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Endocrinology, doi:10.1210/en.2007-1568
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Endocrinology Vol. 149, No. 5 2261-2269
Copyright © 2008 by The Endocrine Society

Premenstrual Regulation of the Pro-Angiogenic Factor CYR61 in Human Endometrium

Isabella Gashaw, Simone Stiller, Carsten Böing, Rainer Kimmig and Elke Winterhager

Institute of Anatomy (I.G., S.S., E.W.) and Department of Obstetrics and Gynaecology (C.B., R.K.), University of Duisburg-Essen, 45122 Essen, Germany

Address all correspondence and requests for reprints to: Isabella Gashaw, Institut für Anatomie II, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany. E-mail: isabella.gashaw{at}uk-essen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pro-angiogenic factor cysteine-rich protein 61 (CYR61/CCN1) mediates different signals in tumorigenesis, angiogenesis and is involved in the pathogenesis of endometriosis. In this study we investigated the temporal and spatial expression pattern in human endometrium during the menstrual cycle and its possible regulation mechanisms in the premenstrual phase. CYR61 transcript expression showed two distinct periods of elevated levels in the proliferative phase and in menstrual effluents. Because the menstrual breakdown of the functionalis is triggered by cytokines, prostaglandins (PGs), as well as hypoxia, we used a benign endometrial cell line to investigate if CYR61 is regulated by these factors. Hypoxic conditions transiently induced CYR61 mRNA levels and enhanced the secretion of the CYR61 protein into the medium. The hypoxia-inducible factor (HIF) 1{alpha} mediated this effect on CYR61 as evidenced by dimethyloxalylglycine treatment and by HIF1{alpha} short interfering RNA. CYR61 mRNA expression was further regulated by IL-1, TNF{alpha}, PGE2, and PGF2{alpha}. In addition, TNF{alpha} and PGE2 elevated significantly CYR61 cellular protein levels in well-oxygenated cells but had only a slight effect on the quantity of secreted protein. Moreover, PGE2 combined with hypoxic conditions increased CYR61 mRNA and protein levels synergistically, whereas the combination with TNF{alpha} abolished the CYR61 levels induced by hypoxia. Together, the up-regulation of CYR61 by hypoxia via HIF1{alpha}, TNF{alpha}, and PGE2 could represent possible mechanisms for the CYR61 increase at the onset of menstruation. The opposite effect of TNF{alpha} combined with hypoxia on CYR61 up-regulation could contribute to a balanced expression level of this angiogenic factor in the endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE MENSTRUAL cycle, the endometrium undergoes distinct morphological and functional changes as a prerequisite for pregnancy. Besides both the ovarian key hormones, estrogen and progesterone, several local cytokines and growth factors are involved in cycling (1). Progesterone withdrawal from the endometrium in midsecretory (MS) phase has been associated with an inflammatory event activating prostaglandins (PGs) and other cytokines such as IL-1, IL-8, and TNF{alpha}, as reviewed elsewhere (2). Moreover, the protein of the hypoxia-inducible transcription factor (HIF) 1 is maximal at progesterone withdrawal during the late secretory (LS) and menstrual phase (3), and is able to induce its angiogenic target gene vascular endothelial growth factor (VEGF) A in endometrial stromal cells (4). VEGF, the best investigated angiogenic factor in the endometrium, is also up-regulated at this stage of the cycle, increasing the permeability of endometrial blood vessels in preparation for endometrial shedding (5, 6). The complex concert of all these factors induces the menstrual breakdown of the functionalis and the beginning of a subsequent cycle. An imbalanced expression of these regulators might direct pathological events in the endometrium such as dysmenorrhea, improper endometrial regeneration, or endometriosis, a common benign chronic condition with a still unexplained pathogenesis (for review, see Ref. 7).

Many angiogenic factors are deregulated during the pathogenesis of endometriosis as reviewed recently (8). Among those, VEGFA was shown to be up-regulated in endometriotic tissues (9, 10, 11, 12). Recently, we found an up-regulation of CYR61 in eutopic and ectopic endometria of women (13) and baboons (12) with endometriosis. These findings support the hypothesis that an adequate vessel supply is required for the development and persistence of endometriotic lesions at ectopic sites.

CYR61 is a member of the CCN family of growth regulators, and acts as a pro-angiogenic factor that mediates different functions in development, cell proliferation, and tumorigenesis (for review, see Ref. 14). Human CYR61 gene encodes a cysteine-rich secreted heparin-binding protein of 381 amino acids with a predicted molecular mass of 42 kDa. Expression of CYR61 mRNA is rapidly induced in an immediate early fashion by a spectrum of stimuli such as growth factors, cytokines, and estrogens (for review, see Ref. 15). CYR61 is a secreted protein that interacts with integrins and heparan sulfate proteoglycans as a link to the extracellular matrix (16, 17). For example, in human skin fibroblasts, CYR61 activates a genetic program for wound healing via cell adhesion through binding to {alpha}6β1 and heparan sulfate proteoglycans, and cell migration via an interaction with {alpha}vβ5, and by inducing cell proliferation through binding to integrin {alpha}vβ3 (18). Proof for the role in angiogenesis comes from Cyr61-deficient mice that die in utero due to vascular defects (19). To understand the possible role of CYR61 in the pathophysiology of the endometrium, we first addressed the question how this factor is regulated during the menstrual cycle in the endometrium. Here, we examined the expression pattern of CYR61 gene and protein in human endometrium. Subsequent investigations of hypoxic and proinflammatory regulation of CYR61 expression in vitro were performed to mimic molecular premenstrual events in the endometrium responsible for the up-regulation of CYR61 in preparation of menstruation and postmenstrual repair. This model system was shown to retain physiological relevant responses to hypoxia and proinflammatory mediators. Our results demonstrate that CYR61 is most expressed in human endometrium during menstruation, and is regulated by hypoxia and inflammatory factors such as TNF{alpha} and PGs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection
Endometrial biopsies (n = 41) were collected from women undergoing gynecological procedures for benign conditions. Patient age ranged from 18–46 yr. All women had regular menstrual cycles (25–35 d) and had not received any hormonal treatments for at least 3 months preceding biopsy. All biopsies were transferred into a buffered saline solution directly after surgery and stored in this buffer for maximal 2 h until further use. An additional 13 menstrual effluents were obtained from six reproductive-aged women (range 22–46; mean age 34) without laparoscopically proven endometriosis. Samples were collected during first 3-d M bleeding, applying the Softcup system (Maxoir BV, Eindhoven, The Netherlands). Written informed consent was obtained from all patients and volunteers before sample collection, and the use of human endometrial tissue was approved by the institutional review board of the University Hospital Essen.

Endometrial biopsies were classified into seven groups according to chronological and histological parameters (20): early proliferative (EP), d 1–5, n = 3; midproliferative (MP), d 6–10, n = 12; late proliferative (LP), d 11–14, n = 11; early secretory (ES), d 15–18, n = 6; MS, d 19–23, n = 4; LS, d 24–28, n = 5; and menstrual, d 1–3, n = 13. For a subpopulation of 16 patients, serum levels of 17β-estradiol, progesterone, LH, and FSH were recorded and considered by staging the biopsies.

Treatment and preparation of endometrial cell line
In vitro studies were performed on human endometrial surface epithelial cell line (HES) (kindly provided by Dr. Asgi T. Fazleabas, University of Illinois, Chicago, IL). These cells were isolated from a proliferative, noncancerous endometrium at hysterectomy and spontaneously immortalized during culture (21).

The cells were subcultured in 50-cm2 flasks in phenol-free DMEM with 10% charcoal-treated newborn calf serum, 10–3 M sodium pyruvate, and 1% penicillin/streptomycin (all reagents are from Invitrogen, Hamburg, Germany).

To investigate hypoxic regulation of CYR61, cells were cultured to 60% confluence in 35- and 60-mm Petri dishes for RNA and protein analyses, respectively. To achieve hypoxic conditions, cells were placed in a Heraeus incubator (ThermoFisher Scientific, Hanau, Germany) with 1% O2, 5% CO2, and N2 as balance for indicated time periods. In parallel, control dishes were incubated under normoxia (21% O2,5% CO2 in air, Heraeus incubator). From all cells, total cellular RNA and protein were extracted. Every experimental approach was performed six times.

Additional tests were performed in six-well plates in triplicates using different cell passages and a range of chemical agents. The following substances were used: dimethyloxalylglycine (DMOG) (10–3 M; Axxora, Lörrach, Germany); PGE2 (10–6 M; Sigma-Aldrich, Steinheim, Germany); PGF2{alpha} (10–6 M; Sigma-Aldrich); TNF{alpha} (10 µg/ml; Peprotech, London, UK); IL-1{alpha} (3 ng/ml; Biovendor, Heidelberg, Germany); and IL-1β, IL-6, and IL-8 (1 ng/ml each, Biovendor). The substances were solved in PBS (DMOG), water (ILs), or ethanol (PGs), and the control groups were supplemented with these vehicles in equal concentration. The incubation times were from 30 min up to 4 h (for details, see Results).

The silencing of HIF1{alpha} with short interfering RNA (siRNA) (Dharmacon, Lafayette, CO) was performed according to Frede et al. (22) by optimizing the conditions for HES cells applying BLOCK-iT fluorescent oligo (Invitrogen). Cells were seeded at 3 x 105 on six-well plates and transfected 1 d later with 10–7 M siRNA using siLentFect reagent (Bio-Rad, München, Germany). Control experiments were performed with siCONTROL Non-Targeting siRNA no. 2 (Dharmacon) using the same conditions. Experiments were started 48 h after transfection, and each experiment was performed three times.

RNA preparation and cDNA synthesis
Tissues were homogenized (Kinematica, Littau-Lucerne, Switzerland) in RLT buffer, and total cellular RNA was prepared using the RNeasy Mini or Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Endometrial cells were processed using the RNeasy Mini Kit. Total RNA (1 µg) was digested with DNase I (Invitrogen) and converted to cDNA as described elsewhere (12).

Quantitative PCR
Quantitative real-time PCR reactions were performed in triplicates using an ABI Prism 7300 Sequence Detector (Applied Biosystems, Weiterstadt, Germany) from a total volume of 20 µl containing 40 ng cDNA, 150 nM gene-specific primers, and Master Green SYBR Green reagent (Applied Biosystems) containing ROX as passive control for signal intensity. CYR61, VEGFA, and HIF1{alpha} were quantified using an external standard cDNA and an additional normalization to β-actin mRNA [actin, β (ACTB)] expression as described elsewhere (12, 22). Melting curve analysis allowed determination of specificity of the PCR fragments.

To investigate the presence of prolyl hydroxylase (PHD) isoforms (PHD2/EGLN1, PHD1/EGLN2, and PHD3/EGLN3) in the endometrium and in HES cells, semiquantitative PCR with 32 cycles and an annealing by 60 C was performed. Primer sequences for PHDs were designed by using the Universal ProbeFinder (Roche Diagnostics, Indianapolis, IN) and are available in Table 1Go. ACTB was coamplified as an internal standard in each experiment, and the signal intensity of the target gene was related to the signal intensity of the ACTB amplicon.


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  		TABLE 1. Oligonucleotide primers used for the semiquantitative PCR

 
Immunofluorescent staining
Immunostainings of CYR61 (1:100, rabbit antihuman antibody, NB100–356; Novus Biologicals, Littleton, CO), CD31 (1:50, mouse antihuman; Dako, Hamburg, Germany), cytokeratin (1:50, mouse antihuman, M0821; Dako), CD163 (1:25, mouse antihuman; HyCult Biotechnology, Uden, The Netherlands), and HIF1{alpha} (1:50, mouse antihuman; Transduction Laboratories, Heidelberg, Germany) were performed on ethanol-fixed cryostat sections from freshly frozen tissues. The following appropriate secondary antibodies were used: donkey antirabbit Alexa Fluor 488 (1:400; MoBiTech, Goettingen, Germany); and Cy3-conjugated goat antimouse (1:400; Dianova, Munich, Germany). Negative controls were performed by omitting the primary antibody and were used to adjust the background fluorescence. Where appropriate, nuclei were identified by 4',6'-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich) using 0.1 µg/ml DAPI in methanol for 15 min at 37 C.

After mounting with Mowiol (Sigma-Aldrich), confocal microscopy was performed using a Zeiss Axiovert 100 microscope and LSM 510 system (Zeiss). CYR61 was detected at 488 nm, CD31, cytokeratin, CD163, as well as HIF1{alpha} at 543 nm, and DAPI at 366 nm, respectively.

Immunoblotting
Tissue lysates were available from 18 endometrial samples from the aforementioned cohort (nine from the proliferative, six from the secretory, and three from the menstrual phase). Whole cell extract preparation from cultured cells was performed according to Frede et al. (22). Cell culture media were collected and centrifuged to discard cellular debris. The supernatant was further processed. Protein samples (50 µg) were separated on a 12% polyacrylamide gel for the analysis of CYR61 expression and on a 7.5% gel for analyzing HIF1{alpha} expression and electrophoretically transferred to polyvinylidene difluoride membranes (Hybond P; GE Healthcare, München, Germany). Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.15% Tween 20 and incubated with primary antibody. The following primary antibodies were used: rabbit polyclonal CYR61 antibody at 1 µg/ml (kindly provided by Dr. N. Schuetze, Wuerzburg, Germany) (23); mouse antihuman HIF1{alpha} (1:100); rabbit antihuman actin antibody (1:600; Sigma-Aldrich); as well as rabbit anti-BSA (1:5000; Sigma-Aldrich). An appropriate secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the ECL kit (Pierce, Rockford, IL) were used for detection using an LAS3000 imaging system (Leica, Bensheim, Germany). For every experimental approach, a minimum of three Western blots was performed, and the precise number is given in the figure legends. The densitometric analyses applied the Scion Image software (Scion Corp., Frederick, MD).

Statistical analysis
The exploratory data analysis, nonparametric Kruskal-Wallis test for group comparisons, as well as the Mann-Whitney U test for the nonparametric independent two-group comparisons, were performed with the program SPSS 14 for Windows (SPSS, Inc., Chicago, IL). Differences with P ≤ 0.05 were regarded as statistically significant and P < 0.01 as highly statistically significant. Values of mRNA quantification are given as mean ± SD or ± SEM for population and in vitro analyses, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CYR61 mRNA and protein throughout the menstrual cycle
CYR61 mRNA was present in all endometrial tissues, biopsies, and menstrual effluents investigated, and the mRNA levels normalized to ACTB varied significantly between the different cycle phases as presented in Fig. 1AGo (Kruskal Wallis test, P < 0.001). CYR61 mRNA expression was in mean 4-fold and significantly higher in the proliferative compared with the secretory phase (Mann-Whitney U test, P = 0.004). The 22-fold decrease of CYR61 mRNA levels from the LP to ES phase was significant (P = 0.002). From the ES phase onwards, CYR61 transcript expression continuously increased with a significant 5-fold change to MS (P = 0.010) and a further significant 7-fold increase from the LS phase to menses (P = 0.003). Maximal levels were detected in menstrual effluents (n = 13, Fig. 1AGo), with a significant, 3-fold up-regulation when compared with all proliferative tissues (n = 26; P < 0.001) and 13-fold increase compared with secretory phase (n = 15; P < 0.001). In menstrual effluents, no significant differences in CYR61 expression from d 1- 3 were found (data not shown).


Figure 1
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  		FIG. 1. Expression of CYR61 mRNA in human endometrium during cycling. A, Endometrial CYR61 mRNA expression of 54 cycling females. Total RNA has been extracted from endometria in the EP (n = 3), MP (n = 12), LP (n = 11), ES (n = 6), MS (n = 4), LS (n = 5), and M (n = 13) of the cycle. The y-axis is scaled logarithmically. Columns represent the mean ratio of CYR61 copy number to ACTB copy number. Error bars represent the SD of the mean. *, P < 0.05. **, P < 0.01. B, Expression levels of CYR61 and VEGFA mRNA in a subset of 35 endometria from the proliferative (P; n = 23), secretory (S; n = 8), and menstrual (M; n = 4) phase of the cycle. The y-axis is scaled logarithmically. Columns represent the mean ratio of CYR61 and VEGFA copy number, respectively, to ACTB copy number. Error bars represent the SD of the mean. *, P < 0.05; **, P < 0.01, significant differences between CYR61 and VEGFA mRNA levels. C, Exemplary immunoblot for CYR61 and actin proteins in tissue extracts from proliferative, secretory, and menstrual endometrium. D, Densitometric analysis of CYR61 protein levels relative to actin expression in 18 tissue samples from proliferative (n = 9), secretory (n = 6), and M (n = 3) endometrium. Columns represent the mean ratio of CYR61 to actin protein amount. Error bars represent the SD of the mean. *, P < 0.05.

 
Because a similar up-regulation during menstruation has already been shown for VEGFA (24, 25), we compared the endometrial expression levels of both CYR61 and VEGFA in a subpopulation of 35 women. Among all investigated samples, we found 6.2-fold higher levels of CYR61 in mean compared with VEGFA (Fig. 1BGo). The major difference in mRNA concentration was observed in menstrual effluents, resulting in a 10-fold higher CYR61 transcript level compared with VEGFA, whereas both pro-angiogenic mediators were expressed at comparable mRNA levels during the secretory phase.

The significant up-regulation of CYR61 protein in menstrual effluents was confirmed by immunoblots as presented in Fig. 1Go, C and D. As shown in Fig. 2Go, CYR61 protein was mainly localized in epithelial and endothelial cells of the endometrium (Fig. 2Go, A–F). Endothelial cells identified by CD31 staining were positive for CYR61 in the proliferative phase (Fig. 2DGo). In secretory and menstrual tissues, not all CD31 stained cells were also positive for CYR61 (Fig. 2Go, E and F). In comparison to endometrial biopsies, menstrual effluents are characterized by a high amount of inflammatory cells, and some of them revealed CYR61 staining (Fig. 2Go, C and F). A colocalization with CD163 identified them as tissue macrophages (Fig. 2FGo, inset).


Figure 2
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  		FIG. 2. Localization of CYR61 protein in human endometrium. A–C, Immunofluorescent colocalization of CYR61 (green) and epithelial cytokeratins (red) in endometrial tissues from the MP (A), LS (B), and M (C) phase of the cycle. D–F, Double staining for CYR61 (green) and the endothelial marker CD31 (red) in endometria from the proliferative (D), secretory (E), and M (F) phase of the cycle. The colocalization of CYR61 and CD31 is indicated by an arrow. Note the CYR61 negative endothelia in the secretory phase (arrowhead). The inset in F presents the immunostaining for CYR61 (green) and the macrophage marker CD163 (red) in the menstrual effluent. The overlay (yellow) represents a colocalization of both proteins (arrow). G–I, Fluorescent labeling of CYR61 (green), HIF1{alpha} (red), and DAPI (blue) in endometrial samples from the LP (G), MS (H), and M (I) phase of the cycle. Note the sporadic HIF1{alpha}-staining in the proliferative sample (G) and the strong nuclear staining of HIF1{alpha} protein in the proliferative gland as well in the menstrual epithelium (arrows).

 
Hypoxia mediated up-regulation of CYR61 expression
The menstrual breakdown is introduced by hypoxic conditions in the superficial zone of functionalis. Endometrial cells express maximal levels of hypoxia-inducible transcription factor HIFI{alpha} upon menstruation (3, 25), and we confirmed these results (Fig. 2Go, G–I) showing nuclear localization of HIF1{alpha} protein in M epithelium. Moreover, we detected the nuclear HIF1{alpha} protein combined with a strong epithelial CYR61 immunostaining (Fig. 2IGo).

To investigate the hypoxic regulation of CYR61, we used in vitro experiments and cultured the benign endometrial epithelial HES cells for up to 24 h under low oxygen (1% O2). HIF1{alpha} protein accumulation was already detected after 1-h hypoxic incubation (Fig. 3AGo).


Figure 3
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  		FIG. 3. CYR61 regulation by hypoxia. A, Exemplary immunoblot for HIF1{alpha} in HES cells incubated for 1, 2, 4, 8, and 24 h under hypoxic conditions compared with normoxia. B, CYR61 expression under hypoxia. Values of mRNA and protein quantification are given as mean expression levels under hypoxia relative to control cells grown under normoxia. Columns represent the means of six independent CYR61 quantifications each in triplicate and bars SEM. *, Significant difference vs. controls (P < 0.05). C, Immunoblot for cellular CYR61 protein relative to actin, which did not differ between cells incubated under hypoxic or normoxic conditions. D, Immunoblot for secreted CYR61 protein (70 kDa). Hypoxic conditions increased the protein concentration.

 
Hypoxic conditions induced a transient significant up-regulation of CYR61 mRNA levels after 2-h incubation (Fig. 3BGo). Shorter treatment or prolonged hypoxia significantly decreased CYR61 transcripts when compared with the expression levels under normoxia.

The intracellular CYR61 protein did not follow the mRNA profile and revealed no significant regulation under hypoxic conditions (Fig. 3Go, B and C). Because CYR61 is a secreted extracellular matrix-associated protein, we next examined the amount of CYR61 protein in the cell culture media. Secreted CYR61 protein was glycosylated and increased in mass from 43–70 kDa. To quantify the amount of protein secreted, we normalized CYR61 to the constant BSA content in the supplemented fetal calf serum (Fig. 3DGo). When incubated under low oxygen, the cells showed an increase in CYR61 secretion into the medium (Fig. 3Go, B and D). The amount of protein secretion now mirrored the mRNA profile with maximal levels 2 h after hypoxia, followed by a steadily decrease after prolonged hypoxia in the culture medium. The protein levels, though decreasing, remained still significantly higher compared with normoxia.

Next, we addressed the question if CYR61 up-regulation under hypoxia might be a result of HIF1{alpha} accumulation. The stability of HIF1{alpha} protein is targeted by oxygen-dependent PHDs (26), which are encoded by three genes: PHD1, PHD2, and PHD3. Because the transcripts are tissue-specific expressed (27), we investigated the expression pattern in endometrial samples and in HES cells under normoxic and hypoxic conditions (Fig. 4AGo). All PHD isoforms were expressed at mRNA level in endometrial tissues and in HES cells as well, giving evidence for a physiologically intact system. DMOG has stabilized HIF1{alpha} through inhibition of HIF prolyl hydroxylation (28, 29). In HES cells the stabilization of HIF1{alpha} protein via DMOG (Fig. 4BGo) increased CYR61 mRNA levels significantly after a 2-h treatment (Fig. 4CGo) and enhanced the protein secretion into the medium after 4-h treatment (Fig. 4Go, C and D). This indirect experimental approach revealed a regulation by HIF1{alpha} on CYR61 expression and protein secretion. This assumption was confirmed using HIF1{alpha} siRNA 48 h before incubation under hypoxic conditions. Application of HIF1{alpha} siRNA abolished the hypoxic stabilization of cellular HIF1{alpha} protein (Fig. 5AGo). The use of HIF1{alpha} siRNA under normoxic conditions led to a 40% decrease of constitutive CYR61 mRNA expression (Fig. 5BGo). This decrease was highest under hypoxic conditions, resulting in a 70% decrease of CYR61 mRNA levels. Moreover, the secretion of CYR61 protein did not change under hypoxia after reduction of HIF1{alpha} protein expression (Fig. 5Go, B–D). Thus, CYR61 expression is regulated downstream of HIF1{alpha} in endometrial epithelial cells.


Figure 4
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  		FIG. 4. CYR61 regulation by HIF1{alpha} protein. A, The PHDs PHD1, PHD2, and PHD3 are expressed in endometrium and in HES cells as presented in this exemplary semiquantitative PCR. ACTB was β-actin cDNA coamplified in the same run. B, Use of DMOG resulted in a time-dependent accumulation of HIF1{alpha} protein. C, Similarly to hypoxic conditions, application of DMOG led to transient increased CYR61 mRNA levels and elevated levels of secreted protein. Values of mRNA and cellular and secreted protein quantification are given as mean expression levels relative to untreated control cells. Columns represent means from four experiments and bars SEM. *, Significant difference vs. controls. D, Exemplary immunoblot of secreted CYR61 protein compared with BSA contained in the medium after treatment with 1 mM DMOG. DMOG treatment slightly increased the amount of CYR61 protein in the cell culture medium. M, Menstrual.

 

Figure 5
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  		FIG. 5. Direct effect of HIF1{alpha} on CYR61 regulation. A, HIF1{alpha} siRNA reduced the level of HIF1{alpha} protein relative to the actin levels in the HES cells. B, Densitometric analysis of CYR61 mRNA levels, cellular protein levels, as well as the amount of secreted CYR61 protein in the cell culture medium after incubations with or without siRNA for HIF1{alpha} under normoxic and hypoxic conditions. Values are given as mean expression levels relative to untreated control cells. Columns represent means from four experiments and bars SEM. *, Significant difference vs. controls treated with control siRNA under normoxic conditions (P < 0.05). C, Immunoblot of cellular CYR61 protein relative to actin after treatment with HIF1{alpha} siRNA under normoxic and hypoxic conditions. D, Immunoblot of secreted CYR61 protein compared with BSA contained in the medium after treatment with HIF1{alpha} siRNA under normoxic and hypoxic conditions. H, 2-h hypoxia (1% O2 in the air); N, normoxia (21% O2 in the air).

 
Cytokine mediated regulation of CYR61 expression
Because a local increase of cytokines and PGs in the endometrium induces the menstrual breakdown, the influence of several proinflammatory mediators on CYR61 expression was investigated (Fig. 6Go). None of the agents tested was able to change significantly CYR61 mRNA levels within 30 min after incubation (data not shown). However, IL-1{alpha}, IL-1β, and TNF{alpha} increased CYR61 mRNA levels with maximal effects after 1-h treatment, whereas incubations with IL-8 had no significant effects (Fig. 6AGo). Both PGs PGE2 and PGF2{alpha} showed an increase on CYR61 transcripts after 2-h treatment.


Figure 6
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  		FIG. 6. CYR61 up-regulation by premenstrual mediators. A, Effects on CYR61 mRNA levels after incubations with IL-1{alpha}, IL-1β, IL-8, TNF{alpha}, PGE2, and PGF2{alpha}. Values are given as mean expression levels relative to vehicle-treated control cells. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). B, Densitometric analysis of CYR61 mRNA levels after incubations with TNF{alpha} under normoxic (black bars), hypoxic conditions (gray bars), and hypoxia (HOX) alone (white bars). Values are given as mean expression levels relative to untreated control cells under normoxic conditions. Same legend applies to D and F. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). C, Densitometric analysis of CYR61 mRNA levels after incubations with PGE2 under normoxic (black bars), hypoxic conditions (gray bars), and hypoxia alone (white bars). Values are given as mean expression levels relative to untreated control cells under normoxic conditions. Same legend applies to E and G. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). D, Densitometric analysis of cellular CYR61 protein levels relative to actin expression after incubations with TNF{alpha} under normoxic (black bars), hypoxic conditions (gray bars), and hypoxia alone (white bars). Values are given as mean expression levels relative to untreated control cells under normoxic conditions. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). E, Densitometric analysis of cellular CYR61 protein levels relative to actin expression after incubations with PGE2 under normoxic (black bars), hypoxic conditions (gray bars), and hypoxia alone (white bars). Values are given as mean expression levels relative to untreated control cells under normoxic conditions. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). Note the significant difference between hypoxia alone and combined effect of hypoxia and PGE2 at 4 h. F, Densitometric analysis of secreted CYR61 protein relative to BSA levels after incubations with TNF{alpha} under normoxic (black bars), hypoxic conditions (gray bars), and hypoxia alone (white bars). Values are given as mean expression levels relative to untreated control cells under normoxic conditions. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). G, Densitometric analysis of secreted CYR61 protein relative to BSA levels after incubations with PGE2 under normoxic (black bars), hypoxic conditions (gray bars), and hypoxia alone (white bars). Values are given as mean expression levels relative to untreated control cells under normoxic conditions. Columns represent means from four experiments and bars SEM. *, Significant difference vs. normoxic, vehicle-treated controls (P < 0.05). H, Exemplary immunoblot of secreted CYR61 protein relative to BSA after treatment with TNF{alpha} under normoxic and hypoxic conditions. I, Exemplary immunoblot of secreted CYR61 protein relative to BSA after treatment with PGE2 under normoxic and hypoxic conditions. NOX, Normoxia.

 
Because of the major effect of TNF{alpha} and the PG PGE2 on CYR61 transcripts, we focused on both in further investigations. TNF{alpha} and PGE2 elevated significantly the expression of CYR61 mRNA and cellular protein under normoxic conditions (Fig. 6Go, B–E) but had no significant impact on CYR61 secretion (Fig. 6Go, F–I). Thus, hypoxia and the proinflammatory mediators seem to act differently on protein expression and secretion. To mimic the physiological situation in the premenstrual endometrium, we combined the treatments with TNF{alpha} and the PG with hypoxic conditions. PGE2 and hypoxia synergistically induced CYR61 mRNA expression (Fig. 6CGo). In addition, the down-regulation of CYR61 after prolonged hypoxia was compensated by the PGE2 supplementation. Now both cellular and secreted CYR61 protein levels were significantly higher under hypoxia compared with normoxic controls (Fig. 6Go, E and G). In contrast, TNF{alpha} treatment combined with low oxygen inhibited the hypoxic up-regulation of CYR61 mRNA, resulting in reduced levels of cellular CYR61 protein (Fig. 6Go, B and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the human endometrium, the pro-angiogenic factor CYR61 is regulated during the menstrual cycle with two distinct periods of elevated mRNA expression in the proliferative phase and menstrual effluents. With the up-regulation upon menstruation, this angiogenic factor follows the same course as VEGFA (24, 25). Thus, both could serve a similar role for in the repair, growth, and maturation of the vasculature in human endometrium after menstruation. For this reason we focused on CYR61 regulation properties in this premenstrual phase using a benign endometrial cell line.

Very recently, another group showed a similar up-regulation of CYR61 mRNA expression in the proliferative phase (30), but in contrast to our findings, the lowest expression of CYR61 transcript was at the MS stage. In our study, CYR61 mRNA levels were continuously increasing from the early to LS phase. The differences observed could be due to differences in the number of biopsies investigated.

CYR61 protein was found mainly in the epithelial cells and in vascular endothelium in the endometrium. which is similar to the spatial pattern of VEGFA (24, 31). However, unlike CYR61, VEGF expression in endothelial cells was only seen during the ES phase (32). This indicates that the pro-angiogenic proteins could serve different roles in the human endometrium. The increase of both pro-angiogenic factors at menstruation could be a result of hypoxic conditions in the endometrium (24, 33). Several studies have previously addressed the link between hypoxia and premenstrual changes in the endometrium. It has been reported that VEGF receptor type 2 (KDR) was dramatically up-regulated during the premenstrual phase (6). A recent study on 16 angiogenic factors noted changes in endometrial VEGF ligands and receptors associated with elevated expression of the hypoxia markers HIF1{alpha} and carboanhydrase IX in the menstrual and EP phases (25). The hypoxia response is mainly mediated by the HIF HIF1{alpha}, as already shown for VEGFA (33, 34). In our cell system, HIF1{alpha} protein was stabilized at 1% O2. Under normoxic conditions, hydroxylation of specific prolyl residues by the oxygen-dependent PHDs targets HIF1{alpha} for proteasomal degradation (28, 35). We could show for the first time that all three PHD isoforms are expressed in the human endometrium and in our in vitro system. We suppose that high expression levels of these enzymes contribute to the high turnover of the HIF1{alpha} protein in endometrial cells, as shown by the absence of the activated HIF1a under normoxia.

CYR61 was previously shown to be regulated via HIF1{alpha} in melanoma cells after 24-h hypoxic conditions at 1% O2 (36). Regulation was evidenced by a HIF1{alpha} interaction with c-Jun/activator protein-1 on CYR61 promoter. Our study presented likewise an involvement of HIF1{alpha} in the initial up-regulation of CYR61 already after 2-h hypoxia in endometrial cells. In contrast to the findings in melanoma cells, prolonged hypoxia of 24 h reduced CYR61 mRNA levels, suggesting a tissue-dependent activation of CYR61 under hypoxia. Moreover, although HIF1{alpha} remained accumulated, CYR61 transcript levels decline upon longer hypoxia indicating additional pathways, which are involved in hypoxic CYR61 suppression.

Because CYR61 is a secreted protein, and it is speculated that the intracellular protein serves other functions than the secreted ones (37), we looked for both and found two discrete regulatory mechanisms. Hypoxic conditions act on an enhanced secretion of CYR61. Thus, endometrial epithelial cells seem to secrete the pro-angiogenic factor at the onset of menstruation, which could fulfill its roles in angiogenesis and postmenstrual repair together with the extracellular matrix. It is likely that CYR61 binds to endometrial integrins such as {alpha}vβ3 or {alpha}4β1 (38) and induces tissue repair processes as already described for the wound healing in the skin (18).

Moreover, the protein seems to be very stable because the levels of secreted CYR61 remain higher than under normoxia for up to 24 h, whereas CYR61 mRNA was significantly down-regulated from 8 h onwards. It has been already described that secreted CYR61 protein has a half-life of greater than 24 h, whereas intracellular CYR61 has an apparent half-life of approximately 30 min (37).

In contrast, both inflammatory mediators TNF{alpha} and PGE2 elevated the cellular protein levels more than the secretion. These differences could point to distinct downstream effects of CYR61 dependent on the mode of activation. We suggest that an up-regulation of the cellular protein levels of CYR61 via proinflammatory mediators directs proliferation of endometrial cells by inducing an intracellular signaling machinery (15).

Although hypoxia is the classical stimulus, HIF1{alpha} gene and protein expression can also be induced through a number of proinflammatory mediators such as nitric oxide, TNF{alpha}, IL-1β, or PGs (39, 40, 41, 42). Critchley et al. (3) demonstrated recently a regulation of HIF1{alpha} by PGE2 and its receptor in endometrial cells. Graubert et al. (24) described a combined effect of hypoxia, TGF{alpha}, and IL-1β on VEGF expression and secretion. Here, we identified a combined effect of PGE2 and HIF1{alpha} on CYR61 expression under normoxic as well as hypoxic conditions. PGE2 is well known to induce angiogenesis (43) and promotes epithelial cell proliferation (44). It has been proposed that it acts upstream of angiogenic processes in the endometrium (3). PGE2 has been recently described to promote endothelial cell migration via ERK activation and angiogenesis (45), and CYR61 has been found downstream of ERK (46). Thus, our results corroborate these findings but need further evaluation if PGE2 and hypoxia synergistically act via CYR61 by inducing angiogenesis in the endometrium. Unexpectedly, incubations of the cells under hypoxia combined with a stimulation by PGE2 or TNF{alpha} led to opposite effects. Only PGE2 potentiated hypoxic effects by inducing CYR61 mRNA and protein levels for a longer period. TNF{alpha} combined with hypoxia significantly reduced CYR61 mRNA levels and abolished the hypoxic up-regulation. The data suggest that TNF{alpha} acts as a physiological antagonist to PGE2 under hypoxia regarding the CYR61 regulation in endometrial epithelial cells. Thus, TNF{alpha} could be responsible for maintaining the CYR61 levels in balance at menstruation under hypoxia.

An imbalanced premenstrual activation of endometrial CYR61 could contribute to pathogenic alterations of the tissue. In a previous study, we presented a feedback mechanism from established endometriotic lesions to induce an increase of CYR61 expression in the eutopic endometrium (12). The paracrine mechanism behind this regulation is thought to involve inflammatory cytokines, which have been described to be amplified in peritoneal fluid and/or sera of patients with endometriosis (47). Interestingly, TNF{alpha}, PGE2, and IL-1 belong to increased factors in endometriosis, and even these mediators induced CYR61 mRNA expression in our in vitro study.

In conclusion, CYR61 up-regulation at the onset of menstruation might direct two distinct signaling pathways involving hypoxia and inflammatory factors. On the one hand, low oxygen combined with PGE2 could serve as a premenstrual preparation of the endometrium by increased production of secreted CYR61, which activates matrix remodeling for tissue shedding. On the other hand, intracellular CYR61 could promote vascular and epithelial growth in the basalis in the course of proliferation and tissue repair during the subsequent cycle.


   Acknowledgments
 
We thank Gabi Sehn, Georgia Rauter, Melanie Gemein, Eva Kusch, and Ulrike Laub for their excellent technical assistance. We also thank Professor Dr. Asgi Fazleabas (University of Illinois, Chicago, IL) for the kind gift of the endometrial cell line used in these studies. We thank Dr. S. Frede and Professor Dr. J. Fandrey from the Institute of Physiology at the University Hospital Essen for their help with our first hypoxia experiments. We also thank Maxoir BV (Eindhoven, The Netherlands) for the donation of the Softcups used in this study.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online January 17, 2008

Abbreviations: ACTB, Actin, β; DAPI, 4',6'-diamidino-2-phenylindole; DMOG, dimethyloxalylglycine; EP, early proliferative; ES, early secretory; HIF, hypoxia-inducible transcription factor; LP, late proliferative; LS, late secretory; MP, midproliferative; MS, midsecretory; PG, prostaglandin; PHD, prolyl hydroxylase; siRNA, short interfering RNA; VEGF, vascular endothelial growth factor.

Received November 13, 2007.

Accepted for publication January 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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