Mol. Hum. Reprod. Advance Access originally published online on May 24, 2007
Molecular Human Reproduction 2007 13(8):595-604; doi:10.1093/molehr/gam032
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Human chorionic gonadotrophin attenuates NF-
B activation and cytokine expression of endometriotic stromal cells
Department of Obstetrics and Gynecology, Medical University of Vienna, AKH, Waehringer Guertel 18-20, A-1090 Vienna, Austria
1 Correspondence address. Tel: +43-1-40400-2842; Fax: +43-1-40400-7842; E-mail: martin.knoefler{at}meduniwien.ac.at
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
Recently, a clinical study provided evidence that treatment of endometriotic women with human chorionic gonadotrophin (hCG) alleviates disease-related pain and sleeplessness suggesting therapeutic effects of the hormone. Since endometriosis is associated with aberrant concentrations of inflammatory mediators in the peritoneal fluid, we investigated whether hCG may affect cytokine-dependent activation of the key-regulatory transcription factor NF-
B and expression of two nuclear factor kappa B (NF-B)-inducible genes, tumour necrosing factor (TNF-
) and interleukin (IL)-1ß, in stromal cells isolated from ectopic endometriotic tissues. Electrophoretic mobility shift assay revealed that treatment of these cultures with the urinary preparation hCG-A suppressed TNF-- or IL-1ß-induced NF-B DNA-binding activity, whereas another urinary hCG preparation (hCG-B) was less effective. Recombinant hCG or epidermal growth factor (EGF), a contaminant of some urinary hCG preparations, did not alter cytokine-dependent NF-B activation. Immunofluorescene of its p65 subunit revealed that pre-incubation with hCG-A strongly decreased TNF--dependent nuclear expression of NF-B. Accordingly, hCG-A diminished IL-1ß-induced TNF- transcript levels and protein release measured by quantitative real-time PCR and enzyme-linked immunosorbent assay. The hormone also attenuated TNF--dependent mRNA expression of IL-1ß. Western blot analyses revealed that hCG-A impaired TNF--mediated phosphorylation and degradation of the inhibitor IB suggesting that the hormone may reduce nuclear import of NF-B by stabilizing its inhibitor. The data suggest that hCG attenuates inflammation-dependent NF-B activation and cytokine expression that could provide one explanation for the beneficial role of the hormone in endometriotic patients.
Key words:
human endometriotic stromal cells/chorionic gonadotrophin/NF-B/cytokine expression
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
Endometriosis is a benign, estrogen-dependent, gynaecological disorder that is defined as the presence of endometrial glands and stroma outside the uterine cavity. Ectopic endometrial tissue is located at the pelvic peritoneum and rarely in the pericardium, pleura or brain, but also often appears as endometriotic ovarian cyst. In particular, the severe forms, deeply infiltrating endometriosis and ovarian cyst endometriosis, are associated with (pelvic) pain as the predominant symptom as well as infertility (Koninckx et al., 1991). As a common disease endometriosis affects
10% of women in their reproductive age and up to 60–80% of women with chronic pelvic pain and/or infertility (Houston, 1984; Koninckx et al., 1991). Despite the fact that endometriosis occurs frequently, pathogenesis of the diseases remains enigmatic.
Many investigators believe that the peritoneal fluid, which mainly originates from the developing follicle/corpus luteum, could play a critical role in the development and preservation of endometriosis (Koninckx et al., 1999). The peritoneal fluid contains high concentrations of steroid hormones, cytokines and other soluble proteins such as CA-125, which serves as one of the clinical markers for the detection of late-stage endometriosis (Bedaiwy and Falcone, 2004). In endometriotic women, the fluid contains increased concentrations of macrophages and activated macrophages with a higher chemotactic activity, which are thought to promote implantation and growth of endometriotic lesions (Weil et al., 1997). Factors involved in the recruitment of macrophages or monocytes/T-cells into the peritoneal cavity such as monocyte chemotactic protein-1 (MCP-1) and RANTES, respectively, are increased in peritoneal fluid of endometriotic women (Akoum et al., 1996a; Khorram et al., 1993). In addition, several other cytokines such as interleukin (IL)-6, IL-1 or tumour necrosing factor (TNF-) were found to be elevated in the fluid and could potentially enhance growth of the ectopic endometrial tissue (Cheong et al., 2002; Kondera-Anasz et al., 2005; Sokolov et al., 2005). Indeed, TNF- was shown to enhance proliferation of endometriotic stromal cells in vitro (Braun et al., 2002). TNF- concentrations in the peritoneal fluid may also have diagnostic value since association with the stage of the disease has been observed (Eisermann et al., 1988; Bedaiwy and Falcone, 2004). Interestingly, treatment with anti-TNF antibodies also reduced the extent of induced endometriosis in baboons (Falconer et al., 2006).
However, in humans, current strategies for the treatment of endometriosis may only temporarily relieve the symptoms of the disease. After laparoscopic excision, endometriotic implants may recur within 2 years in up to 75% of women and further surgery is needed (Candiani et al., 1991). The most widely used medical agents for the treatment of endometriosis are oral contraceptives/progestagens and gonadotrophin-releasing hormone (GnRH) agonists, which suppress ovarian function and thereby menstrual cycle/estrogen-dependent growth of the ectopic endometriotic implants (Giudice and Kao, 2004). However, autonomous production of estrogen and diminished inactivation of the hormone in endometriotic tissue (Zeitoun and Bulun, 1999) cannot be overcome by treatment with GnRH analogue. Suppression of ovarian estrogen production also does not relieve pain in many patients (Lessey, 2000). Moreover, relative progesterone resistance of endometriotic implants has been observed, which could lead to further escalation of estradiol actions on these lesions (Giudice and Kao, 2004). Therefore, due to the heterogeneity of the disease and the high recurrence rate, optimal medical and surgical treatment of individual patients has not yet been clearly defined (Olive and Pritts, 2002).
Recent evidence from our clinics suggested that treatment of endometriotic women with an urinary human chorionic gonadotrophin (hCG) preparation, which is commonly used in ovulation induction and to promote follicle maturation in assisted reproduction, could diminish disease-associated symptoms (Huber et al., 2004). The molecular basis of the putative therapeutic effect, however, is currently unknown. Upon binding to the LH/hCG receptor hCG maintains production of steroid hormones and other growth factors in the corpus luteum until the 7th week of gestation before the luteo-placental shift in progesterone production occurs (Tulchinsky and Hobel, 1973). On the other hand, hCG is known to exert pleiotrophic effects on diverse reproductive cell types such as trophoblasts and endometrial stromal/epithelial cells thereby promoting implantation, decidualization and placental differentiation (Han et al., 1999; Reshef et al., 1990; Shi et al., 1993; Fazleabas et al., 1999). In vitro studies demonstrated that hCG may promote or suppress cell proliferation and motility suggesting cell type-specific actions of the hormone (Horiuchi et al., 2000; Ku et al., 2002; Rao et al., 2004; Shi et al., 2006; Saleh et al., 2007). Expression of the LH/hCG receptor has also been detected in ectopic endometrial implants (Lincoln et al., 1992); however; the biological effects of the hormone on endometriotic cells have not been elucidated so far. Since hCG was recently shown to diminish NF-B activity in several cancer cell lines (Manna et al., 2000), we envisage the possibility that the hormone may also affect the inflammatory response in endometriotic cells. Therefore, we here investigated whether different hCG preparations can modulate cytokine-induced NF-B activation and expression of NF-B-dependent genes in primary stromal cells isolated from ectopic endometrial tissues.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
Collection of endometriotic tissue
Ectopic endometrial tissues were obtained by laparoscopic surgery of women suffering from endometriosis. Isolation of the material was approved by the ethical committee of the Medical University of Vienna and required informed consent of the patients. Women did not receive any medication before laparoscopic excision. Stage of menstrual cycle at the time of surgery was identified. In total, 31 endometriotic tissues (19 ovarian cysts and 12 ectopic, peritoneal lesions) were obtained for primary cell isolation. Of the 19 ovarian cysts, 8 and 11 tissue samples were derived from the proliferative and secretory phase of the menstrual cycle, respectively. Of the 12 peritoneal tissues, 4 and 8 samples were obtained from the proliferative and secretory phase, respectively.
Preparation and stimulation of primary endometriotic stromal cells
Endometriotic stromal cells were prepared by enzymatic digestion as described by different authors (Osteen et al., 1989; Iwabe et al., 2000; Iba et al., 2004). Briefly, endometriotic specimens were washed in Hank's Balanced Salt Solution (HBSS), minced in 3 mm3 pieces and digested with 2 mg/ml DispaseI (2.2 IU/ml), 2 mg/ml Collagenase (484 IU/ml) and 0.5 mg/ml DNAseI in HBSS (containing 25 mM HEPES) by shaking at 37°C for 60–90 min. Supernatants were routinely checked every 15 min for accumulation of dispersed cells. Subsequently, supernatant was filtered through a 40-µm nylon mesh to remove undigested tissue pieces/endometrial glands. Isolated cells were washed twice in HBSS and seeded in DMEM/F-12 supplemented (Biochrom, Berlin, Germany) with 50 µg/ml gentamycin and 10% fetal bovine serum (Biochrom) for further cultivation. All cell preparations were routinely seeded in chamber slides and stained by immunofluorescene at the beginning and end (passage 2–3) of the culture period using antibodies against epithelial (cytokeratin 7, DAKO, 15.4 µg/ml), stromal (vimentin, DAKO, 0.65 µg/ml), immune (CD45, DAKO, 129 µg/ml; CD56, NeoMarkers/Lab Vision Corporation, Fremont, CA, 2 µg/ml) and endothelial (von Willebrand factor, DAKO, 15 µg/ml) cell markers. All primary isolates were 99% vimentin-positive, 1% cytokeratin-7-positive, but negative for CD45, CD56 or von Willebrand factor. To prove endometriotic origin of cultures, aliquots of all cell preparations were seeded in 24 wells and incubated with 0.5 mM 8-bromo-cAMP (Sigma, St Louis, MO, USA) for 3, 6, 9 and 12 days (medium containing fresh 8-bromo-cAMP was changed every 3rd day) for induction of decidualization as described (Klemmt et al., 2006). Subsequently, prolactin mRNA was measured using semi-quantitative PCR, as shown previously (Kimatrai et al., 2003), as well as quantitative real-time PCR. Basal and cAMP-inducible expression of prolactin mRNA could be detected in all stromal cell preparations (data not shown). For inflammatory stimulation, cultures were grown to 80% confluence and incubated with recombinant 1 ng/ml TNF- (Strathmann Biotech GmbH, Hannover, Germany) or 1 ng/ml IL-1ß (R&D systems, Minneapolis, MN, USA) for indicated time periods. To analyse hormone-dependent effects, endometriotic stromal cells were pre-incubated with 50 or 500 IU/ml hCG for 12 h. Pre-incubation with hCG-A for 24 or 48 h did not improve the suppressive effects of the hormone (not shown). The following hCG preparations were utilized: hCG-A isolated from urine of pregnant women (Pregnyl, Organon, UK), hCG-B from urine of pregnant women (C-0434, Sigma) and recombinant -subunit of hCG (Ovidrel, Serono, Boston, MA, USA). Twelve hours of pre-incubation with either 10 or 20 ng/ml recombinant epidermal growth factor (EGF) (Strathman Biotech GmbH) were used as a control.
Immunocytochemistry
Cells were seeded in 8 well chamber slides (Nunc, Wiesbaden, Germany), pre-incubated with 500 IU/ml hCG-A as described above and treated with 1 ng/ml TNF- for 30 min. Subsequently, cells were fixed with 4% formaldehyde and 0.1% Triton-X, blocked with 0.5% gelatin from cold water fish skin (Sigma) and incubated for 1 h with 10 µg/ml goat anti-NFB-p65 antibody (C-20, sc-372X, Santa Cruz) at 37°C. Signal detection was performed by adding 2 µg/ml of Alexa Flour 488 donkey anti-goat immunoglobulin G (A-11055, Molecular Probes) for 30 min at 37°C. Nuclei were counterstained with DAPI (4',6-diamidine-2'-phenylindole dihydrochloride, Roche), and the slides were mounted with Flouromont G (Southern Biotechnology, Birmingham, UK). Slides were analysed by fluorescence microscopy (Olympus BX50) and digitally photographed. Each four different fields of 150–300 cells were counted under the fluorescence microscope, and the ratio of NF
B-p65-positive nuclei to DAPI-stained nuclei was evaluated.
Semi-quantitative and quantitative real-time PCR
After pre-treatment with 500 IU/ml hCG-A for 12 h, cells (seeded in 24 wells) were incubated with 1 ng/ml TNF- for an additional 3 h. Initial investigations at 1.5, 3, 4 and 6 h revealed that inducible cytokine expression was highest at 3 h of stimulation. Total RNA was extracted by direct lysis in the culture dishes using TriFast Reagent (PeqLab, Erlangen, Germany) according to the manufacturer's instructions. The RNA amount and integrity were evaluated using the Agilent Bioanalyzer 2100 (Agilent, Palo Alo, CA, USA). RNA (2 µg) was reverse transcribed using 200 U of RevertAid H Minus M.MuLV Reverse Transcriptase (Fermentas, St Leon-Rot, Germany), 0.4 µl Hexanucleotide Mix (Roche) and 0.1 mM dNTP (Fermentas) in a final volume of 20 µl.
Semi-quantitative PCR amplification (45 s 96°C, 1 min annealing temperature, 1 min 72°C) was performed in a RoboCycler Gradient 96 Cycler (Stratagene, Amsterdam, Netherlands) using 1 µl of cDNA, 0.5 U Taq Polymerase (Fermentas), 0.25 µl sense and antisense primer (25 pmol), 1.5 mM MgCl2 and 2.5 mM dNTP mix (Fermentas) for each 25 µl PCR reaction. Cycle numbers were optimized within the linear range of individual PCR reactions. Oligonucleotide primers, annealing temperatures, cycle numbers and fragment lengths were as follows: TNF--sense: 5'-GACACCACCTGAACGTCTCTTC-3', TNF--antisense: 5'-GAGGAGAGGCACATGGAAGG-3' (50°C, 42 cycles, 445 bp); 18SrRNA-sense: 5'-AGGAATTGACGGAAGGGC-ACC-3', 18SrRNA-antisense: 5'-GGACATCTAAGGGCATCACAG-3' (50°C, 18 cycles, 394 bp); IL-1ß-sense: 5'-GAAGTGCTCCTTCCAGGACC-3', IL-1ß-antisense: 5'-GGGCAGACTCAAATTCCAGC-3' (55°C, 28 cycles, 599 bp); prolactin-sense: 5'-GGGTTCATTACCAAGGCCATC-3' prolactin-antisense: 5'-TTCAGGATGAACCTGGCTGAC-3' (54°C, 33 cycles, 276 bp). In all experiments, a possible DNA contamination was checked by negative control RT–PCR in which reverse transcriptase was omitted in the reverse transcription step. The PCR products were analysed on 1% agarose gels containing ethidium bromide and photographed under UV radiation. All PCR fragments were sequence-verified on a 16-capillary sequencer by using the non-radioactive ABI PRISM Terminator Cycle Sequencing Ready Reaction Kit as specified by the supplier (Applied Biosystems, Foster City, CA, USA).
Real-time PCR was performed on the ABI 5700 Sequence Detection System (Applied Biosystems) using Taq Man Gene Expression Assays (TaqMan Universal PCR Master Mix, 20x Taq Man Gene Expression Assay Mix for TNF-, Hs0017128_m1, IL-1ß, Hs00174097_m1, LH/hCG-receptor, Hs 00174885_m1, prolactin, Hs 00168730_m1, TATA-box binding protein (TBP), TaqMan Endogenous Control) according to the manufacturer's instructions.
Calculation of signals was done as suggested in the PE Biosystems Sequence Detector User Bulletin and elsewhere (Leisser et al., 2006). Briefly, Ct (threshold cycle) is defined as the first fluorescent signal reaching statistical significance above background. For each individual condition 
Ct (the difference of CtTNF/Il-1ß/CGreceptor and CtTBP) values are calculated representing normalization to the housekeeping gene. Subsequently, Ct values are built indicating normalization to controls. The amount of target normalized to an endogenous reference and relative to the unstimulated control is then given by 2–Ct.
Electrophoretic mobility shift assay
After incubation with 500 IU/ml Pregnyl for 12 h, cells were stimulated with 1 ng/ml TNF- or 1 ng/ml IL-1ß for 30 min as mentioned (Manna et al., 2000). Initial experiments with 0.1, 1 and 10 ng/ml of the cytokines revealed that 1 ng/ml was sufficient to provoke optimal induction of NF-B binding activity. Nuclear/cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagent according to the manufacturer's instructions (Pierce, Rockford, IL, USA). The protein content was quantified by a Bradford assay (Pierce). Annealing/labelling of complementary oligonucleotide sequences and electrophoretic mobility shift assay (EMSA) were performed as described elsewhere (Knöfler et al., 2004). Oligonucleotide sequences were as follows (binding site underlined (Hou et al., 2004)): NF-B-consensus-sense, 5'-AGTTGAGGGGACTTTCCCAGGC-3', NF-B-consensus-antisense, 5'-GCCTGGGAAAGTCCCCTCAACT-3'. Binding reactions (30 min, RT) were performed in a buffer containing each 4 µg of nuclear extract, 4fm 32P-labelled, double-stranded oligonucleotide, 16 mM Hepes, 0.5% Triton, 0.8 mM DTT, 60 mM KCl, 20 ng/µl dIdC (Roche), 0.1 mM ZnCl2 and 6% glycerin. In supershift experiments, binding reactions were incubated for an additional 30 min with the NFB p65 antibody (C-20) (Santa Cruz Biotech, 0.6 µg/0.1 ml). Electrophoresis of protein–DNA complexes was carried out on 4% polyacrylamide gels (3% glycerin) in the cold (25 mA). Gels were dried and exposed to films (Hyperfilm MP, Amersham Pharmacia Biotech). Quantification of NF-B signals on films was done by densitometrical scanning using alphaEaserFC software (Alpha Innotech, CA, USA).
Western blot analyses
Cells were serum starved for 12 h in the absence or presence of 500 IU/ml hCG-A and subsequently stimulated with 1 ng/ml TNF- for 5, 10, 15 and 30 min. Isolation of total protein was performed by freeze (liquid nitrogen)-thawing in a buffer containing 50 mM Tris–HCl, 125 mM NaCl, 0.1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM DTT, protease inhibitor cocktail 1:100 and HALT Phosphatase Inhibitor Cocktail 1:100 (Pierce). Each 50 µg of protein lysate was separated on 12% SDS/polyacrylamide gels and transferred onto 0.45 µm nitrocellulose (Schleicher & Schuell, Dassel, Germany) in a buffer containing 25 mM Tris · Cl, pH 8.3, 0.5% SDS, 192 mM glycine, 20% methanol. Equal loading of protein was monitored by Ponceau S staining of membranes. Subsequently, membranes were blocked with 5% non-fat dry milk/TBS/0.1% Tween-20 for 1 h at room temperature and incubated with the primary antibody in 5%BSA/TBS, 0.1%Tween-20 at 4°C with gentle shaking over night. The following primary antibodies were used: IB, phospho-IB (Ser32) (1:1000, Cell Signaling, Danvers, MA), anti-actin (1 µg/ml A-2066, Sigma) After 1 h of treatment (room temperature) with secondary antibody (anti-rabbit Ig horse-radish peroxidase linked, Amersham; 1:50.000), signals were developed by using the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. For reprobing, blots were stripped for 30 min in a buffer containing 100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris chloride (pH 6.7) at room temperature and subsequently incubated with a different primary antibody. IB and phospho-IB (Ser32) detected specific signals at 41 kD. Quantification of total IB signals on films was done by densitometrical scanning using alphaEaserFC software (Alpha Innotech).
Enzyme-linked immunosorbent assay
For the quantitative determination of secreted TNF- levels, Quantikine human TNF- immunoassay was utilized according to the manufacturer's instructions (R&D systems). Intra- and inter-assay CV were 5.3 and 6.8%, respectively.
Statistics
Statistical analyses were performed by non-parametric Wilcoxon test or Student's paired t-test using SPSS 14 (SPSS Inc., Chicago, IL, USA). A P-value <0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
The urinary hCG-A was shown to have beneficial effects in the treatment of endometriotic women (Huber et al., 2004). We speculated that the hormone could potentially alter the inflammatory response of endometriotic tissues which might be provoked by activated macrophages and elevated cytokine concentrations. Therefore, we have tested whether hCG-A may affect NF-
B activation in endometriotic stromal cells. EMSA revealed that pre-incubation of different primary cultures with hCG-A diminished the TNF--dependent induction of NF-B (Fig. 1A). Specificity of DNA-binding to the NF-B consensus sequence was verified by supershift analyses using an antibody against the p65 subunit of NF-B. Treatment with hCG-A alone did not provoke activation of NF-B. Similarly, hCG-A also reduced IL-1ß-mediated induction of the inflammatory transcription factor (Fig. 1B). Recent evidence suggested that some urinary hCG preparations, such as hCG-A, contain EGF as a contaminant (Hofmann et al., 1989), which may alter the biological effects of hCG in vitro (Saleh et al., 2007). Therefore, we analysed whether another urinary cell preparation, recently shown to lack EGF (Saleh et al., 2007), may also repress NF-B. Down-regulation of TNF--dependent NF-B activation could also be observed in the presence of hCG-B, however to a lesser extent (Fig. 1C). In contrast, recombinant EGF alone or the recombinant subunit of hCG, hCG, could not suppress inducible binding of NF-B (Fig. 1D). Densitometrical scanning of films revealed that hCG-A and hCG-B diminished NF-B binding activity to 24 and 55%, respectively, at a concentration of 500 IU/ml (Fig. 2).
|
|
Immunocytochemical analyses revealed that stimulation with TNF-
strongly increased the number of nuclei positive for the p65 subunit of NF-B, whereas in the presence of hCG-A few p65-positive nuclei could be detected (Fig. 3A). Quantification of immunofluorescent signals indicated that 80.7% of stromal cell nuclei were positive for NF-B after supplementation of TNF- (Fig. 3B). In contrast, 12.3% p65-positive nuclei were observed upon treatment with both hCG-A and TNF- (Fig. 3B). Of these 82% displayed only weak nuclear p65 staining (not shown) suggesting that the majority of cells responded to the hCG-A treatment.
|
Subsequently, hCG-A-mediated decrease of cytokine expression was investigated using semi-quantitative RT–PCR and quantitative real-time PCR (Fig. 4). In agreement with the NF-
B activation, semi-quantitative RT–PCR showed that recombinant TNF--induced IL-1ß mRNA expression after 3 h of stimulation (Fig. 4A). Similarly, recombinant IL-1ß elevated TNF- transcript levels, whereas hCG-A alone had no effects. Elevated expression of both mRNAs was attenuated in the presence of hCG-A. Quantitative real-time PCR analyses revealed a 27- and 44-fold increase of IL-1ß and endogenous TNF- transcript levels, respectively, after 3 h of incubation with recombinant TNF- (Fig. 4B). Compared with inducible expression, supplementation of hCG-A significantly decreased IL-1ß and TNF- mRNA, 2.7- and 7-fold, respectively, whereas EGF did not alter TNF--mediated cytokine induction. Similarly, recombinant IL-1ß increased IL-1ß and TNF- mRNA levels 275- and 116-fold, respectively (Fig. 4C). In the presence of hCG-A, IL-1ß mRNA was 1.7-fold and TNF- mRNA was 3.9-fold reduced. Again, recombinant EGF did not significantly influence mRNA expression levels. We also compared three different lots of hCG-A with respect to their repressive effects in three different experiments/cell preparations. Real-time PCR demonstrated that the extent of IL-1ß mRNA suppression was not significantly different between distinct preparations (not shown).
|
Interestingly, hCG-A-mediated down-regulation of inducible NF-
B activation and cytokine mRNA expression could only be detected in 11 out of 16 and 14 out of 18 endometriotic cell preparations, respectively. Therefore, we have tested whether the extent of hCG-A-mediated suppression of TNF--induced IL-1ß mRNA levels could be explained by different concentrations of LH/hCG receptor. Indeed, quantitative real-time PCR revealed that non-responsive cell preparations contained significantly less LH/hCG receptor mRNA than responsive cultures (Fig. 5). Therefore, all cell preparations were routinely checked by real-time PCR for hCG-A-mediated repression and non-responsive cultures were excluded from further investigations.
|
Furthermore, protein concentration of the inflammatory mediators was measured in supernatants of endometriotic stromal cell cultures using enzyme-linked immunosorbent assay (ELISA). Compared with negative controls, soluble TNF-
increased 4.7-fold at 6 h of IL-1ß stimulation (Fig. 6). Pre-incubation with hCG-A significantly diminished the inducible TNF- concentration 3.1-fold. Surprisingly, two different high sensitivity ELISAs failed to detect soluble IL-1ß in supernatants of six different stromal cell preparations after stimulation (1.5, 3, 6, 12, 24 and 48 h) with 1 ng/ml TNF-, although all cultures showed inducible/hCG-A-attenuated IL-1ß mRNA expression (not shown). Also, cytokine-inducible mRNA levels of IL-1ß were 64–256-fold higher than those of TNF- (i.e. IL-1ß mRNA appeared 6–8 cycles earlier in real-time PCRs) suggesting that absent IL-1ß protein levels cannot be explained by the relative mRNA concentration.
|
To gain insights into the suppressive mechanism of hCG, one inhibitor of NF-
B, IB was investigated using western blotting (Fig. 7A). In the absence of hCG-A, phosphorylation and degradation of IB could be observed at 10 min of TNF- treatment. IB, a well-known target gene of NF-B (Sun et al., 1993), reappeared at 60 min of TNF- stimulation indicating transient inflammatory response. In contrast, upon pre-incubation with hCG-A, IB expression was largely unchanged at 10 and 15 min of TNF- incubation. Phosphorylation and degradation could only be detected at 30 and 60 min of cytokine stimulation suggesting diminished/delayed NF-B response in the presence of hormone. Quantification of IB signals revealed that the inhibitor significantly decreased to 29.7, 18.1 and 15.9% at 10, 15 and 30 min, respectively, in the absence of hCG (Fig. 7B). Hormone treatment did not significantly change IB levels at 10 and 15 min of TNF- stimulation, but the inhibitor decreased to 78.5 and 71.5%, respectively, at 30 and 60 min of incubation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
Although the definitive cause of endometriosis still remains enigmatic, increased concentrations of activated macrophages, cytokines, chemokines and angiogenic factors in the peritoneal fluid are thought to play a critical role in the maintenance of endometriotic tissues (Weil et al., 1997; Taylor et al., 2002; Hastings and Fazleabas, 2006). Endometriosis can therefore be regarded as a local inflammatory disease. Since proliferation of endometriotic tissues is estrogen-dependent, common treatments of the disease are directed at inhibiting estrogen action or its production from the ovaries using oral contraceptives and GnRH analogues (Valle and Sciarra, 2003). However, creation of an hypoestrogenic milieu in the peritoneal cavity might be impaired by the fact that endometriotic cells produce the steroid hormone in an autonomous manner (Giudice and Kao, 2004). Therefore, alternative therapies such as inhibition of expression of inflammatory and angiogenic mediators are used or currently being developed (Olive et al., 2004). For example, danazol treatment may exert its therapeutic effects by reducing secretion of MCP-1 and IL-6 from endometriotic cells (Akoum et al., 1996b; Jolicoeur et al., 2001). Another potential beneficial substance that may have less side effects than other inflammatory inhibitors could be hCG which affects ovarian function but also exhibits anti-inflammatory properties (Manna et al., 2000). The hormone significantly reduced dyspareunia and dysmenorrhoea and other disease-related parameters such as sleeplessness, irritability and depressive moods in endometriotic women (Huber et al., 2004).
To gain first insights into the molecular properties of hCG, we here investigated whether it may affect the inflammatory response of endometriotic stromal cells. DNA binding activity assays suggested that hCG suppresses TNF- and IL-1ß-mediated induction of the key-inflammatory transcription factor NF-B. These data were confirmed by immunofluorescent staining of its p65-subunit indicating that the hormone strongly reduced cytokine-induced, nuclear translocation of NF-B. Urinary hCG-A (Pregnyl) was predominantly utilized in our in vitro studies since this drug is commonly used in clinical practice. However, urinary hCG preparations such as hCG-A may contain varying amounts of EGF as a major contaminant, which upon application in vitro could considerably affect cellular function (Yarram et al., 2004; Saleh et al., 2007). As a control, we therefore utilized recombinant EGF at a concentration similar to the doses that had been detected in 500 IU/ml of hCG-A as well as urinary hCG-B (Sigma), which was recently shown to lack EGF (Saleh et al., 2007). However, recombinant EGF was unable to impair TNF- and IL-1ß-induced NF-B binding activity as well as NF-B-dependent cytokine mRNA expression. Also, IL-1ß-induced TNF- secretion was not altered by EGF. In accordance, EGF-free hCG-B repressed NF-B activity in endometriotic stromal cells. Suppression of NF-B was noticed at 500 IU/ml of hCG-A and hCG-B, whereas hCG-B was not effective at a concentration of 50 IU/ml. This is in agreement with a previous study using a purified hCG preparation: inhibition of TNF--induced NF-B binding activity in tumour cells was observed at doses of 745 IU/ml (50 ng/ml) and 1490 IU/ml (100 ng/ml), whereas 14.9 IU/ml (1 ng/ml) and 149 IU/ml (10 ng/ml) had no or little effect (Manna et al., 2000). The data therefore suggest that hCG-mediated suppression of NF-B activation is not due to a contaminating component and is operational in all cell types expressing the LH/hCG receptor. In addition, the recombinant -subunit of hCG (Ovidrel) was investigated, which was shown to be similarly effective as urinary hCG in inducing follicular maturation and luteinization in women undergoing assisted reproduction (Chang et al., 2001). However, the substance failed to suppress cytokine-mediated NF-B activation at a concentration of 500 IU/ml, which may question its therapeutic usefulness for the treatment of endometriosis.
Inhibition of NF-B binding activity likely involves the well-characterized IB-dependent inhibitory mechanism. Upon inflammatory stimulation IB becomes phosphorylated and degraded allowing NF-B to enter the nucleus (Thanos and Maniatis, 1995). Besides activation of a large number of inflammation-associated genes, IB is rapidly induced following initial NF-B activation (Sun et al., 1993). The newly synthesized IB protein translocates to the nucleus, removes NF-B from its target promoters and returns it to the cytoplasm thereby controlling the transient NF-B response through an inhibitory feedback mechanism (Arenzana-Seisdedos et al.,1997). TNF--dependent degradation of IB and re-synthesis could also be detected in endometriotic stromal cells. As previously noticed, this response was strongly reduced in the presence of hCG providing an explanation for the inhibitory action of the hormone (Manna et al., 2000). It is probable that stabilization of IB is achieved through hCG-mediated increase in cAMP levels since it was shown that the effects were mimicked upon treatment of cells with a cAMP analogue (Manna et al., 2000).
Since the effects of hCG-dependent NF-B suppression had not been investigated so far, two classical target genes, TNF- and IL-1ß, were chosen for further analyses. Semi-quantitative and quantitative real-time PCR suggested that inhibition of NF-B binding activity significantly reduced TNF- and IL-1ß mRNA expression. With respect to maintenance of endometriotic tissues, TNF- might be of particular interest. The cytokine is produced by peritoneal macrophages, endometriotic stromal and epithelial cells and was found to be elevated in the peritoneal fluid of endometriotic women (Bergqvist et al., 2000; Cheong et al., 2002; Richter et al., 2005). Although TNF- may not increase adhesion of endometriotic tissue to the pelvic mesothelium (Debrock et al., 2006), the cytokine could be an important promoter of cell growth since it was shown to increase proliferation of endometriotic stromal cultures (Braun et al., 2002). Inhibition of cytokine-mediated TNF- secretion by hCG observed in this study may therefore impair autocrine control of endometriotic stromal cell growth. Although TNF- and IL-1ß-dependent induction of IL-1ß mRNA and its repression through hCG has been noticed, significant release of the IL could not be detected. This is in agreement with previous observations indicating that soluble IL-1ß can only be found in a minority of endometriotic cell samples (Bergqvist et al., 2000). We suspect that endometriotic stromal cells either display defects in IL-1ß expression at the translational or post-translational step or require additional factors for IL-1ß secretion, which could be missing in the purified stromal cell preparation. Besides the cytokines investigated here, other soluble inflammatory mediators such as IL-6 or IL-8 are thought to be involved in endometriosis (Calhaz-Jorge et al., 2003; Sokolov et al., 2005). In particular, IL-8, which acts as an autocrine growth factor to the endometrium, could also play a role in maintenance of implanted tissue (Arici et al., 1998). It may well be that expression and secretion of these cytokines are also affected by hCG. On the other hand, published literature suggests that hCG treatment alone can also modulate endometrial expression of cytokines such as LIF, IL-8, IL-6 or IL-1ß, which are thought to play a role in implantation (Uzumcu et al., 1998; Perrier d'Hauterive et al., 2004; Strakova et al., 2005). We envisage the possibility that cytokine expression is finely tuned by hCG. Whereas moderate elevation by hCG may promote implantation and decidualization of normal endometrium, down-regulation of aberrant cytokine expression, such as in endometriotic tissues, could be a mechanism limiting proliferation and cell invasion. Further in vitro studies are needed to delineate hCG-dependent effects of eutopic versus ectopic endometrial tissue.
In conclusion, this study shows that hCG attenuates NF-B activation and expression/release of inflammatory cytokines, which are thought to play a critical role in the maintenance of endometriotic tissues. Further in vitro studies are required to delineate the biological consequences of hCG treatment on endometriotic cell adhesion, proliferation and invasion. Besides anti-inflammatory actions, inhibition of these cellular processes might be an explanation for the beneficial role of the hormone in endometriotic patients. So far, numerous substances such as oral contraceptives and GnRH analogues are used in the treatment of endometriosis, but none of these substances has proven to eradicate the disease. Also, common therapies repressing inflammation and/or ovarian function are usually limited to 6 months due to considerable metabolic side effects (Crosignani et al., 2006). Additional clinical trials are required to investigate whether long-term treatment with hCG might be favourable as compared with the current therapies.
|
Acknowledgement |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
We thank G. Puller for preparation of graphics.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
|---|
Akoum A, Lemay A, McColl S, et al. Elevated concentration and biologic activity of monocyte chemotactic protein-1 in the peritoneal fluid of patients with endometriosis. Fertil Steril (1996a) 66:17–23.[Web of Science][Medline]
Akoum A, Lemay A, Paradis I, et al. Secretion of interleukin-6 by human endometriotic cells and regulation by proinflammatory cytokines and sex steroids. Hum Reprod (1996b) 11:2269–2275.
Arenzana-Seisdedos F, Turpin P, Rodriguez M, et al. Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci. 110:369–378.
Arici A, Seli E, Zeyneloglu HB, et al. Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor. J Clin Endocrinol Metab (1998) 83:1201–1205.
Bedaiwy MA, Falcone T. Laboratory testing for endometriosis. Clin Chim Acta (2004) 340:41–56.[CrossRef][Web of Science][Medline]
Bergqvist A, Nejaty H, Froysa B, et al. Production of interleukins 1beta, 6 and 8 and tumor necrosis factor alpha in separated and cultured endometrial and endometriotic stromal and epithelial cells. Gynecol Obstet Invest (2000) 50:1–6.[Web of Science][Medline]
Braun DP, Ding J, Dmowski WP. Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-alpha in women with endometriosis. Fertil Steril (2002) 78:727–732.[CrossRef][Web of Science][Medline]
Calhaz-Jorge C, Costa AP, Santos MC, et al. Peritoneal fluid concentrations of interleukin-8 in patients with endometriosis depend on the severity of the disorder and are higher in the luteal phase. Hum Reprod (2003) 18:593–597.
Candiani GB, Fedele L, Vercellini P, et al. Repetitive conservative surgery for recurrence of endometriosis. Obstet Gynecol (1991) 77:421–424.[Web of Science][Medline]
Chang P, Kenley S, Burns T, et al. Recombinant human chorionic gonadotropin (rhCG) in assisted reproductive technology: results of a clinical trial comparing two doses of rhCG (Ovidrel) to urinary hCG (Profasi) for induction of final follicular maturation in in vitro fertilization-embryo transfer. Fertil Steril (2001) 76:67–74.[CrossRef][Web of Science][Medline]
Cheong YC, Shelton JB, Laird SM, et al. IL-1, IL-6 and TNF-alpha concentrations in the peritoneal fluid of women with pelvic adhesions. Hum Reprod (2002) 17:69–75.
Crosignani P, Olive D, Bergqvist A, et al. Advances in the management of endometriosis: an update for clinicians. Hum Reprod Update (2006) 12:179–189.
Debrock S, De Strooper B, Vander Perre S, et al. Tumour necrosis factor-alpha, interleukin-6 and interleukin-8 do not promote adhesion of human endometrial epithelial cells to mesothelial cells in a quantitative in vitro model. Hum Reprod (2006) 21:605–609.
Eisermann J, Gast MJ, Pineda J, et al. Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil Steril (1988) 50:573–579.[Web of Science][Medline]
Falconer H, Mwenda JM, Chai DC, et al. Treatment with anti-TNF monoclonal antibody (c5 N) reduces the extent of induced endometriosis in the baboon. Hum Reprod (2006) 21:1856–1862.
Fazleabas AT, Donnelly KM, Srinivasan S, et al. Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity. Proc Natl Acad Sci USA (1999) 96:2543–2548.
Giudice LC, Kao LC. Endometriosis. Lancet (2004) 364:1789–1799.[CrossRef][Web of Science][Medline]
Han SW, Lei ZM, Rao CV. Treatment of human endometrial stromal cells with chorionic gonadotropin promotes their morphological and functional differentiation into decidua. Mol Cell Endocrinol (1999) 147:7–16.[CrossRef][Web of Science][Medline]
Hastings JM, Fazleabas AT. A baboon model for endometriosis: implications for fertility. Reprod Biol Endocrinol (2006) 4((Suppl 1)):S7.[CrossRef][Medline]
Hofmann J, Holzel F, Hackenberg R, et al. Characterization of epidermal growth factor-related proteins from human urinary chorionic gonadotrophin. J Endocrinol (1989) 123:333–340.
Horiuchi A, Nikaido T, Yoshizawa T, et al. HCG promotes proliferation of uterine leiomyomal cells more strongly than that of myometrial smooth muscle cells in vitro. Mol Hum Reprod (2000) 6:523–528.
Hou B, Eren M, Painter CA, et al. Tumor necrosis factor alpha activates the human plasminogen activator inhibitor-1 gene through a distal nuclear factor kappaB site. J Biol Chem (2004) 279:18127–18136.
Houston DE. Evidence for the risk of pelvic endometriosis by age, race and socioeconomic status. Epidemiol Rev (1984) 6:167–191.
Huber AV, Huber JC, Kolbus A, et al. Systemic HCG treatment in patients with endometriosis: a new perspective for a painful disease. Wien Klin Wochenschr (2004) 116:839–843.[CrossRef][Web of Science][Medline]
Iba Y, Harada T, Horie S, et al. Lipopolysaccharide-promoted proliferation of endometriotic stromal cells via induction of tumor necrosis factor alpha and interleukin-8 expression. Fertil Steril (2004) 82((Suppl 3)):1036–1042.[CrossRef][Web of Science][Medline]
Iwabe T, Harada T, Tsudo T, et al. Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab (2000) 85:824–829.
Jolicoeur C, Lemay A, Akoum A. Comparative effect of danazol and a GnRH agonist on monocyte chemotactic protein-1 expression by endometriotic cells. Am J Reprod Immunol (2001) 45:86–93.[CrossRef][Web of Science][Medline]
Khorram O, Taylor RN, Ryan IP, et al. Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol (1993) 169:1545–1549.[Web of Science][Medline]
Kimatrai M, Oliver C, Abadia-Molina AC, et al. Contractile activity of human decidual stromal cells. J Clin Endocrinol Metab (2003) 88:844–849.
Klemmt PA, Carver JG, Kennedy SH, et al. Stromal cells from endometriotic lesions and endometrium from women with endometriosis have reduced decidualization capacity. Fertil Steril (2006) 85:564–572.[CrossRef][Web of Science][Medline]
Knöfler M, Saleh L, Bauer S, et al. Transcriptional regulation of the human chorionic gonadotropin beta gene during villous trophoblast differentiation. Endocrinology (2004) 145:1685–1694.
Kondera-Anasz Z, Sikora J, Mielczarek-Palacz A, et al. Concentrations of interleukin (IL)-1alpha, IL-1 soluble receptor type II (IL-1 sRII) and IL-1 receptor antagonist (IL-1 Ra) in the peritoneal fluid and serum of infertile women with endometriosis. Eur J Obstet Gynecol Reprod Biol (2005) 123:198–203.[CrossRef][Web of Science][Medline]
Koninckx PR, Meuleman C, Demeyere S, et al. Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril (1991) 55:759–765.[Web of Science][Medline]
Koninckx PR, Kennedy SH, Barlow DH. Pathogenesis of endometriosis: the role of peritoneal fluid. Gynecol Obstet Invest (1999) 47((Suppl 1)):23–33.[CrossRef][Web of Science][Medline]
Ku SY, Choi YM, Suh CS, et al. Effect of gonadotropins on human endometrial stromal cell proliferation in vitro. Arch Gynecol Obstet (2002) 266:223–228.[CrossRef][Medline]
Leisser C, Saleh L, Haider S, et al. Tumour necrosis factor-alpha impairs chorionic gonadotrophin beta-subunit expression and cell fusion of human villous cytotrophoblast. Mol Hum Reprod (2006) 12:601–609.
Lessey BA. Medical management of endometriosis and infertility. Fertil Steril (2000) 73:1089–1096.[CrossRef][Web of Science][Medline]
Lincoln SR, Lei ZM, Rao CV, et al. The expression of human chorionic gonadotropin/human luteinizing hormone receptors in ectopic human endometrial implants. J Clin Endocrinol Metab (1992) 75:1140–1144.[Abstract]
Manna SK, Mukhopadhyay A, Aggarwal BB. Human chorionic gonadotropin suppresses activation of nuclear transcription factor-kappa B and activator protein-1 induced by tumor necrosis factor. J Biol Chem (2000) 275:13307–13314.
Olive DL, Pritts EA. The treatment of endometriosis: a review of the evidence. Ann NY Acad Sci (2002) 955:360–372.[Web of Science][Medline]
Olive DL, Lindheim SR, Pritts EA. New medical treatments for endometriosis. Best Pract Res Clin Obstet Gynaecol (2004) 18:319–328.[CrossRef][Medline]
Osteen KG, Hill GA, Hargrove JT. Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens. Fertil Steril (1989) 52:965–972.[Web of Science][Medline]
Perrier d'Hauterive S, Charlet-Renard C, Berndt S, et al. Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium. Hum Reprod (2004) 19:2633–2643.
Rao ChV, Li X, Manna SK, et al. Human chorionic gonadotropin decreases proliferation and invasion of breast cancer MCF-7 cells by inhibiting NF-kappaB and AP-1 activation. J Biol Chem (2004) 279:25503–25510.
Reshef E, Lei ZM, Rao CV, Pridham DD, et al. The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes, and decidua. J Clin Endocrinol Metab (1990) 70:421–430.
Richter ON, Dorn C, Rosing B, Flaskamp C, et al. Tumor necrosis factor alpha secretion by peritoneal macrophages in patients with endometriosis. Arch Gynecol Obstet (2005) 271:143–147.[CrossRef][Medline]
Saleh L, Prast J, Haslinger P, et al. Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation. Placenta (2007) 28:199–203.[CrossRef][Web of Science][Medline]
Shi QJ, Lei ZM, Rao CV, et al. Novel role of human chorionic gonadotropin in differentiation of human cytotrophoblasts. Endocrinology (1993) 132:1387–1395.
Shi SQ, Xu L, Zhao G, et al. Apoptosis and tumor inhibition induced by human chorionic gonadotropin beta in mouse breast carcinoma. J Mol Med (2006) 84:933–941.[CrossRef][Web of Science][Medline]
Sokolov DI, Solodovnikova NG, Pavlov OV, et al. Study of cytokine profile and angiogenic potential of peritoneal fluid in patients with external genital endometriosis. Bull Exp Biol Med (2005) 140:541–544.[CrossRef][Web of Science][Medline]
Sun SC, Ganchi PA, Ballard DW. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science (1993) 259:1912–1915.
Strakova Z, Mavrogianis P, Meng X, et al. In vivo infusion of interleukin-1beta and chorionic gonadotropin induces endometrial changes that mimic early pregnancy events in the baboon. Endocrinology (2005) 146:4097–4104.
Taylor RN, Lebovic DI, Mueller MD. Angiogenic factors in endometriosis. Ann NY Acad Sci (2002) 955:89–100.[Web of Science][Medline]
Thanos D, Maniatis T. NF-kappa B: a lesson in family values. Cell (1995) 80:529–532.[CrossRef][Web of Science][Medline]
Tulchinsky D, Hobel CJ. Plasma human chorionic gonadotropin, estrone, estradiol, estriol, progesterone, and 17 alpha-hydroxyprogesterone in human pregnancy. 3. Early normal pregnancy. Am J Obstet Gynecol (1973) 117:884–893.[Web of Science][Medline]
Uzumcu M, Coskun S, Jaroudi K, et al. Effect of human chorionic gonadotropin on cytokine production from human endometrial cells in vitro. Am J Reprod Immunol (1998) 40:83–88.[Web of Science][Medline]
Valle RF, Sciarra JJ. Endometriosis: treatment strategies. Ann NY Acad Sci (2003) 997:229–239.[CrossRef][Web of Science][Medline]
Weil SJ, Wang S, Perez MC, et al. Chemotaxis of macrophages by a perit- oneal fluid protein in women with endometriosis. Fertil Steril (1997) 67:865–869.[CrossRef][Web of Science][Medline]
Yarram SJ, Jenkins J, Cole LA, et al. Epidermal growth factor contamination and concentrations of intact human chorionic gonadotropin in commercial preparations. Fertil Steril (2004) 82:232–233.[Web of Science][Medline]
Zeitoun KM, Bulun SE. Aromatase: a key molecule in the pathophysiology of endometriosis and a therapeutic target. Fertil Steril (1999) 72:961–969.[CrossRef][Web of Science][Medline]
Submitted on January 18, 2007; resubmitted on March 23, 2007; accepted on March 26, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Sonderegger, P. Haslinger, A. Sabri, C. Leisser, J. V. Otten, C. Fiala, and M. Knofler Wingless (Wnt)-3A Induces Trophoblast Migration and Matrix Metalloproteinase-2 Secretion through Canonical Wnt Signaling and Protein Kinase B/AKT Activation Endocrinology, January 1, 2010; 151(1): 211 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Prast, L. Saleh, H. Husslein, S. Sonderegger, H. Helmer, and M. Knofler Human Chorionic Gonadotropin Stimulates Trophoblast Invasion through Extracellularly Regulated Kinase and AKT Signaling Endocrinology, March 1, 2008; 149(3): 979 - 987. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||