2 in the presence or absence of Wort; 20 µg of protein was loaded in each well.

 
Inhibiting PI3K activity also blocks recruitment of ER{alpha} to the VEGF promoter as well as other target gene promoters
Phosphorylation of ER{alpha} on Ser167 by Akt and other kinases in response to E2 and nonligand ER{alpha} activators has been reported (41, 65, 66, 67, 68, 69, 70, 71). Phosphorylation on Ser167 has also been shown to be essential for ER{alpha} binding to target gene promoters (66, 70, 72, 73). We determined therefore whether blocking the PI3K/Akt pathway would prevent the binding of ER{alpha} to the VEGF promoter, as it had of HIF-1{alpha}. As shown in Fig. 7Go, in vivo ChIP analysis showed that E2 induced an increase in ER{alpha} binding to the GC-rich proximal region of the VEGF promoter, in agreement with previous findings (10). This increase was also blocked by inhibition of the PI3K pathway, with no change in total ER{alpha} levels (Fig. 7EGo). No product was obtained when normal serum was substituted for the ER{alpha} antibody. When PCR was carried out using primer pairs encompassing the HRE-containing region of the promoter (–944 to –611), a product was obtained in some samples, but unlike the binding of HIF-1{alpha} to this region, or of ER{alpha} to the proximal GC-rich region, there was no pattern relative to treatment (Fig. 6BGo). Furthermore, amplification of a more distant downstream segment of the 3' untranslated region of the gene (+2537 to +2799) yielded no product at all (Fig. 7DGo).

To determine whether this requirement for PI3K/Akt was specific for the VEGF gene or applied to other estrogen-induced genes as well, the expression of the CKB and progesterone receptor genes was also examined. As shown in Fig. 8Go, E2 induced an increase in the levels of both mRNAs by 1 h, and, like VEGF, this increase was prevented by wortmannin. These data suggest that phosphorylation of ER{alpha} as a result of PI3K/Akt activation is required for interaction with many target gene promoters in the uterus, as has been demonstrated previously in vitro (66, 70, 72, 73). We have previously shown that CKB expression is associated with the rapid binding of ER{alpha} and Sp1 to an Sp1-variant estrogen response element (ERE) site on the promoter (10). A significant increase in the binding of both ER{alpha} and Sp1 to this site was again detected in this study (Fig. 9Go). Treatment with wortmannin blocked the E2-induced binding of both factors with this region, indicating that the PI3K pathway again plays a role in the recruitment of both factors to the CKB promoter. No E2-induced binding of either factor was detected when primers to a downstream region of the gene were used (Fig. 9DGo).


Figure 8
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 8. Effect of E2 and wortmannin (Wort) on CKB mRNA and PR-A/B mRNA expression in the rat uterus. Immature female rats were treated as previously described in Fig. 7Go. Total RNA was extracted from uteri, and CKB mRNA and PR-A/B mRNA were analyzed using conventional RT-PCR (representative gels, A and C) and real time RT-PCR (B and D, respectively). Real-time RT-PCR results are expressed as fold increase in CKB mRNA or PR-A/B mRNA levels, compared with uteri not exposed to either Wort or E2 (DMSO vehicle-0 h E2) after normalization to 18S rRNA (means ± SEM, n = 4 uteri/group). a, P < 0.01 vs. DMSO-0 h E2, Wort-0 h E2, and Wort-1 h E2.

 

Figure 9
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 9. Effect of E2 and wortmannin (Wort) on recruitment of ER{alpha} and Sp1 to the CKB promoter in the uterus. Immature female rats were treated as previously described in Fig. 7Go. Immunoprecipitation was carried out using an antibody to either ER{alpha} or Sp1 or an equivalent volume of normal serum (NS). Primers for the –678 to –319 region of the rat CKB promoter (A), which contains a variant ERE (vERE) flanked by two Sp1 sites, were used for PCR. B, Representative ChIP gels. C, ChIP real-time PCR results expressed as the fold increase, compared with uteri not exposed to either Wort or E2 (DMSO vehicle-0 h E2; means ± SEM, n = 4 uteri/group). a, P < 0.05 vs. DMSO-0 h E2 and Wort-1 h E2; b, P < 0.05 vs. Wort-1 h E2; c, P < 0.01 vs. Wort-0 h E2. D, PCR results using primers targeting a downstream region of the CKB gene (+502 to +865); the expected product was obtained only in the input samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrate that the PI3K pathway plays an essential role in one of estrogen’s earliest and most important effects on the endometrium: the induction of stromal edema. This exudation of serum into the interstitium, which is mediated by VEGF (2), precedes and plays an essential role in the cyclic growth and remodeling of the endometrium as well as in implantation (2, 3, 4). These observations add to the rapidly growing evidence that many of estrogen’s effects are mediated through cytoplasmic signaling pathways acting in conjunction with nuclear ER{alpha}-mediated transcriptional mechanisms. Our data indicate that both processes are required for estrogen induction of VEGF expression in the endometrium. Specifically, estrogen simultaneously causes the recruitment of HIF-1{alpha} and ER{alpha} to distinct sites on the VEGF promoter, and both of these events require the PI3K/Akt pathway (10) (Fig. 10Go). As previously shown, HIF-1{alpha} is present in the uterus before E2 treatment and does not increase until after 1 h, by which time it has already been recruited to the VEGF promoter and VEGF mRNA expression has peaked (10). PI3K activation of basal HIF-1{alpha}, leading to gene transcription, has also been reported by Kaluz et al. (74).


Figure 10
View larger version (55K):
[in this window]
[in a new window]

  		FIG. 10. Model illustrating steps in E2 induction of VEGF expression in the rat uterus in vivo. Previous ChIP results (7 ) indicate that estrogen simultaneously induces the recruitment of both HIF-1 ({alpha} and ß) to the upstream HRE and ER{alpha} to the proximal GC-rich region of the VEGF promoter, probably via interaction with Sp proteins; p300 binds to both transcription factor complexes. The current results show that the activation and recruitment of both HIF-1{alpha} and ER{alpha} is mediated by PI3K. This may involve activation of a membrane form of the ER (here designated ERm), possibly through c-Src (28 29 32 39 45 83 ).

 
This mechanism, recruitment of both ER{alpha} and other transcription factors to distinct sites on gene promoters triggered by both cytoplasmic pathways and the binding of E2 to nuclear ER{alpha}, is consistent with a convergence model of estrogen action (29). We predict that other hormones and growth factors will be shown to induce tissue- or condition-specific VEGF expression via combinations of HIF-1 and other transcription factors. HIF-1 is the primary regulator of expression of genes required for cellular adaptation to hypoxia. Control by hormones may have become superimposed on this basic survival mechanism during evolution to regulate expression of the same genes during developmental processes, such as estrogen-induced growth of the endometrium. We proposed that HIF-1 is essential for estrogen-induced VEGF expression (10). Because the PI3K/Akt pathway was found to affect the binding of both HIF-1{alpha} and ER{alpha} to the VEGF promoter, however, this could not be confirmed in this study. Other recent work from our laboratory, however, supports this hypothesis (75). In that study, E2 induced no VEGF expression by endometrial cancer cells (ECC-1) cultured in 95% air-5% CO2, and therefore 20% oxygen, which causes complete HIF-1{alpha} degradation (10). When HIF-1{alpha} was artificially elevated by treating the cells with CoCl2 (which suppresses degradation), however, a strong estrogen induction was unmasked. This suggests that ER{alpha} alone is not sufficient to drive VEGF transcription and that HIF-1{alpha} is also required. It is also possible that ER{alpha} can interact with the VEGF promoter only when HIF-1 is also bound.

Blocking PI3K with wortmannin has also been shown to inhibit estrogen-induced adherence of leukocytes to the walls of blood vessels in vivo (30). This could be relevant to the current results because VEGF is known to stimulate leukocyte adhesion to endothelial cells in vivo (76). Furthermore, estrogen induces a large influx of leukocytes into the uterus (77). Thus, PI3K activation may be central to the coordinated induction of estrogen’s early inflammatory-like sequelae in the uterus, increased microvascular permeability (via VEGF), increased blood flow (via nitric oxide synthase/NO), and increased leukocyte influx, thereby creating an environment optimal for endometrial growth and remodeling.

Marked changes in Akt localization and activation in the uterus during the cycle and in response to exogenous estrogen are consistent with a role for PI3K/Akt in estrogen’s uterotrophic effects. In adult rats, phospho-Akt (the activated form) actually decreases during estrus, which immediately follows the proestrus peak in E2, compared with the other stages of the cycle (46), but what remains is localized at the epithelial cell membrane, whereas it is cytoplasmic and nuclear at other stages. Both Akt activation and inactivation occur at the cell membrane. Thus, this change in phospho-Akt distribution is indicative of changes in Akt activity around the time of estrogen exposure. Furthermore, E2 induces marked increases in Akt phosphorylation in uteri of ovariectomized rats (46, 47) and in the endometrial epithelial cells of mice (49). Finally, the level of Akt phosphorylation in human endometrial epithelial cells also correlates positively with E2 levels (50).

Estrogen activation of cytoplasmic signaling pathways is now widely attributed to membrane-associated forms of ER{alpha}, which interact with other membrane signaling molecules such as c-Src kinase and PI3K (28, 29, 32, 39, 41, 43, 45, 78, 79). The linkage of PI3K/Akt to estrogen-induced activation of HIF-1{alpha} and the expression of VEGF in the uterus further support the involvement of such receptors in estrogen action. It has been suggested that Akt may serve as the link between membrane and nuclear ER{alpha} (69). Our results are entirely consistent with that proposal. Others have postulated that activation of cytoplasmic pathways by estrogen is indirect. For example, it has been proposed that estrogen may induce the synthesis of IGF-I by endometrial stromal cells (49), which in turn binds to IGF-I receptors (IGF-IR) on epithelial cells to activate cytoplasmic pathways. This cannot explain the rapid induction of VEGF gene transcription, however, which is not blocked by protein synthesis inhibitors (5, 6). On the other hand, activation of IGF-IR may not require ligand because estrogen has been shown to trigger the direct interaction of IGF-IR and ER{alpha} in cells in vitro (80). Furthermore, estrogen-induced formation of an ER{alpha}-IGF-IR-PI3K complex has been described (81). Finally, estrogen’s rapid effects have also been attributed to the G protein-coupled receptor GPR30, but its role is still controversial (82, 83).

Our results are consistent with studies that link PI3K/Akt-mediated phosphorylation of ER{alpha} to the transcription of a number of genes in cells in vitro (41, 65, 66, 67, 68, 69, 70, 71, 72, 73). Binding of ER{alpha} to target gene promoters has been shown to require phosphorylation on Ser167, the primary Akt target (66, 70, 72, 73), consistent with our ChIP results, which show that PI3K inhibitors block ER{alpha} recruitment to both the VEGF and CKB promoters. Recently it was shown that more than 100 genes known to be regulated by ER{alpha} are similarly up- or down-regulated in the uteri of Pten+/– mice, which have elevated PI3K/Akt activity [phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is the phosphatase that counteracts PI3K activity] and develop endometrial cancer (84). Thus, activation of PI3K/Akt mimics many of estrogen’s effects on the endometrium, indicating that this pathway lies downstream of an ER. Conversely, reduction of endometrial ER{alpha} dramatically reduces the neoplastic effect of PTEN loss on the endometrium, indicating that PI3K/Akt-induced gene expression is mediated in large part through ER{alpha} (71). This interdependency of estrogen signaling and the PI3K/Akt pathway is consistent with the latter lying downstream of a membrane ER and upstream of nuclear ER{alpha} (Fig. 10Go).

Recruitment of ER{alpha} to both the VEGF and CKB promoters involves interaction with Sp1 (10). In this study, inhibiting the PI3K pathway blocked the estrogen-induced recruitment of both ER{alpha} and Sp1 to the CKB promoter (Fig. 9Go) but only ER{alpha} to the VEGF promoter (Fig. 7Go; data not shown). PI3K/Akt involvement in the binding of Sp1 to some genes, including VEGF, has been previously reported (85, 86).

The mechanism of HIF-1{alpha} activation is poorly understood at this time. Several studies have concluded that HIF-1{alpha} activity is regulated through phosphorylation (25, 87). On the other hand, it was reported that active HIF-1{alpha} is not phosphorylated in the testis (88), and we see no change in the gel mobility of uterine HIF-1{alpha} after either estrogen exposure or lambda phosphatase treatment (data not shown). Phosphorylation of the coactivator p300, rather than HIF-1{alpha} itself, has also been proposed to be the key event in HIF-1{alpha} activation (89). Finally, it is possible that estrogen could regulate the expression or activity of factor inhibiting HIF-1{alpha}, an oxygen-regulated asparagine hydroxylase, which blocks binding of p300 to HIF-1under normoxic conditions (90). Clearly, further work on the mechanism of HIF-1{alpha} activation is required.

The processes that lead to tumor growth and spread generally reflect normal developmental processes that have become deregulated or hyperactivated. The frequent association between increased activity of the PI3K/Akt pathway and cancers, particularly those influenced by estrogen, again probably reflects a central role for this pathway in normal estrogen-induced growth of tissues such as the endometrium. Increased production of phosphatidylinositol-3,4,5-triphosphate is a potent oncogenic signal, leading to the activation of a variety of kinases, including Akt, phosphoinositide-dependent kinase-1 (PDK1), and mammalian target of rapamycin (mTOR). Constitutive activation of these kinases, which regulate cell proliferation, apoptosis, cell size, migration, genetic stability, and angiogenesis, leads to tumor initiation and progression. Several recent studies implicated the PI3K/Akt pathway in elevated VEGF expression, increased microvascular permeability, and angiogenesis in tumors (91, 92, 93, 94, 95). Activation of PI3K/Akt has also been linked to estrogen-induced proliferation of cancer cells (39, 44, 96). Conversely, activation of the PI3K/Akt pathway for other reasons (e.g. PTEN loss) could cause tumor growth in estrogen-target tissues through ligand-independent nuclear ER{alpha} activation. As mentioned earlier, the absence of ER{alpha} dramatically reduces the neoplastic effect of PTEN loss on the endometrium (71). The close connection between PI3K/Akt activity and cancer makes elements of the pathway promising targets for the development of new anticancer drugs, a number of which are already in clinical evaluation (97). The demonstration that PI3K/Akt is central to estrogen action means that such drugs might be particularly effective for the treatment of estrogen-dependent tumors, particularly in combination with drugs targeting estrogen action or synthesis.

Interestingly, PI3K activation by ER activators other than E2 has also recently been shown. The plant compound diosgenin, a steroid-like furostanol saponin, was reported to induce HIF-1 activation and VEGF expression in an ER-mediated, PI3K- and p38 MAPK-dependent manner in cultured osteoblasts (98). This is in agreement with our previous (10) and current results, although we have not examined the role of p38 MAPK. Second, the protein hormone relaxin, which we have shown to be a ligand-independent activator of ERs in the uterus (99), stimulates VEGF expression in THP-1 cells and mouse mesangial cells, both of which express ER{alpha}, and this effect is blocked by the PI3K inhibitor LY294002 (100).

To our knowledge, this is the first demonstration of MAPK activation by estrogen in the normal uterus. Although the ERK1/2 MAPK pathway does not appear to be involved in estrogen regulation of VEGF gene expression there, its activation clearly indicates a role for it in other effects of estrogen on the endometrium. MAPK activation has been shown, for example, to be involved in estrogen induction of early growth factor 1 in MCF-7 cells (64) and eNOS in endothelial cells (101).

In summary, we have now shown that estrogen-induced VEGF expression and uterine edema are mediated via the PI3K/Akt pathway, which is required for the recruitment of both HIF-1{alpha} and ER{alpha} to the VEGF gene promoter. This observation sheds new light on estrogen action in normal target tissues in vivo. It also has important implications for cancer biology because increased PI3K/Akt activity has been linked to a variety of cancers, particularly endometrial carcinoma.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Cooperative Agreement U54 HD36207 (as part of the Specialized Cooperative Centers Program in Reproduction Research), NIH R21 ES013061, and a Pioneer Award from the School of Medicine (to R.D.K.). A.A.K. was supported by NIH Institutional Training Grant HD07170.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 1, 2007

Abbreviations: Akt/PKB, Protein kinase B; BW, body weight; ChIP, chromatin immunoprecipitation; CKB, creatine kinase B; DMSO, dimethyl sulfoxide; E2, 17ß-estradiol; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; ERE, estrogen response element; HIF, hypoxia-inducible factor; HRE, hypoxia response element; IGF-IR, IGF-I receptor; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PR-A/B, progesterone receptor A/B; PTEN, phosphatase and tensin homolog deleted on chromosome 10; RT, reverse transcription; SDS, sodium dodecyl sulfate; Sp1, specificity protein 1; TBS, Tris-buffered saline; TE, Tris/EDTA; VEGF, vascular endothelial growth factor.

Received October 13, 2006.

Accepted for publication January 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ferrara N 2004 Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25:581–611[Abstract/Free Full Text]
  2. Rockwell LC, Pillai S, Olson CE, Koos RD 2002 Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents. Biol Reprod 67:1804–1810[Abstract/Free Full Text]
  3. Hastings JM, Licence DR, Burton GJ, Charnock-Jones DS, Smith SK 2003 Soluble vascular endothelial growth factor receptor 1 inhibits edema and epithelial proliferation induced by 17ß-estradiol in the mouse uterus. Endocrinology 144:326–334[Abstract/Free Full Text]
  4. Koos RD, Rockwell LC 2006 The microvasculature of the endometrium. In: Shepro D, ed. Microvascular research: biology and pathology. San Diego: Academic Press/Elsevier; 587–594
  5. Cullinan-Bove K, Koos RD 1993 Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology 133:829–837[Abstract/Free Full Text]
  6. Hyder SM, Stancel GM, Chiappetta C, Murthy L, Boettger-Tong HL, Makela S 1996 Uterine expression of vascular endothelial growth factor is increased by estradiol and tamoxifen. Cancer Res 56:3954–3960[Abstract/Free Full Text]
  7. Shweiki D, Itin A, Neufeld G, Gitay-Goren H, Keshet E 1993 Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J Clin Invest 91:2235–2243[Medline]
  8. Karuri AR, Kumar AM, Mukhopadhyay D 1998 Differential expression and selective localization of vascular permeability factor/vascular endothelial growth factor in the rat uterus during the estrous cycle. J Endocrinol 159:489–499[Abstract]
  9. Yi XJ, Jiang HY, Lee KK, O WS, Tang PL, Chow PH 1999 Expression of vascular endothelial growth factor (VEGF) and its receptors during embryonic implantation in the golden hamster (Mesocricetus auratus). Cell Tissue Res 296:339–349[CrossRef][Medline]
  10. Kazi AA, Jones JM, Koos RD 2005 Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor {alpha} and hypoxia-inducible factor 1 to the vascular endothelial growth factor promoter. Mol Endocrinol 19:2006–2019[Abstract/Free Full Text]
  11. Semenza GL 2003 Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3:721–732[CrossRef][Medline]
  12. Hellwig-Burgel T, Rutkowski K, Metzen E, Fandrey J, Jelkmann W 1999 Interleukin-1ß and tumor necrosis factor-{alpha} stimulate DNA binding of hypoxia-inducible factor-1. Blood 94:1561–1567[Abstract/Free Full Text]
  13. Zelzer E, Levy Y, Kahana C, Shilo BZ, Rubinstein M, Cohen B 1998 Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1{alpha}/ARNT. EMBO J 17:5085–5094[CrossRef][Medline]
  14. Richard DE, Berra E, Pouyssegur J 2000 Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1{alpha} in vascular smooth muscle cells. J Biol Chem 275:26765–26771[Abstract/Free Full Text]
  15. Gorlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, Kietzmann T, Busse R 2001 Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22(phox)-containing NADPH oxidase. Circ Res 89:47–54[Abstract/Free Full Text]
  16. Spinella F, Rosano L, Di C, V, Natali PG, Bagnato A 2002 Endothelin-1 induces vascular endothelial growth factor by increasing hypoxia-inducible factor-1{alpha} in ovarian carcinoma cells. J Biol Chem 277:27850–27855[Abstract/Free Full Text]
  17. Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, Semenza GL 2002 Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem 277:38205–38211[Abstract/Free Full Text]
  18. Fukuda R, Kelly B, Semenza GL 2003 Vascular endothelial growth factor gene expression in colon cancer cells exposed to prostaglandin E2 is mediated by hypoxia-inducible factor 1. Cancer Res 63:2330–2334[Abstract/Free Full Text]
  19. Mabjeesh NJ, Willard MT, Frederickson CE, Zhong H, Simons JW 2003 Androgens stimulate hypoxia-inducible factor 1 activation via autocrine loop of tyrosine kinase receptor/phosphatidylinositol 3'-kinase/protein kinase B in prostate cancer cells. Clin Cancer Res 9:2416–2425[Abstract/Free Full Text]
  20. Qian D, Lin HY, Wang HM, Zhang X, Liu DL, Li QL, Zhu C 2004 Normoxic induction of the hypoxic-inducible factor-1{alpha} by interleukin-1ß involves the extracellular signal-regulated kinase 1/2 pathway in normal human cytotrophoblast cells. Biol Reprod 70:1822–1827[Abstract/Free Full Text]
  21. Kasuno K, Takabuchi S, Fukuda K, Kizaka-Kondoh S, Yodoi J, Adachi T, Semenza GL, Hirota K 2004 Nitric oxide induces hypoxia-inducible factor 1 activation that is dependent on MAPK and phosphatidylinositol 3-kinase signaling. J Biol Chem 279:2550–2558[Abstract/Free Full Text]
  22. Gao N, Nester RA, Sarkar MA 2004 4-Hydroxy estradiol but not 2-hydroxy estradiol induces expression of hypoxia-inducible factor 1{alpha} and vascular endothelial growth factor A through phosphatidylinositol 3-kinase/Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells. Toxicol Appl Pharmacol 196:124–135[CrossRef][Medline]
  23. Stroka DM, Burkhardt T, Desbaillets I, Wenger RH, Neil DA, Bauer C, Gassmann M, Candinas D 2001 HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J 15:2445–2453[Abstract/Free Full Text]
  24. Berra E, Pages G, Pouyssegur J 2000 MAP kinases and hypoxia in the control of VEGF expression. Cancer Metastasis Rev 19:139–145[CrossRef][Medline]
  25. Minet E, Michel G, Mottet D, Raes M, Michiels C 2001 Transduction pathways involved in hypoxia-inducible factor-1 phosphorylation and activation. Free Radic Biol Med 31:847–855[CrossRef][Medline]
  26. Semenza G 2002 Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol 64:993–998[CrossRef][Medline]
  27. Wenger RH 2002 Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 16:1151–1162[Abstract/Free Full Text]
  28. Shupnik MA 2004 Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation. Oncogene 23:7979–7989[CrossRef][Medline]
  29. Bjornstrom L, Sjoberg M 2005 Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol 19:833–842[Abstract/Free Full Text]
  30. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407:538–541[CrossRef][Medline]
  31. Simoncini T, Rabkin E, Liao JK 2003 Molecular basis of cell membrane estrogen receptor interaction with phosphatidylinositol 3-kinase in endothelial cells. Arterioscler Thromb Vasc Biol 23:198–203[Abstract/Free Full Text]
  32. Haynes MP, Li L, Sinha D, Russell KS, Hisamoto K, Baron R, Collinge M, Sessa WC, Bender JR 2003 Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. J Biol Chem 278:2118–2123[Abstract/Free Full Text]
  33. Yallampalli C, Dong YL 2000 Estradiol-17ß inhibits nitric oxide synthase (NOS)-II and stimulates NOS-III gene expression in the rat uterus. Biol Reprod 63:34–41[Abstract/Free Full Text]
  34. Han G, Magee T, Khorram O 2005 Regulation of nitric oxide synthase isoforms by estrogen in the human endometrium. Fertil Steril 84(Suppl 2):1220–1227
  35. Collins PD, Connolly DT, Williams TJ 1993 Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo. Br J Pharmacol 109:195–199[Medline]
  36. Chaves MC, Ribeiro RA, Rao VS 1993 Possible involvement of nitric oxide in estrogen-induced uterine edema in the immature rat. Braz J Med Biol Res 26:853–857[Medline]
  37. Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S 2000 Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. J Neurosci Res 60:321–327[CrossRef][Medline]
  38. Ivanova T, Mendez P, Garcia-Segura LM, Beyer C 2002 Rapid stimulation of the PI3-kinase/Akt signalling pathway in developing midbrain neurones by oestrogen. J Neuroendocrinol 14:73–79[CrossRef][Medline]
  39. Castoria G, Migliaccio A, Bilancio A, Di Domenico M, de Falco A, Lombardi M, Fiorentino R, Varricchio L, Barone MV, Auricchio F 2001 PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. EMBO J 20:6050–6059[CrossRef][Medline]
  40. Marquez DC, Pietras RJ 2001 Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells. Oncogene 20:5420–5430[CrossRef][Medline]
  41. Sun M, Paciga JE, Feldman RI, Yuan Z, Coppola D, Lu YY, Shelley SA, Nicosia SV, Cheng JQ 2001 Phosphatidylinositol-3-OH kinase (PI3K)/Akt2, activated in breast cancer, regulates and is induced by estrogen receptor {alpha} (ER{alpha}) via interaction between ER{alpha} and PI3K. Cancer Res 61:5985–5991[Abstract/Free Full Text]
  42. Duan R, Xie W, Li X, McDougal A, Safe S 2002 Estrogen regulation of c-fos gene expression through phosphatidylinositol-3-kinase-dependent activation of serum response factor in MCF-7 breast cancer cells. Biochem Biophys Res Commun 294:384–394[CrossRef][Medline]
  43. Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER 2004 Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol 18:2854–2865[Abstract/Free Full Text]
  44. Lee YR, Park J, Yu HN, Kim JS, Youn HJ, Jung SH 2005 Up-regulation of PI3K/Akt signaling by 17ß-estradiol through activation of estrogen receptor-{alpha}, but not estrogen receptor-ß, and stimulates cell growth in breast cancer cells. Biochem Biophys Res Commun 336:1221–1226[CrossRef][Medline]
  45. Cabodi S, Moro L, Baj G, Smeriglio M, Di Stefano P, Gippone S, Surico N, Silengo L, Turco E, Tarone G, Defilippi P 2004 p130Cas interacts with estrogen receptor {alpha} and modulates non-genomic estrogen signaling in breast cancer cells. J Cell Sci 117:1603–1611[Abstract/Free Full Text]
  46. Dery MC, Leblanc V, Shooner C, Asselin E 2003 Regulation of Akt expression and phosphorylation by 17ß-estradiol in the rat uterus during estrous cycle. Reprod Biol Endocrinol 1:47[CrossRef][Medline]
  47. Lengyel F, Vertes Z, Kovacs KA, Kornyei JL, Sumegi B, Vertes M 2004 Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus. J Steroid Biochem Mol Biol 91:285–288[CrossRef][Medline]
  48. Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS 2002 Requirement of estrogen receptor-{alpha} in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277:8531–8537[Abstract/Free Full Text]
  49. Chen B, Pan H, Zhu L, Deng Y, Pollard JW 2005 Progesterone inhibits the estrogen-induced phosphoinositide 3-kinase–>Akt–>GSK-3ß–>cyclin D1–>pRB pathway to block uterine epithelial cell proliferation. Mol Endocrinol 19:1978–1990[Abstract/Free Full Text]
  50. Guzeloglu Kayisli O, Kayisli UA, Luleci G, Arici A 2004 In vivo and in vitro regulation of Akt activation in human endometrial cells is estrogen dependent. Biol Reprod 71:714–721[Abstract/Free Full Text]
  51. Bokemeyer D, Panek D, Kramer HJ, Lindemann M, Kitahara M, Boor P, Kerjaschki D, Trzaskos JM, Floege J, Ostendorf T 2002 In vivo identification of the mitogen-activated protein kinase cascade as a central pathogenic pathway in experimental mesangioproliferative glomerulonephritis. J Am Soc Nephrol 13:1473–1480[Abstract/Free Full Text]
  52. Einat H, Yuan P, Gould TD, Li J, Du J, Zhang L, Manji HK, Chen G 2003 The role of the extracellular signal-regulated kinase signaling pathway in mood modulation. J Neurosci 23:7311–7316[Abstract/Free Full Text]
  53. Singh VP, Saluja AK, Bhagat L, van Acker GJ, Song AM, Soltoff SP, Cantley LC, Steer ML 2001 Phosphatidylinositol 3-kinase-dependent activation of trypsinogen modulates the severity of acute pancreatitis. J Clin Invest 108:1387–1395[CrossRef][Medline]
  54. Masunaga S, Takahashi A, Ohnishi K, Ohnishi T, Suzuki M, Nagata K, Kinashi Y, Ono K 2003 Effects of p53 status and wortmannin treatment on potentially lethal damage repair, with emphasis on the response of intratumor quiescent cells. Radiat Med 21:120–127[Medline]
  55. Kang WS, Tamarkin FJ, Wheeler MA, Weiss RM 2004 Rapid up-regulation of endothelial nitric-oxide synthase in a mouse model of Escherichia coli lipopolysaccharide-induced bladder inflammation. J Pharmacol Exp Ther 310:452–458[Abstract/Free Full Text]
  56. Hu L, Zaloudek C, Mills GB, Gray J, Jaffe RB 2000 In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res 6:880–886[Abstract/Free Full Text]
  57. Wolfrum S, Dendorfer A, Rikitake Y, Stalker TJ, Gong Y, Scalia R, Dominiak P, Liao JK 2004 Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol 24:1842–1847[Abstract/Free Full Text]
  58. Schabbauer G, Tencati M, Pedersen B, Pawlinski R, Mackman N 2004 PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler Thromb Vasc Biol 24:1963–1969[Abstract/Free Full Text]
  59. Gupta AK, Cerniglia GJ, Mick R, Ahmed MS, Bakanauskas VJ, Muschel RJ, McKenna WG 2003 Radiation sensitization of human cancer cells in vivo by inhibiting the activity of PI3K using LY294002. Int J Radiat Oncol Biol Phys 56:846–853[CrossRef][Medline]
  60. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 15:1292–1300[Medline]
  61. Neugarten J, Medve I, Lei J, Silbiger SR 1999 Estradiol suppresses mesangial cell type I collagen synthesis via activation of the MAP kinase cascade. Am J Physiol 277:F875–F881
  62. Bi R, Broutman G, Foy MR, Thompson RF, Baudry M 2000 The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus. Proc Natl Acad Sci USA 97:3602–3607[Abstract/Free Full Text]
  63. Song RX, McPherson RA, Adam L, Bao Y, Shupnik M, Kumar R, Santen RJ 2002 Linkage of rapid estrogen action to MAPK activation by ER{alpha}-Shc association and Shc pathway activation. Mol Endocrinol 16:116–127[Abstract/Free Full Text]
  64. Chen CC, Lee WR, Safe S 2004 Egr-1 is activated by 17ß-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1. J Cell Biochem 93:1063–1074[CrossRef][Medline]
  65. Arnold SF, Obourn JD, Jaffe H, Notides AC 1994 Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor. Mol Endocrinol 8:1208–1214[Abstract/Free Full Text]
  66. Castano E, Vorojeikina DP, Notides AC 1997 Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation. Biochem J 326:149–157[Medline]
  67. Joel PB, Smith J, Sturgill TW, Fisher TL, Blenis J, Lannigan DA 1998 pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167. Mol Cell Biol 18:1978–1984[Abstract/Free Full Text]
  68. Martin MB, Franke TF, Stoica GE, Chambon P, Katzenellenbogen BS, Stoica BA, McLemore MS, Olivo SE, Stoica A 2000 A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I. Endocrinology 141:4503–4511[Abstract/Free Full Text]
  69. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H 2001 Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor {alpha}: a new model for anti-estrogen resistance. J Biol Chem 276:9817–9824[Abstract/Free Full Text]
  70. Shah YM, Rowan BG 2005 The Src kinase pathway promotes tamoxifen agonist action in Ishikawa endometrial cells through phosphorylation-dependent stabilization of estrogen receptor {alpha} promoter interaction and elevated steroid receptor coactivator 1 activity. Mol Endocrinol 19:732–748[Abstract/Free Full Text]
  71. Vilgelm A, Lian Z, Wang H, Beauparlant SL, Klein-Szanto A, Ellenson LH, Di Cristofano A 2006 Akt-mediated phosphorylation and activation of estrogen receptor {alpha} is required for endometrial neoplastic transformation in Pten+/– mice. Cancer Res 66:3375–3380[Abstract/Free Full Text]
  72. Arnold SF, Obourn JD, Jaffe H, Notides AC 1995 Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: consequence on DNA binding. J Steroid Biochem Mol Biol 55:163–172[CrossRef][Medline]
  73. Tzeng DZ, Klinge CM 1996 Phosphorylation of purified estradiol-liganded estrogen receptor by casein kinase II increases estrogen response element binding but does not alter ligand stability. Biochem Biophys Res Commun 223:554–560[CrossRef][Medline]
  74. Kaluz S, Kaluzova M, Chrastina A, Olive PL, Pastorekova S, Pastorek J, Lerman MI, Stanbridge 2002 Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1{alpha} stabilization: a role for phosphatidylinositol 3'-kinase. Cancer Res 62:4469–4477[Abstract/Free Full Text]
  75. Happ KE, Kazi AA, Koos RD, Inhibition of hypoxia-inducible factor-1{alpha} degradation with cobalt chloride unmasks estradiol induction of vascular endothelial growth factor expression in endometrial cancer cells in vitro. Proc 39th Annual Meeting of the Society for the Study of Reproduction, Omaha, NE, 2006, p 106 (Abstract 154)
  76. Detmar M, Brown LF, Schon MP, Elicker BM, Velasco P, Richard L, Fukumura D, Monsky W, Claffey KP, Jain RK 1998 Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 111:1–6[CrossRef][Medline]
  77. Tibbetts TA, Conneely OM, O’Malley BW 1999 Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus. Biol Reprod 60:1158–1165[Abstract/Free Full Text]
  78. Pedram A, Razandi M, Levin ER 2006 Nature of functional estrogen receptors at the plasma membrane. Mol Endocrinol 20:1996–2009[Abstract/Free Full Text]
  79. Barletta F, Wong CW, McNally C, Komm BS, Katzenellenbogen B, Cheskis BJ 2004 Characterization of the interactions of estrogen receptor and MNAR in the activation of cSrc. Mol Endocrinol 18:1096–1108[Abstract/Free Full Text]
  80. Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, Grohe C 2000 Estrogen receptor {alpha} rapidly activates the IGF-1 receptor pathway. J Biol Chem 275:18447–18453[Abstract/Free Full Text]
  81. Mendez P, Azcoitia I, Garcia-Segura LM 2003 Estrogen receptor {alpha} forms estrogen-dependent multimolecular complexes with insulin-like growth factor receptor and phosphatidylinositol 3-kinase in the adult rat brain. Brain Res Mol Brain Res 112:170–176[Medline]
  82. Filardo EJ, Thomas P 2005 GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release. Trends Endocrinol Metab 16:362–367[CrossRef][Medline]
  83. Levin ER 2005 Integration of the extranuclear and nuclear actions of estrogen. Mol Endocrinol 19:1951–1959[Abstract/Free Full Text]
  84. Lian Z, De Luca P, Di Cristofano A 2006 Gene expression analysis reveals a signature of estrogen receptor activation upon loss of Pten in a mouse model of endometrial cancer. J Cell Physiol 208:255–266[CrossRef][Medline]
  85. Garcia A, Cereghini S, Sontag E 2000 Protein phosphatase 2A and phosphatidylinositol 3-kinase regulate the activity of Sp1-responsive promoters. J Biol Chem 275:9385–9389[Abstract/Free Full Text]
  86. Safe S, Abdelrahim M 2005 Sp transcription factor family and its role in cancer. Eur J Cancer 41:2438–2448[CrossRef][Medline]
  87. Brahimi-Horn C, Mazure N, Pouyssegur J 2005 Signalling via the hypoxia-inducible factor-1{alpha} requires multiple posttranslational modifications. Cell Signal 17:1–9[CrossRef][Medline]
  88. Powell JD, Elshtein R, Forest DJ, Palladino MA 2002 Stimulation of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) protein in the adult rat testis following ischemic injury occurs without an increase in HIF-1{alpha} messenger RNA expression. Biol Reprod 67:995–1002[Abstract/Free Full Text]
  89. Sang N, Stiehl DP, Bohensky J, Leshchinsky I, Srinivas V, Caro J 2003 MAPK signaling up-regulates the activity of hypoxia-inducible factors by its effects on p300. J Biol Chem 278:14013–14019[Abstract/Free Full Text]
  90. Dayan F, Roux D, Brahimi-Horn MC, Pouyssegur J, Mazure NM 2006 The oxygen sensor factor-inhibiting hypoxia-inducible factor-1 controls expression of distinct genes through the bifunctional transcriptional character of hypoxia-inducible factor-1{alpha}. Cancer Res 66:3688–3698[Abstract/Free Full Text]
  91. Zhong H, Chiles K, Feldser D, Laughner E, Hanrahan C, Georgescu MM, Simons JW, Semenza GL 2000 Modulation of hypoxia-inducible factor 1{alpha} expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/Akt/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 60:1541–1545[Abstract/Free Full Text]
  92. Wen S, Stolarov J, Myers MP, Su JD, Wigler MH, Tonks NK, Durden DL 2001 PTEN controls tumor-induced angiogenesis. Proc Natl Acad Sci USA 98:4622–4627[Abstract/Free Full Text]
  93. Huang J, Kontos CD 2002 PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects. J Biol Chem 277:10760–10766[Abstract/Free Full Text]
  94. Abe T, Terada K, Wakimoto H, Inoue R, Tyminski E, Bookstein R, Basilion JP, Chiocca EA 2003 PTEN decreases in vivo vascularization of experimental gliomas in spite of proangiogenic stimuli. Cancer Res 63:2300–2305[Abstract/Free Full Text]
  95. Gomez-Manzano C, Fueyo J, Jiang H, Glass TL, Lee HY, Hu M, Liu JL, Jasti SL, Liu TJ, Conrad CA, Yung WK 2003 Mechanisms underlying PTEN regulation of vascular endothelial growth factor and angiogenesis. Ann Neurol 53:109–117[CrossRef][Medline]
  96. Song RX, Zhang Z, Santen RJ 2005 Estrogen rapid action via protein complex formation involving ER{alpha} and Src. Trends Endocrinol Metab 16:347–353[CrossRef][Medline]
  97. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB 2005 Exploiting the PI3K/Akt pathway for cancer drug discovery. Nat Rev Drug Discov 4:988–1004[CrossRef][Medline]
  98. Yen ML, Su JL, Chien CL, Tseng KW, Yang CY, Chen WF, Chang CC, Kuo ML 2005 Diosgenin induces hypoxia-inducible factor-1 activation and angiogenesis through estrogen receptor-related phosphatidylinositol 3-kinase/Akt and p38 mitogen-activated protein kinase pathways in osteoblasts. Mol Pharmacol 68:1061–1073[Abstract/Free Full Text]
  99. Pillai SB, Rockwell LC, Sherwood OD, Koos RD 1999 Relaxin stimulates uterine edema via activation of estrogen receptors: blockade of its effects using ICI 182,780, a specific estrogen receptor antagonist. Endocrinology 140:2426–2429[Abstract/Free Full Text]
  100. Dessauer CW, Nguyen BT 2005 Relaxin stimulates multiple signaling pathways: activation of cAMP, PI3K, and PKC{zeta} in THP-1 cells. Ann NY Acad Sci 1041:272–279[CrossRef][Medline]
  101. Chen DB, Bird IM, Zheng J, Magness RR 2004 Membrane estrogen receptor-dependent extracellular signal-regulated kinase pathway mediates acute activation of endothelial nitric oxide synthase by estrogen in uterine artery endothelial cells. Endocrinology 145:113–125[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
A. A. Kazi, K. H. Molitoris, and R. D. Koos
Estrogen Rapidly Activates the PI3K/AKT Pathway and Hypoxia-Inducible Factor 1 and Induces Vascular Endothelial Growth Factor A Expression in Luminal Epithelial Cells of the Rat Uterus
Biol Reprod, August 1, 2009; 81(2): 378 - 387.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
M. R. Kim, H. S. Choi, J. W. Yang, B. C. Park, J.-A. Kim, and K. W. Kang
Enhancement of vascular endothelial growth factor-mediated angiogenesis in tamoxifen-resistant breast cancer cells: role of Pin1 overexpression
Mol. Cancer Ther., August 1, 2009; 8(8): 2163 - 2171.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
M. Wang, J. Tan, A. Coffey, J. Fehrenbacher, B. R. Weil, and D. R. Meldrum
Signal transducer and activator of transcription 3-stimulated hypoxia inducible factor-1{alpha} mediates estrogen receptor-{alpha}-induced mesenchymal stem cell vascular endothelial growth factor production
J. Thorac. Cardiovasc. Surg., July 1, 2009; 138(1): 163 - 171.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Yoshie, E. Miyajima, S. Kyo, and K. Tamura
Stathmin, a Microtubule Regulatory Protein, Is Associated with Hypoxia-Inducible Factor-1{alpha} Levels in Human Endometrial and Endothelial Cells
Endocrinology, May 1, 2009; 150(5): 2413 - 2418.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
S. P. Yun, M. Y. Lee, J. M. Ryu, C. H. Song, and H. J. Han
Role of HIF-1{alpha} and VEGF in human mesenchymal stem cell proliferation by 17{beta}-estradiol: involvement of PKC, PI3K/Akt, and MAPKs
Am J Physiol Cell Physiol, February 1, 2009; 296(2): C317 - C326.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Bake, L. Ma, and F. Sohrabji
Estrogen Receptor-{alpha} Overexpression Suppresses 17{beta}-Estradiol-Mediated Vascular Endothelial Growth Factor Expression and Activation of Survival Kinases
Endocrinology, August 1, 2008; 149(8): 3881 - 3889.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kazi, A. A.
Right arrow Articles by Koos, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kazi, A. A.
Right arrow Articles by Koos, R. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals