Secondary Logo

Journal Logo

Original article

Expression of Annexin-1 in patients with endometriosis

LI, Chun-yan; LANG, Jing-he; LIU, Hai-yuan; ZHOU, Hui-mei

Author Information
  • Free

Abstract

Endometriosis is a common gynecological disorder, frequently associates with pelvic pain and infertility. It affects 15%—20% of all women in their reproductive life.1 The pathogenesis of endometriosis remains elusive, however, the most widely accepted theory is Sampson's theory of retrograde menstruation, which could explain the presence of endometrial cells in ectopic sites. But retrograde menstruation is a universal phenomenon with viable endometrial cells found in peritoneal fluids of 76% —90% of women, a prevalence much higher than that of endometriosis.2 This finding indicates that there must be further ‘permissive’ factors that promote the implantation and growth of refluxed endometrial cells in women with endometriosis. Various factors are implicated in the pathogenesis of endometriosis, including the characteristics of eutopic endometrium, the biochemical and cellular composition of the peritoneal fluids and the local and systemic immune response.3,4

In our previous study (unpublished), using a two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we compared protein expression profiles in human eutopic endometrium between patients with endometriosis and those without endometriosis, and identified several endometriosis-related proteins. Among them, the protein Annexin-1, a family member of calcium-dependent phospholipid-binding proteins with diverse functions, was dysregulated in women with endometriosis. Annexin-1, was originally described as a glucocoticoid-inducible protein with phospholipase inhibitory actions.5,6 It has been shown to be an endogenous regulator of inflammation in cells of the immune system.7 Recent studies also demonstrate that Annexin-1 exerts significant effects on several other physiological and pathological processes, including cell growth,8 differentiation,9 apoptosis10 and signal transduction.11 All these processes may play an important role in the pathogenesis of endometriosis.

In the present study, to investigate whether Annexin-1 is involved in the pathogenesis of endometriosis, we examined the expression of Annexin-1 in eutopic endometrium of women with or without endometriosis. We also investigated whether Annexin-1 presented in the peritoneal fluids of women with endometriosis.

METHODS

Tissue collection

Eutopic endometriums of the study group were obtained from 25 patients (mean age (34.48±5.93) years; range 26 —43 years) with endometriosis undergoing laparoscopy or laparotomy. All the patients were surgically and histologically diagnosed with endometriosis at stages II to IV according to the revised American Society for Reproductive Medicine Classification (1997). Peritoneal fluids were obtained from 10 such patients who underwent laparoscopy at the beginning of the procedure. In a control group, endometrial tissues were collected from patients (mean age 34.55±7.61 years; range 26—46 years) undergoing laparoscopy or laparotomy for benign gynecological conditions. Sixteen patients with benign ovarian cyst or cervical intraepithelial neoplasma were included in the control groups. All the patients were surgically proven to be free of endometriosis. Consent forms were signed by each participant and the project was approved by the Ethic Committee of Peking Union Medical College. No patients included in our study received any hormonal preparations within 6 months before surgery and all of them had normal menstrual cycles. Once obtained, the tissue samples were immediately frozen in liquid nitrogen. Peritoneal fluids were centrifuged at 1700 × g for 10 minutes and the supernatants were stored at — 80°C until used for Western blotting.

The day of the menstrual cycle was established from patients’ menstrual history and was confirmed by endometrial histology. All endometrial samples were grouped according to the menstrual cycle phase: proliferative (days 1 — 14 of the cycle) and secretory (days 15—28). Among eutopic endometrium of patients with endometriosis, 14 samples were in the proliferative and 11 in the secretory phase, whereas in the control group, 8 were in the proliferative and 8 in the secretory phase of the cycle.

RNA isolation and complementary DNA synthesis

Total RNA was extracted from endometrial tissues using TRIzol reagent (Invitrogen Life Technologies, Inc, UK) according to the manufacturer's instructions. First strand cDNA was reverse-transcribed from total RNA using the Superscrip First-Strand Synthesis System for RT-PCR ((Invitrogen Life Technologies, Inc, UK) following the manufacture's instructions, and was used as the template in the real-time quantitive PCR analysis.

Real-time PCR

Real-time PCR analysis were performed with a 7300 Fast Real-Time PCR System (Applied Biosystems), using the relative standard curve method. Annexin-1 and β-actin expression was detected in a 20 μl volume, containing 5×real-time PCR buffer 4 μl, 0.25 mmol/L dNTP mixture, 6.25 mmol/L Mg2+ solution, 50 U/ml TaKaRa Ex Taq HS, 10×SYBR Green I 1 μ1, 1 μl cDNA template, 0.5 μmol/L of each primer. Annexin-1 forward primer is 5′-GCAGGCCTGGTTTATTGAAA-3′, and reverse primer is 5′-GCTGTGCATTGTTTCGCTTA-3′; β-actin forward primer is 5′-CGGGAAATCGTGCGTGACATT-3′, and reverse primer is 5′-GGAGTTGAAGGTAGTTTCGTGG-3′. Amplification conditions include holding for 10 minutes at 95°C, 40 cycles of denaturing for 15 seconds at 95°C, annealing for 20 seconds at 60°C and extending for 30 seconds at 72°C. β-actin represents a non-regulated gene, and its expression served as an internal control and was used to normalize for variance in input cDNA. All measurements were performed in triplicate. Data were present as the relative average value of the investigated gene and normalized with the average value of the housekeeping gene β-actin. Melting curves were analyzed to confirm amplification specificity.

Immunohistochemistry

Endometrial tissues were fixed in 10% neutral buffered formalin and embedded into paraffin blocks. Tissue blocks were sectioned at 5 μm and mounted on 3-aminopropyltriethoxysilane (APES)-coated slides. The following procedures were performed using DakoCytomation EnVision+system-HRP labeled polymer (DakoCytomation Inc, California, USA) according to the instructions. Antigen retrieval was performed by microwave, and endogenous peroxide activity was quenched with Peroxidase Block for 5 minutes. Sections were then incubated with mouse polyclonal antibody raised against human Annexin-1 (dilution 1:50, BD Biosciences, NJ, USA) for 30 minutes at room temperature. Peroxidase labeled polymer was incubated for 30 minutes at room temperature, followed by the substrate-chromogen for 3 minutes. Sections were counterstained with Harris’ hematoxylin. All stainings were performed with the same procedure but with the omission of the primary antibody as a negative control. Immunostaining was evaluated by two independent pathologists, blind to the identity of the subject groups.

Western blot analysis

Equal amounts of total protein extracted from eutopic endometrium specimens and peritoneal fluids were separated by electrophoresis on a 12% gradient polyacrylamide gel and transferred onto a nitrocellulose membrane (GIBCOBRL Life Technologies Inc, USA). The membrane was blocked with 5% dry nonfat skimmed milk powder (Santa Cruz Biotechnology, USA) in 0.1% Tween-20-TBS for 1 hour, then incubated with mouse anti-human Annexin-1 (BD Biosciences, NJ, USA) diluted in 1:2000 for 2 hours. After rinsing in 0.1% Tween-20-TBS the membrane was incubated with HRP-labeled anti-mouse IgGs (Santa Cruz Biotechnology) for 1.5 hours, then incubated with an enhanced chemiluminescence system (Santa Cruz Biotechnology) for 1 minute and exposed to Kodak BioMax film for 1—2 minutes till all bands were visible but not overexposed. β-actin was used as an internal control for protein loading and transfer. All the samples were tested in triplicate.

Statistical analysis

Cycle threshold (CT) values were used as a measure of mRNA expression. Final results (ΔCT) were transformed from exponential into linear space through 2(-ΔCT) calculation method. The relative copy numbers were evaluated by 2(ΔCT) values. Relative protein level of Annexin-1 was evaluated by the values of blot brightness. Data were expressed as means ± standard deviation (SD). Differences in mRNA and protein were analyzed by nonparametric Mann-Whitney U test. Differences were considered as statistically significant for P <0.05.

RESULTS

Quantitive expression of Annexin-1 mRNA in eutopic human endometrium

In real-time PCR analysis, the Annexin-1 transcript was detected in all 41 eutopic endometrial specimens. Annexin-1 mRNA expression was significantly higher in eutopic endometriums of patients with endometriosis than those without endometriosis (Table, t=2.36, P <0.05). The abundance of Annexin-1 mRNA was lower in the proliferative phase and tended to increase in the secretary phase in both groups, but there were no statistical differences (t=1.25, P >0.05). Thus, the abundance of Annexin-1 mRNA in eutopic endometrium was not significantly changed during the menstrual cycle.

Table
Table:
The relative copy numbers (2(-ΔCT)) for Annexin-1 mRNA levels in two groups

Annexin-1 protein localization and expression in eutopic endometrium

Annexin-1 protein expression was quite strong in the cytoplasm and cytomembrane of endometrial glandular cells throughout the menstrual cycle in both groups. But little immunoreactivity was evident in stromal cells throughout the menstrual cycle (Figure 1).

Figure 1.
Figure 1.:
Annexin-1 was expressed in cytoplasm and membrane of glandular cells in eutopic endometrium. A: eutopic endometrium of women with endometriosis in secretory phase; B: eutopic endometrium of women without endometriosis in secretory phase. (Immunohistochemistry, original magnification × 200).

In Western blot analysis, Annexin-1 protein was detected at 38 kD at a single band in proteins extracts from 32 endometrium specimens, including 8 in the proliferative phase and 8 in the secretary phase in both the endometriosis and control groups. Annexin-1 protein expression was higher in the eutopic endometrium of women with endometriosis than in the control group (Figure 2, P <0.05). And protein expression in the endometrium showed no difference between the proliferative phase and secretory phase in both groups (Figure 2, P >0.05).

Figure 2.
Figure 2.:
Mean values of relative levels of Annexin-1 protein in EP (endometriosis patients in proliferative phase, n=8), ES (endometriosis patients in secretory phase, n=8), CP (controls in proliferative phase, n=8), CS (controls in secretory phase, n=8), EM (patients with endometriosis, n=16) and CON (patients without endometriosis, n=16). * P <0.05, EM vs CON (analyzed by Mann-Whitney U test). The expression of Annexin-1 protein was detected by Western blotting.

Expression of Annexin-1 protein in peritoneal fluids of women with endometriosis

Annexin-1 expression was examined in peritoneal fluids of ten patients with endometriosis by Western blotting. The protein was detected at 38 kD as a single band in all the ten samples (Figure 3).

Figure 3.
Figure 3.:
Western blot analysis of Annexin-1 in peritoneal fluids of all the patients with endometriosis.

DISCUSSION

Annexin-1, the first characterized member of Annexin superfamily, was found to have calcium and phospholipids binding properties. A research12 suggests that Annexin-1 is a multifunctional protein that regulates a variety of cellular processes. In the present study, we demonstrated that Annexin-1 mRNA and protein were overexpressed in eutopic endometrium of endometriosis using real-time PCR and Western blot analysis, without a significant difference between the proliferative and secretory phases. These findings suggest that Annexin-1 is overexpressed commonly in endometriosis and may play a role in the pathogenesis of endometriosis. As a multifunctional protein, Annexin-1 may facilitate the growth and implantation of the endometrial cells in ectopic sites in several ways.

The overexpression of Annexin-1 in the eutopic endometrium of patients with endometriosis may protect the refluxed endometrial cells from necrosis and promote their proliferation in ectopic sites. The possible importance of Annexin-1 in cell survival was first demonstrated in postlactating mammary ducts undergoing apoptosis where Annexin-1 expression was increased.13 Research also showed that exogeneous Annexin-1 can protect cells from necrosis induced by hydrogen peroxide.14 Thus, increased Annexin-1 levels may inhibit the necrosis of refluxed endometrial cells and keep them viable, which is necessary for the development of endometriosis. Annexin-1 acts as a substrate for the EGF receptor tyrosine kinase, which plays an important role in cell proliferation and differentiation.15 Furthermore, it possesses phosphorylation sites for important proliferative signaling molecules, such as various signal transducing kinase associated hepatocyte growth factor receptor11 and protein kinase C.16 The overexpression of Annexin-1 may promote the proliferation of endometrial cells by modulating these signal transduction pathways.

The role of Annexin-1 in the immune system and its presence in the peritoneal fluids might allow the peritoneal microenvironment to be a relevant “permissive” condition for implantation and growth of refluxed endometrial cells. Researches have demonstrated that immunological factors are involved in the genesis and development of endometriosis, and have suggested that endometriosis is a local pelvic inflammatory process with altered function of immune-related cells in the peritoneal environment.17 Available data indicate that macrophages in the peritoneal fluid play an active role in the initiation, maintenance and progression of endometriosis.18 It has also been proved that T cell-mediated immunity changes in patients with endometriosis.19 Annexin-1 has been demonstrated to regulate the activities of both the innate and adaptive immune cells such as macrophages and T lymphocytes. Annexin-1 modulates the phagocytic potential of macrophages and its production of TNF-α and IL-6.20 In prototype Th1 and Th2-type human T cell responses, Annexin-1-derived peptides can inhibit antigen-driven cellular proliferation and cytokine production.21 Annexin-1 augments anti-CD3/CD28-mediated CD25 and CD69 expression, and increases the activation and proliferation of T cells in response to anti-CD3 plus anti-CD28 stimulation.22 High levels of Annexin-1 in endometrium and its presence in peritoneal fluids may change the constituents of the peritoneal fluids and the local immune microenvironment. Endometriosis may develop when a defective “disposal system” permits the implantation and growth of endometrial cells or fragments.17

Thus, the overexpression of Annexin-1 in eutopic endometrium, and its presence in the peritoneal fluids of women with endometriosis, might increase the survival and proliferation of refluxed endometrial cells, and may also make the pelvic environment become “permissive” to their adherence and implantation.

In our study, immunohistochemistry showed that Annexin-1 protein was expressed mainly in endometrial glandular cells throughout the menstrual cycle. This indicates that Annexin-1 may act on endometrial glandular and the stromal cells by autocrine or paracrine mechanisms, without significant change with the hormone levels. The actual mechanism of Annexin-1 on endometrial cells should be further investigated.

In conclusion, Annexin-1 is overexpressed in eutopic endometrium of women with endometriosis and presents in the peritoneal fluids of patients with endometriosis. It may play an important role in the pathogenesis of endometriosis in different ways. But the mechanism of how Annexin-1 promotes the growth and implantation of shed endometrial cells in ectopic sites should be further studied.

REFERENCES

1. Cramer DW, Missmer SA. The epidemiology of endometriosis. Ann N Y Acad Sci 2002; 955: 11–22.
2. Bartosik D, Jacobs SL, Kelly LJ. Endometrial tissue in peritoneal fluid. Fertil Steril 1986; 46: 796–800.
3. Oral E, Arici A. Pathogenesis of endometriosis. Obstet Gynecol Clin North Am 1997; 24: 219–233.
4. Melega C, Balducci M, Bulletti C, Galassi A, Jasonni VM, Flamigni C. Tissue factors influencing growth and maintenance of endometriosis. Ann N Y Acad Sci 1991; 622: 256–265.
5. Goulding NJ, Godolphin JL, Sampson MB, Maddison PJ, Flower RJ. Hydrocortisone induces lipocortin 1 production by peripheral blood mononuclear cells in vivo in man. Biochem Soc Trans 1990; 18: 306–307.
6. Cirino G, Flower RJ. Human recombinant lipocortin 1 inhibits prostacyclin production by human umbilical artery in vitro. Prostaglandins 1987; 34: 59–62.
7. Kamal AM, Flower RJ, Perretti M. An overview of the effects of annexin 1 on cells involved in the inflammatory process. Mem Inst Oswaldo Cruz 2005; 100 Suppl 1: 39–47.
8. Croxtall JD, Gilroy DW, Solito E, Choudhury Q, Ward BJ, Buckingham JC, et al. Attenuation of glucocorticoid functions in an Anx-A1−/− cell line. Biochem J 2003; 371(Pt 3): 927–935.
9. Violette SM, King I, Browning JL, Pepinsky RB, Wallner BP, Sartorelli AC. Role of lipocortin I in the glucocorticoid induction of the terminal differentiation of a human squamous carcinoma. J Cell Physiol 1990; 142: 70–77.
10. Solito E, Kamal A, Russo-Marie F, Buckingham JC, Marullo S, Perretti M. A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils. FASEB J 2003; 17: 1544–1546.
11. Skouteris GG, Schröder CH. The hepatocyte growth factor receptor kinase-mediated phosphorylation of lipocortin-1 transduces the proliferating signal of the hepatocyte growth factor. J Biol Chem 1996; 271: 27266–27273.
12. Lim LH, Pervaiz S. Annexin 1: the new face of an old molecule. FASEB J 2007; 21: 968–975.
13. McKanna JA. Lipocortin 1 in apoptosis: mammary regression. Anat Rec 1995; 242: 1–10.
14. Sakamoto T, Repasky WT, Uchida K, Hirata A, Hirata F. Modulation of cell death pathways to apoptosis and necrosis of H2O2-treated rat thymocytes by lipocortin I. Biochem Biophys Res Commun 1996; 220: 643–647.
15. Radke S, Austermann J, Russo-Marie F, Gerke V, Rescher U. Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain. FEBS Lett 2004; 578 (1-2): 95–98.
16. Varticovski L, Chahwala SB, Whitman M, Cantley L, Schindler D, Chow EP, et al. Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C. Biochemistry 1988; 27: 3682–3690.
17. Dmowski WP. Immunological aspects of endometriosis. Int J Gynaecol Obstet 1995; 50 (Suppl 1): S3–S10.
18. Senturk LM, Arici A. Immunology of endometriosis. J Reprod Immunol 1999; 43: 67–83.
19. Ho HN, Wu MY, Yang YS. Peritoneal cellular immunity and endometriosis. Am J Reprod Immunol 1997; 38: 400–412.
20. Yona S, Heinsbroek SE, Peiser L, Gordon S, Perretti M, Flower RJ. Impaired phagocytic mechanism in annexin 1 null macrophages. Br J Pharmacol 2006; 148: 469–477.
21. Kamal AM, Smith SF, De Silva Wijayasinghe M, Solito E, Corrigan CJ. An annexin 1 (ANXA1)-derived peptide inhibits prototype antigen-driven human T cell Th1 and Th2 responses in vitro. Clin Exp Allergy 2001; 31: 1116–1125.
22. D'Acquisto F, Paschalidis N, Sampaio AL, Merghani A, Flower RJ, Perretti M. Impaired T cell activation and increased Th2 lineage commitment in Annexin-1-deficient T cells. Eur J Immunol 2007; 37: 3131–3142.
Keywords:

Annexin-1; endometriosis; endometrium; ascitic fluid

© 2008 Chinese Medical Association