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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 6112-6118
Copyright © 2004 by The Endocrine Society

Prostaglandin E2 Mediates Phosphorylation and Down-Regulation of the Tuberous Sclerosis-2 Tumor Suppressor (Tuberin) in Human Endometrial Adenocarcinoma Cells via the Akt Signaling Pathway

Kurt J. Sales, Sharon Battersby, Alistair R. W. Williams, Richard A. Anderson and Henry N. Jabbour

MRC Human Reproductive Sciences Unit (K.J.S., S.B., R.A.A., H.N.J.), Centre for Reproductive Biology, and Department of Pathology (A.R.W.W.), The University of Edinburgh Academic Center, Edinburgh EH16 4SB, United Kingdom

Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, MRC Human Reproductive Sciences Unit, Center for Reproductive Biology, The University of Edinburgh Academic Center, 49 Little France Crescent, Old Dalkeith Road, Edinburgh EH16 4SB, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin (PG) E2 promotes tumor growth via interaction with its G protein-coupled receptors and activation of intracellular signaling. Tuberous sclerosis 2 (tuberin) is a tumor suppressor, which negatively regulates cell growth. Its phosphorylation results in its inactivation and targeted down- regulation, thus lifting the growth inhibition effects. This study investigated the expression and localization of tuberin in neoplastic and normal endometrium and the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Quantitative RT-PCR and Western blot analysis demonstrated reduced expression of tuberin in neoplastic tissue, compared with normal endometrial tissue. Tuberin expression was localized by immunohistochemistry to the glandular epithelial compartment in neoplastic and normal endometrium. We investigated the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Treatment of neoplastic and normal endometrium with 100 nM PGE2 enhanced phosphorylated tuberin immunoreactivity in the glandular epithelium. PGE2 also phosphorylated Akt and tuberin in Ishikawa endometrial adenocarcinoma cells, leading to a reduction in expression of total tuberin protein. Cotreatment of cells with wortmannin or LY294002 inhibited the PGE2-induced phosphorylation of Akt and tuberin. These data suggest that PGE2 signaling may promote endometrial tumorigenesis by inactivation of tuberin after its phosphorylation via the Akt signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIAL ADENOCARCINOMA IS one of the leading causes of cancer-related death in the Western world and has a major impact on morbidity (1, 2, 3). Endometrial carcinomas arise from several cell types, with adenocarcinoma arising from the glandular epithelium being the most common type, accounting for 80–90% of all uterine tumors. Prostaglandins (PGs), including PGE2, have been implicated in the etiology of endometrial disorders, including dysfunctional uterine bleeding, endometriosis, and tumorigenesis (4).

PGE2 is biosynthesized from arachidonic acid by cyclooxygenase (COX) enzymes and terminal prostanoid E synthase enzymes (4, 5). Most tumors that highly express COX enzymes, including reproductive tract cancers such as endometrial adenocarcinoma and cervical carcinoma, have also been found to have elevated expression of prostanoid E synthase and biosynthesis of PGE2 (4, 6, 7, 8). PGE2 exerts its autocrine/paracrine effects on target cells by coupling to four subtypes of G protein-coupled receptors, which have been pharmacologically classified as EP1, EP2, EP3, and EP4 (9). Clinical and experimental data indicate that elevated biosynthesis of PGE2 promotes tumorigenesis by enhancing the invasiveness and tumorigenic potential of epithelial cells, promoting vascularization, and inhibiting apoptosis (10, 11, 12). In addition, a negative relationship between PGE2 and tumor suppression has been ascertained. In an in vitro model system of mouse embryo fibroblasts, cells expressing a mutant inactive p53 tumor suppressor gene demonstrated an increase in COX enzyme expression and synthesis of PGE2, compared with those expressing active wild-type p53 (13). Similarly, elevated COX enzyme expression and loss of tumor suppressor function is observed in the tuberous sclerosis complex (TSC) 2 gene mutant rat (Eker rat) (14). Eker rats with an inactivating mutation in TSC2 tumor suppressor function show spontaneous carcinomas coincident with elevated COX enzyme expression (14).

TSC is a familial autosomal multisystem disorder caused by inactivation of the TSC1 and/or TSC2 tumor suppressor genes, which encode the proteins hamartin and tuberin, respectively. The critical role for TSC1 and TSC2 in tumorigenesis has been ascertained from gene ablation studies. Both TSC1+/– and TSC2+/– rodent models develop malignancies in multiple organs, including uterine neoplasms (15, 16, 17). In vivo, the TSC1 and TSC2 gene products, hamartin, and tuberin form active heteromers (18), and their interaction is dependent on the state of phosphorylation of tuberin (19, 20) via intracellular signal transduction pathways, such as the phosphatidylinositol 3 kinase (PI3K)/Akt (protein kinase B) pathway (21). Phosphorylation of tuberin thus leads to its dissociation from the hamartin-tuberin complex and its degradation, abolishing its tumor suppressor function.

As part of a series of ongoing investigations of the mechanisms by which PGs may regulate endometrial function/dysfunction, the present study was designed to investigate the spatial expression and localization of tuberin within normal and neoplastic human endometrium. In addition, localization of phosphorylation of tuberin was investigated by immunohistochemistry in endometrial tissue explants treated with PGE2. Finally, we investigated a possible autocrine/ paracrine regulation of tuberin phosphorylation in endometrial adenocarcinoma cells by PGE2 via the Akt signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

All culture medium was purchased from Life Technologies, Inc. (Paisley, UK). Penicillin-streptomycin and fetal calf serum were purchased from PAA Laboratories Ltd. (Middlesex, UK). The goat antihuman ß- actin antibody (sc-1616) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (Autogenbioclear, Wiltshire, UK). Tuberin (3612), phosphotuberin (3611), Akt (9272), and phospho-Akt (92715) rabbit polyclonal antibodies were purchased from Cell Signaling Technologies (New England Biolabs, Herts, UK). Antirabbit alkaline phosphatase secondary antibodies, indomethacin, PBS, BSA, and PGE2 were purchased from Sigma Chemical Co. (Dorset, UK). An ECF chemiluminescence system was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). Wortmannin (200 µM stock in dimethylsulfoxide) and LY294002 (5 mM stock in dimethylsulfoxide) were purchased from Calbiochem (Nottingham, UK) and stored at –20 C.

Patients and tissue collection

Endometrial adenocarcinoma tissue (n = 24) was collected from women undergoing hysterectomy who had been prediagnosed to have adenocarcinoma of the uterus. All women were postmenopausal and had received no treatment before surgery. The ages of the patients ranged from 50 to 71 yr of age with a median age of 60.5 yr. Hysterectomy specimens for adenocarcinoma were collected from the operating theater and placed on ice. With minimal delay, the specimens were opened by a gynecological pathologist. Small samples (approximately 5 mm to 3 cm) of adenocarcinoma tissue were collected into neutral buffered formalin and wax embedded (for immunohistochemistry studies) or snap frozen in dry ice and stored at –70 C (for RNA extraction) or placed in RPMI 1640 culture medium containing 2 mM L-glutamine, 100 U penicillin, 100 µg/ml streptomycin, and 3 µg/ml indomethacin (to inhibit endogenous COX activity) for in vitro culture. The diagnosis of adenocarcinoma was confirmed histologically in all cases. Normal endometrial tissue (n = 61) at different stages of the menstrual cycle was collected from women undergoing surgery for minor gynecological procedures with no underlying endometrial pathology with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) from women with regular menstrual cycles (25–35 d) and processed exactly as described above. The ages of the control women ranged from 21 to 39 yr of age with a median age of 30.5 yr. None of the control women had received a hormonal preparation in the 3 months preceding biopsy collection. Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to criteria of Noyes et al. (22). Samples obtained on d 1–10, 11–14, 15–18, 19–24, and 25–28 of the menstrual cycle were staged as early-midproliferative, late proliferative, early secretory, midsecretory, and late secretory phases, respectively. Ethical approval was obtained from the Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.

Cell culture

Ishikawa (human endometrial epithelial adenocarcinoma) cells (European Collection of Cell Culture, Centre for Applied Microbiology, Wiltshire, UK) were routinely maintained in DMEM nutrient mixture F-12 with glutamax-1 and pyridoxine, supplemented with 10% fetal calf serum, and 1% antibiotics (stock 500 IU/ml penicillin and 500 µg/ml streptomycin) at 37 C and 5% CO2 (vol/vol).

Taqman quantitative RT-PCR

Endometrial RNA samples were extracted from early-midproliferative (n = 10), late proliferative (n = 7), early secretory (n = 10), midsecretory (n = 10), and late secretory phases (n = 3) of the menstrual cycle and poorly differentiated (n = 3), moderately differentiated (n = 3), and well differentiated (n = 3) endometrial adenocarcinoma tissue using Tri-reagent (Sigma) following the manufacturer’s guidelines. Once extracted and quantified, RNA samples were reverse transcribed using MgCl2 (5.5 mM), deoxynucleotide triphosphates (0.5 mM each), random hexamers (2.5 µM), RNAase inhibitor (0.4 U/µl), and multiscribe reverse transcriptase (1.25 U/µl; all from PE Biosystems, Warrington, UK). The mix was aliquoted into individual tubes (16 µl/tube) and template RNA was added (4 µl/tube of 100 ng/µl RNA). After mixing by brief centrifugation, samples were incubated for 90 min at 25 C, 45 min at 48 C, and 95 C for 5 min. Thereafter cDNA samples were stored at –20 C. A tube with no reverse transcriptase was included to control for any DNA contamination.

To measure cDNA expression, a reaction mix was prepared containing Taqman buffer (5.5 mM MgCl2, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM deoxyuridine 5-triphosphate), ribosomal 18S forward and reverse primers and probe (all at 50 nM), forward and reverse primers for tuberin (300 nM), tuberin probe (200 nM), AmpErase UNG (0.01 U/µl), and AmpliTaq Gold DNA polymerase (0.025 U/µl; all from PE Biosystems). After mixing, 48 µl were aliquoted into separate tubes and 2 µl/replicate (40 ng) of cDNA added and mixed before placing duplicate 24-µl samples into a PCR plate. A no template control (containing water) was included in triplicate. Wells were sealed with optical caps and the PCR carried out using an ABI Prism 7700. Tuberin primers and probe for quantitative PCR were designed using the PRIMER express program (PE Biosystems). The sequence of the tuberin primers and probe were: forward, 5'-TGA AGC AGG AGT CTG ACT GGA A-3'; reverse, 5'-CAC TTT ATA GCG CAG GGA CTC A-3'; and probe (FAM labeled, 6-carboxyfluorescein), 5'-CTG AAG CTG GTT CTG GGC AGG CTG-3'. The ribosomal 18S primers and probe sequences were: forward, 5'-CGG CTA CCA CAT CCA AGG AA-3'; reverse, 5'-GCT GGA ATT ACC GCG GCT-3'; and probe (VIC-labeled, PE Biosystems), 5'-TGC TGG CAC CAG ACT TGC CCT C-3'. Data were analyzed and processed using Sequence Detector (version 1.6.3, PE Biosystems) as instructed by the manufacturer. Expression of the tuberin was normalized to RNA loading for each sample using the 18S ribosomal RNA as an internal standard. Results are expressed relative to an internal standard of normal endometrial RNA as mean ± SEM.

Protein extraction

Cells. For the effect of PGE2 on phosphorylation of tuberin and Akt, 3 x 106 cells were seeded in 10-cm dishes and allowed to attach and grow overnight. The following day, culture medium was aspirated and the cells washed with PBS and incubated in serum-free culture medium containing penicillin/streptomycin and 3 µg/ml indomethacin (a dual COX-enzyme inhibitor used to inhibit endogenous prostanoid biosynthesis) overnight. The next day, cells were either stimulated with 100 nM PGE2 for 0 or 1–5 h or pretreated with specific inhibitors for the PI3K/Akt pathways [wortmannin (20 nM) or LY294002 (5 µM)] for 1 h before stimulation with 100 nM PGE2 for 1 h. After stimulation with PGE2, cells were washed with PBS. Proteins were extracted from cells by lysis on ice in protein lysis buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris/HCl (pH7.4), 1 mM EDTA, 5 mM EGTA, 0.1% sodium dodecyl sulfate (SDS) containing 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 5 µg/ml aprotinin]. Thereafter insoluble material was pelleted by centrifugation at 14,000 x g for 20 min at 4 C. The clarified lysate was removed to a new tube for protein quantification and SDS-PAGE. The protein content in the supernatant fraction was determined using protein assay kits (Bio-Rad Laboratories, Hemel Hempstead, UK). Data represent four independent experiments.

Tissue

Tuberin expression was examined in normal (n = 9) and carcinoma (n = 6) tissue. Tissue was homogenized in protein lysis buffer, clarified by centrifugation, and assayed as described above by Western blot analysis.

Immunoprecipitation and Western blot analysis

For immunoprecipitation, equal amounts of protein were incubated with either tuberin or Akt antibodies overnight at 4 C with gentle rotation. The following day 20 µl protein-A-agarose slurry were added and the samples incubated at 4 C with gentle rotation for 4 h. The beads were washed extensively with lysis buffer and immune complexes eluted in Laemmli buffer [125 mM Tris-HCl (pH6.8), 4% SDS, 5% 2- mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue], boiled for 5 min, and microcentrifuged. Proteins were resolved on 4–12% Tris-glycine gels (NOVEX, Invitrogen, De Schelp, The Netherlands), transferred onto polyvinylidene difluoride membrane (Millipore, Watford, UK), and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25 C in 4% BSA diluted in Tris-buffered saline (TBS) and Tween 20 [50 mM Tris-HCl, 150 mM NaCl, and 0.05% (vol/vol) Tween 20] and incubated with specific primary antibodies. After washing and incubating with secondary antibodies, immunoreactive proteins were visualized by the ECF chemiluminescence system following the manufacturer’s instructions. Proteins were revealed and quantified by PhosphorImager analysis using the Typhoon 9400 system (Molecular Dynamics, Amersham Biosciences, Buckinghamshire, UK). Where appropriate, membranes were stripped and reprobed with antibody recognizing total protein to control for protein loading. Semiquantitative analysis was carried out by dividing the relative density of phosphorylated protein by the relative density obtained from total protein. To control for loading for tuberin, approximately 20 µg protein were taken from the immunoprecipitate lysate to confirm that equal amounts of protein were used for immunoprecipitation. Data are presented as mean ± SEM from four independent experiments.

Immunohistochemistry

Localization of tuberin and phosphorylated tuberin protein expression in normal endometrial tissues (n = 12) and endometrial adenocarcinomas (n = 9) was examined by immunohistochemistry. To determine the site of phosphorylation of tuberin in normal and carcinoma tissue, endometrial explants were incubated overnight in serum free culture medium containing penicillin/streptomycin and 3 µg/ml indomethacin. The next day, explants were treated with vehicle or 100 nM PGE2 for 1 h before paraffin wax embedding. Five-micrometer paraffin wax-embedded tissue sections were cut and mounted onto 3-aminopropyl triethoxysilane-coated slides (Sigma). Sections were dewaxed in xylene, rehydrated in graded ethanol, and washed in water followed by TBS [50 mM Tris-HCl, 150 mM NaCl (pH7.4)] and blocked for endogenous endoperoxidase (1% H2O2 in methanol). Antigen retrieval was performed by pressure cooking (23) for 2 min in 0.01 M sodium citrate (pH6). Sections were blocked using 5% normal swine serum diluted in TBS. Subsequently the tissue sections were incubated with polyclonal rabbit antituberin or rabbit anti-phospho-tuberin antibody at a dilution of 1:200 at 4 C for 18 h. Control tissue was incubated with rabbit IgG. Thereafter the tissue sections were incubated with secondary swine antirabbit antibody followed by streptavidin-peroxidase complex (Dako Corp., High Wycombe, UK) at 25 C for 20 min. Color reaction was developed by incubation with 3,3'-diaminobenzidine (Dako). The tissue sections were counterstained in aqueous hematoxylin, followed by sequential dehydration using graded ethanol and xylene, before mounting and coverslipping.

Statistics

Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 5.0; Abacus Concepts Inc., Carpinteria, CA) and statistical significance accepted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The pattern of expression of tuberin in human endometrium across the menstrual cycle and endometrial adenocarcinoma tissue was investigated by quantitative RT-PCR (Fig. 1Go, A and B) and Western blot (Fig. 1CGo) analysis. The relative mRNA expression of tuberin was significantly reduced during d 11–14 of the menstrual cycle (late proliferative phase; 0.09 ± 0.03; n = 7; P < 0.05), compared with d 1–10 (early-midproliferative phase; 0.19 ± 0.05; n = 10; P < 0.05), d 15–18 (early secretory phase; 0.23 ± 0.03; n = 10; P < 0.05), d 19–24 (midsecretory phase; 0.23 ± 0.05; n = 10, P < 0.05), and d 25–28 (late secretory phase; 0.14 ± 0.02; n = 3; P < 0.05) of the menstrual cycle (Fig. 1AGo). Tuberin mRNA was detected in 70% of all cases of poorly differentiated (P; n = 4), moderately differentiated (M; n = 3), and well-differentiated (W; n = 3) endometrial adenocarcinomas investigated, with no correlation observed between the different grades of adenocarcinoma. The relative expression of tuberin in endometrial adenocarcinoma tissue was determined to be 10.1 ± 0.2-fold lower in endometrial adenocarcinomas (n = 10), compared with normal endometrium across the menstrual cycle (Fig. 1BGo; n = 40; P < 0.05). Western blot analysis of total endometrial protein showed reduced expression of tuberin in the human endometrium during d 12–14 (N1-N3; late proliferative phase) of the menstrual cycle, compared with d 1, 3, and 5 (N4-N6; early-midproliferative phase), d 17 and 18 (N7 and N8; early secretory phase), and d 27 (N9; late secretory phase) of the menstrual cycle (Fig. 1CGo), and confirmed reduced expression of tuberin protein in P, M, and W endometrial adenocarcinoma tissue, compared with normal endometrial tissue across the menstrual cycle (Fig. 1CGo).



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  		FIG. 1. The pattern of expression of tuberin in endometrial biopsies across the menstrual cycle (n = 40) and endometrial adenocarcinomas (n = 10). A, Histogram of relative mRNA expression from early-midproliferative (d 1–10; n = 10), late proliferative (d 11–14; n = 7), early secretory (d 15–18; n = 10), midsecretory (d 19–24; n = 10), and late secretory (d 25–28; n = 3) phases of the menstrual cycle and endometrial adenocarcinomas (n = 4, P; n = 3, M; and n = 3, W adenocarcinomas) as determined by real-time quantitative RT-PCR analysis. B, Fold difference in tuberin mRNA expression in endometrial adenocarcinomas, compared with normal endometrial tissue as determined by real-time quantitative RT-PCR analysis. C, Western blot analysis of protein isolated from d 12–14 (N1-N3; late proliferative phase), d 1, 3, and 5 (N4-N6; early-midproliferative phase), d 17 and 18 (N7 and N8; early secretory phase), and d 27 (N9; late secretory phase) endometrium and P, M, and W adenocarcinoma tissue. The proteins were immunoprecipitated as described in Materials and Methods, loaded onto a 4–12% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred to a polyvinylidene difluoride membrane. The immunoblot was probed with antibody raised against the COOH terminus of human tuberin. (b represents significance from a; P < 0.05). IP, Immunoprecipitation; WB, Western blotting.

 
Localization of tuberin was determined by immunohistochemistry (Fig. 2Go). Tuberin expression was detected across the menstrual cycle in glandular epithelial cells in the early-midproliferative (Figs. 2AGo, d 3; 2B, d 6; and 2C, d 9), late proliferative (Fig. 2DGo, d 12), and midsecretory (Fig. 2EGo, d 21) phase of the menstrual cycle. Expression was generally cytoplasmic with stromal and perivascular cell immunoreactivity (indicated by the arrowhead) present in a high percentage of tissues. In contrast, minimal tuberin immunoreactivity was detected in the glandular epithelium and stromal compartment in P (Fig. 2FGo), M (Fig. 2GGo), and W (Fig. 2HGo) endometrial adenocarcinomas. Incubating sections with nonimmune rabbit IgG (Fig. 2AGo, inset) in place of the primary antibody abolished the positive staining.



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  		FIG. 2. Localization of expression of tuberin protein by immunohistochemistry in d 3, d 6, d 9 (A, B, and C; early-midproliferative phase), d 12 (D; late proliferative phase), and d 21 (E, midsecretory phase) of the menstrual cycle and in P (F), M (G), and W (H) endometrial adenocarcinomas. Inset is shown for immunohistochemistry negative control (A). Scale bar, 100 µm.

 
Localization of phosphorylated tuberin in normal and endometrial adenocarcinoma tissue explants in response to administration of 100 nM PGE2 was investigated by immunohistochemistry (Fig. 3Go). Expression of phosphorylated tuberin in normal and neoplastic tissues was localized to the glandular epithelial compartment. Basal levels of phosphorylation were noticeably lower in carcinoma tissues (Fig. 3CGo), compared with normal endometrium (Fig. 3AGo). Treatment of normal (Fig. 3BGo) and neoplastic (Fig. 3DGo) endometrial tissues with 100 nM PGE2 caused a punctate and granular phospho-tuberin immunoreactivity. Incubating sections with nonimmune rabbit IgG (Fig. 3Go, A and C, inset) in place of the primary antibody abolished the positive staining in the normal and neoplastic tissue respectively.



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  		FIG. 3. Localization of phosphorylated tuberin expression by immunohistochemistry in normal (A and B) and neoplastic (C and D) endometrial tissues in response to treatment with vehicle (A and C) or 100 nM PGE2 (B and D). Insets are shown for the negative controls of normal (A) and neoplastic (C) tissues, respectively. Scale bar, 100 µm.

 
Recent studies suggested that the modulation of the hamartin-tuberin complex is regulated by the PI3K/Akt signaling pathway by phosphorylation of tuberin (18, 21, 24). We investigated PGE2-mediated phosphorylation of Akt and tuberin in endometrial epithelial cells using the Ishikawa neoplastic endometrial epithelial cell line. Ishikawa cells were treated with 100 nM PGE2 for 0, 1, 2, 3, 4, and 5 h, and Akt and tuberin phosphorylation was assessed by Western blotting. Stimulation of Ishikawa cells with PGE2 phosphorylated Akt (Fig. 4AGo) and tuberin (Fig. 4BGo), with maximal phosphorylation observed after 1 h. Coincident with this increase in Akt and tuberin phosphorylation, we observed a time-dependent decrease in the expression of total tuberin protein (Fig. 4BGo, center panel) in response to PGE2 treatment. To determine whether PGE2 phosphorylation of tuberin in endometrial epithelial cells occurs by activation of the Akt signaling pathway, we treated Ishikawa cells with 100 nM PGE2 or vehicle for 1 h in the absence or presence of the PI3K/Akt signaling pathway inhibitors wortmannin or LY294002. A significant increase in phosphorylation of Akt (Fig. 5AGo; P < 0.05) and tuberin (Fig. 5BGo; P < 0.05) was observed in Ishikawa cells after treatment with PGE2 for 1 h, compared with cells stimulated with vehicle alone. Coincident with this increase in phosphorylation of tuberin was a decrease in total tuberin protein expression (Fig. 5BGo). Preincubation of cells with chemical inhibitors of PI3K/Akt signaling (wortmannin or LY294002) abolished the PGE2-induced phosphorylation of both Akt and tuberin and reversed the PGE2-induced reduction of total tuberin protein (P < 0.05).



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  		FIG. 4. A, Representative Western blots demonstrating the effect of PGE2 on Akt signaling and phosphorylation of tuberin in Ishikawa cells. Ishikawa cells were stimulated with 100 nM PGE2 for 0, 1, 2, 3, 4, and 5 h. After lysis, cells were immunoprecipitated with antibody recognizing Akt and subjected to immunoblot analysis using an antibody against phosphorylated Akt. The total amount of Akt in cell lysates was determined by reprobing the same blot with antibody recognizing total Akt protein (lower panel). B, Phosphorylation of tuberin in Ishikawa cells in response to treatment with 100 nM PGE2 for 0, 1, 2, 3, 4, and 5 h. After lysis cells were immunoprecipitated with tuberin antisera and subjected to immunoblot analysis using an antibody against phosphorylated tuberin. The total amount of tuberin in cells after PGE2 stimulation was determined by reprobing the same blot with antibody recognizing total tuberin protein (middle panel). Approximately 20 µg protein, taken from the immunoprecipitate after centrifugation of the agarose as described in Materials and Methods, were immunoblotted for actin as a control to confirm that equal amounts of protein were used for immunoprecipitation (lower panel). Semiquantitative analysis of Akt and tuberin phosphorylation and total tuberin was determined by scanning densitometry software as described in Materials and Methods. Data are presented as mean ± SEM from four independent experiments [b is significantly different from a (P < 0.05); c is significantly different from a and b (P < 0.05); and d is significantly different from a, b, and c (P < 0.05)]. IP, Immunoprecipitation; WB, Western blotting.

 


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  		FIG. 5. Representative Western blots demonstrating the effect of the PI3K/Akt inhibitors wortmannin and LY294002 on PGE2-induced Akt (A) and tuberin phosphorylation (B) in Ishikawa cells. Ishikawa cells were pretreated for 1 h with inhibitors or vehicle followed by stimulation with vehicle, 100 nM PGE2, 100 nM PGE2 + wortmannin, or 100 nM PGE2 + LY294002 for 1 h. After lysis, cells were immunoprecipitated and subjected to immunoblot analysis using antibody against phosphorylated Akt (A), phosphorylated tuberin, or tuberin (B). The total amount of Akt and tuberin in cell lysates was determined by reprobing the same blot with antibody recognizing total protein (lower panel, A; middle panel, B). Actin was used as an additional control to confirm that equal amounts of protein were immunoprecipitated (B). Semiquantitative analysis of Akt and tuberin phosphorylation was determined by scanning densitometry software as described in Materials and Methods. Data are presented as mean ± SEM from four independent experiments (b is significantly different from a, P < 0.05; – denotes absence of agent; + denotes presence of agent). IP, Immunoprecipitation; WB, Western blotting.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Over the past decade, there has been much evidence in support of a role for COX enzymes, prostanoids, and prostanoid receptors in uterine disorders, including uterine carcinoma and benign endometrial pathologies (4, 25). COX-1 and/or COX-2 expression are elevated coincident with elevated prostanoid receptor expression and biosynthesis and signaling of PGE2 (6, 7, 26), suggesting an autocrine/paracrine control of endometrial cell function by prostaglandins. Recently elevated COX-1 expression has been reported in renal carcinomas of the TSC2 mutant rat, suggesting a relationship among the absence of tuberin, aberrant activation of the COX-1 biosynthetic signaling pathway, and tumorigenesis (14). In humans, inactivation of tuberin manifests an autosomal disorder TSC, a tumor susceptibility syndrome affecting approximately 1 in 6000 individuals, characterized by a high incidence of benign and malignant neoplasms (18, 27, 28). To date the possible involvement of the COX/PGE biosynthetic signaling pathway in regulation of tuberin in the human endometrium remains to be determined. To our knowledge, this is the first study to report on the expression and localization of tuberin in human endometrium across the menstrual cycle and human endometrial adenocarcinomas and its possible autocrine/paracrine regulation by PGE2.

The PG biosynthetic signaling pathway has been linked to diverse phenotypic effects on target cells. These include the promotion of angiogenesis, cellular invasion, metastasis, adhesion, and proliferation (11, 29, 30). The data presented in this study demonstrate a significant reduction in expression of tuberin during the late proliferative phase of the menstrual cycle (d 11–14). Furthermore, tuberin expression is dramatically reduced in endometrial adenocarcinoma tissue, compared with normal endometrial tissue. Previous data from our laboratory demonstrated that PGE2 receptor expression and signaling is highest during the mid-late proliferative phase of the menstrual cycle (31) and is further enhanced in endometrial adenocarcinomas, compared with normal endometrium (6). Taken together, these data suggest that proliferation of normal endometrial epithelial cells during the menstrual cycle and aberrant proliferation of endometrial tumor cells may be regulated by PGE2 via a mechanism involving the inactivation of tuberin. Evidence in support of a role for tuberin in aberrant proliferation of uterine cells has been documented in a recent study, in which loss of tuberin function in the uterus promotes unregulated proliferation of mesenchymal cells in uterine leiomyomas by inducing the expression of high mobility group proteins (28), a group of architectural factors that are dormant in normal tissues but reactivated in neoplastic tissues. Loss or inactivation of functional tuberin has also been shown to induce uncontrolled proliferation in several other experimental systems (32). Inactivation of tuberin has been shown to enhance cell growth in TSC2-negative cells (33) as well as in vivo in TSC2 mutant Drosophila melanogaster (34). Transfection of wild-type tuberin into NIH-3T3 cells has been shown to reverse the proliferative effects associated with loss of tuberin (35). Further investigation is needed to address the role of tuberin in cell growth and proliferation, specifically with regard to the endometrium, and in the regulation of uncontrolled growth, characteristic of the TSC.

In vivo, the TSC consists of tuberin and hamartin heteromers, which associate to form a complex (18). Phosphorylation of tuberin, leading to the inactivation and dissociation of the hamartin-tuberin complex, can occur by intracellular activation of the PI3K/Akt signaling pathway (21, 24, 34). Because PGE2 has been shown to activate PI3K/Akt signaling in other model systems (36), we examined whether PGE2 could induce tuberin phosphorylation in endometrial epithelial cells via the Akt signaling pathway. We have shown that PGE2 can phosphorylate tuberin in an Akt-dependent manner and that the phosphorylation of tuberin by Akt in response to PGE2 administration leads to a coincident time-dependent reduction in total tuberin protein. Hence, it is feasible to suggest that after phosphorylation of tuberin by Akt, the tuberin-hamartin complex dissociates and tuberin is targeted for degradation. This phenomenon has been reported in other model systems whereby Akt activation triggers the ubiquitination of tuberin leading to its proteasomal degradation (24). Likewise, in human cervical epithelial cells, Dan et al. (21) report that Akt decreases the levels of tuberin protein, after its phosphorylation, due to protein degradation. The targeting of tuberin for proteasomal degradation could result in subcellular redistribution of tuberin and could explain the punctate granulization of phosphorylated tuberin that we observed in the endometrial tissue explants treated with PGE2.

In conclusion, we have determined that the expression of tuberin is reduced in endometrial adenocarcinoma tissues, compared with normal endometrium, across the menstrual cycle and is phosphorylated in response to PGE2. Furthermore, we have demonstrated that tuberin phosphorylation is regulated in endometrial adenocarcinoma cells by PGE2 via the Akt signaling pathway and that phosphorylation of tuberin leads to a time-dependent reduction in total tuberin protein. Taken together, these findings suggest that a reduction in tuberin expression in endometrial epithelial cells by PGE2 may promote an environment, which is ideal for supporting aberrant neoplastic cell proliferation in endometrial cancer. Furthermore, these data suggest that inhibition of PGE2 function or PI3K/Akt signaling in endometrial carcinomas may be a potential therapeutic target for restoring the growth inhibition effects of tumor suppressors like tuberin. However, caution needs to be exercised with the type of nonsteroidal antiinflammatory drug used because indomethacin is known to be a potent agonist of the prostaglandin D2 receptor (37) and could potentially activate Akt signaling on its own. However, in the light of the present study, it is not known whether human endometrial epithelial cells express either or both DP receptor subtypes or whether DP receptor signaling can activate the Akt pathway.


   Acknowledgments
 
The authors thank Ms. J. Creiger and Ms. S. C. Boddy for sample collection and technical assistance.


   Footnotes
 
Abbreviations: COX, Cyclooxygenase; M, moderately differentiated; P, poorly differentiated; PG, prostaglandin; PI3K, phosphatidylinositol 3 kinase; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TSC, tuberous sclerosis complex; W, well differentiated.

Received May 12, 2004.

Accepted September 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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