Differential Proteome Profiling of Eutopic Endometrium from Women

Differential Proteome Profiling of Eutopic Endometrium from Women with Endometriosis To Understand Etiology of Endometriosis Priyanka Rai,† Venkatesh Kota,† Mamata Deendayal,‡ and Sisinthy Shivaji*,† Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India, and Infertility Institute and Research Centre, Hyderabad, India Received February 23, 2010

The identification of molecular differences in the endometrium of women with endometriosis is an important step toward understanding the pathogenesis of this condition and for developing novel strategies for the treatment of associated infertility and pain. In this study, we investigated protein expression analysis of eutopic endometrium from women with and without endometriosis. The proteomic analysis revealed molecular dysregulation of more than 70 proteins in the proliferative phase of eutopic endometrium in stage IV and secretory phase of stage II, III and IV endometriosis. Using mass spectrometry, 48 proteins spots which were consistently differentially expressed from stage II to IV endometriosis were identified. The differentially expressed proteins include structural proteins, proteins involved in stress response, protein-folding and protein-turnover, immunity, energy production, signal transduction, RNA biogenesis, protein biosynthesis, and nuclear proteins. Immunoblot and immunohistochemical analyses confirmed the observed changes in eight representative proteins. The present study provides identification of new players that have a potential role in the initiation and progression of endometriosis and also sets a framework for further investigations on mechanisms underlying the pathogenesis of endometriosis. Keywords: endometriosis • endometrium • 2D-PAGE • proteome • proliferative phase • secretory phase • menstrual cycle

Introduction Endometriosis is a benign, estrogen-dependent complex gynecological disorder which affects approximately 6-10% of women in the reproductive age and 35-40% of the affected women have episodes of pain and/or infertility.1 Its defining feature is the presence of endometrial like stromal and epithelial cells at sites outside the uterine cavity, primarily on the pelvic peritoneum and ovaries.1,2 These endometriotic deposits are hormonally responsive,3,4 stimulate angiogenesis, and are highly invasive.5 The etiology of endometriosis is debatable, but probably multifactorial and is not explained by any single theory. Susceptibility to endometriosis is thought to depend on the complex interaction of genetic, immunological, hormonal, and environmental factors. However, it is accepted that retrograde menstruation is the permissive factor in endometriosis.6 The formation of a lesion is, therefore, likely to involve the dissemination of uterine endometrium into the peritoneal cavity at the time of menstruation and its survival, attachment, growth, proliferation, neoangiogenesis, and invasion into ectopic sites within the peritoneal cavity. This may be due to * To whom correspondence should be addressed. Dr. S Shivaji, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. E-mail: [email protected]. Telephone: 00-91-40-27192504. Fax: 00-91-4027160311. † Centre for Cellular and Molecular Biology. ‡ Infertility Institute and Research Centre. 10.1021/pr100657s

 2010 American Chemical Society

abnormalities of the eutopic endometrium itself, predisposing the cells to survive and implant ectopically.7 To have a better understanding of the disease, newer approaches such as genomics8-11 and proteomics12-14 are now being used to compare eutopic endometrium from women having endometriosis and normal controls. These studies have led to the identification of several genes and proteins which could be implicated in endometriosis. Earlier studies on differential protein expression in endometriosis using proteomics approach have given us clues about the aberration in the protein expression profiling of eutopic endometrium from women with endometriosis.12-14 However, due to limitation in obtaining tissue samples and small quantity of sample, not many differentially expressing proteins were identified. As on date, only three studies have been carried out to analyze the protein expression in the eutopic endometrium of women with endometriosis using 2D-PAGE and mass spectrometry, either in secretory phase12,13 or both in secretory and proliferative phases of the menstrual cycle.14 Zhang et al.12 have identified 11 out of 129 differentially expressed protein spots that are involved in cell cycle, signal transduction, and immunological function using eutopic endometrium from women having endometriosis in stage II, III, and IV. Further, Have et al.13 reported the identification of 50 protein spots out of 119 differentially expressing protein spots in stage II endometriosis, most of which can be grouped as cell structure, immune Journal of Proteome Research 2010, 9, 4407–4419 4407 Published on Web 07/21/2010

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reaction, and transcription proteins. Later, another group reported the effects of endometriosis (stage II) on the proteome of human eutopic endometrium both in secretory and proliferative phase.14 This group has reported the identification of 9 of the most abundant and consistently altering spots including molecular chaperones, proteins involved in cellular redox state, molecules involved in protein and DNA formation/ breakdown, and secreted proteins in secretory and proliferative phase, respectively. By using two-dimensional gel electrophoresis (2D-PAGE) combined with semiquantitative computerized analysis and immunoblotting, we have previously established the proteome of human endometrium and investigated the differential expression of proteins in human endometrium in the proliferative and secretory phase of the menstrual cycle.15 In the present study, attempts have been made to get a more consistent profile of aberrant protein expression both in proliferative and secretory phase of eutopic endometrium from women having endometriosis using 2D-PAGE and mass spectrometry. More than 70 proteins were found to be differentially expressed in the proliferative phase of eutopic endometrium in stage IV and secretory phase of stage II, III, and IV endometriosis. Among these, 48 protein spots which were consistently differentially expressed from stage II to IV endometriosis, were successfully identified by MALDI MS and/or MS/MS. Thus, the present study identifies many more differential proteins in the proliferative and secretory phase of eutopic endometrium compared to the earlier studies.12-14 This combined study on the differential protein analysis in the proliferative and secretory phase of eutopic endometrium may provide new insights into endometrial developmental defects that could be responsible for endometriosis.

Materials and Methods Human Endometrial Tissue Samples. Endometrial tissue samples were obtained from women admitted to Infertility Institute and Research Centre, Hyderabad, India, for diagnostic purposes, including infertility, tubal re-enastomosis, or pelvic pain. Eutopic endometrial tissue samples were collected from women with moderate-severe (II-IV) endometriosis, staged using the revised American Fertility Society (rAFS) classification system.16 All the women had a trans-vaginal ultrasound scan (TVS) at screening followed by laparoscopy to confirm the diagnosis (rAFS II-12; III-12; IV-33). Their mean age was 27.5 ( 4.4 (range 20-35) years. All the patients had different forms of endometriosis such as peritoneal lesions, adhesions and endometrioma. Women with normal menstrual cycle, normal hormonal profiles, and free of uterine abnormalities constituted the control sample group (n ) 42, secretory phase; n ) 17, proliferative phase). Their mean age was 26.7 ( 3.9 (range 22-36) years. Irregularly cycling, amenorrhic, postmenopausal women and those who had received steroid hormone therapy in the last 3 months were excluded from the study. All tissue samples were collected during routine surgical procedures. Written consent was obtained for the sampling of eutopic endometrium for research purposes. The samples were frozen in liquid nitrogen and preserved at -80 °C until the protein was extracted. The accurate phasing of the endometrial samples was done by histopathology using a standard procedure.17 All experiments were performed in accordance with the guidelines of the Institutional Review Board of the Centre for Cellular and Molecular Biology, Hyderabad, India. 4408

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Figure 1. Two dimensional electrophoresis map of human secretory phase eutopic endometrium tissue proteins. The first dimension was performed by IEF on pH 4-7 (7 cm) IPG strips, the second dimension on 10% SDS-PAGE gels and the proteins were visualized by CBB G250. The protein spots with endometriosis-related alterations are indicated as 1-48 and were excised from the gel and identified by MALDI MS and/or MSMS as listed in Table 1.

Proteins Extraction and 2D-PAGE. The frozen endometrial tissue samples were thawed, washed with PBS, and lysed with lysis buffer [containing 7 M urea, 2 M thiourea, 2% NP40, 50 mM DTT, 0.5% pharmalytes 3-10, and protease inhibitor cocktail (Roche, Mannheim, Germany)]. The suspension was then homogenized for approximately 5 min and kept at 4 °C for 2 h. Subsequently, the lysate was centrifuged at 12 5000g for 1 h at 4 °C and the solubilized protein was recovered carefully without disturbing the sediment. The supernatant was precipitated using trichloroacetic acid/acetone and resultant pellet was resuspended in lysis buffer and protein was quantified by amido black method.18 The protein (100 µg) was then loaded onto a commercially available IPG strip (7 cm, pH 4-7; Bio-Rad, Hercules, CA) by passive rehydration for 12 h. Later, IEF was performed at 50 mA/IPG strips at 4000 V for 20 000 Vh using a Protean IEF cell (Bio-Rad). After the IEF run was completed, strips were equilibrated in buffer I (containing 6 M urea, 0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol, and 2% DTT), followed by a second incubation in buffer II which contained all the ingredients of buffer I except that DTT was replaced with 2.5% iodoacetamide. Each equilibration step was carried out for 20 min under gentle agitation. Strips were then transferred onto a 10% SDS-PAGE gel (10 × 10.5 × 10 cm3) and embedded into the gel with 1% agarose containing a trace amount of bromophenol blue. SDS-PAGE was performed using vertical gel electrophoresis system (GE Healthcare Bio-Sciences, Piscataway, NJ) at 20 mA/gel. The gels were stained with colloidal Coomassie stain.19 Image Capture and Analysis. To compare the 2D gels of cases and controls, the stained gels were scanned using Quantity one-4.6.8 (Basic) software in VersaDoc (Bio-Rad) and transferred to PDQuest Advanced 2D Analysis Software Version 8.0.1 (Bio-Rad). The semiautomated routines available in this

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Differential Proteome Profiling of Eutopic Endometrium

Table 1. List of Eutopic Endometrial Protein Spots Showing Differences in the Pattern of Changes in Expression from Stage II to Stage IV (Secretory) and Stage IV (Proliferative) Phases in Women with and without Endometriosis fold differencec s. no.

spot no.a

accession numberb

protein name

Alpha 2 type VI collagen isoform 2C2 precursor Vinculin, isoform CRA_a Gelsolin isoform b Gelsolin isoform a precursor VIM Tubulin, beta Vimentin Collagen, type VI, alpha 1 precursor Vimentin Vimentin Actin, beta Beta actin variant Tropomyosin 4 isoform 2 F-actin capping protein beta subunit

stage III (sec)

stage IV (sec)

stage IV (pro)

1.22

1.67

3.15

1.98

gi|119574932 gi|38044288 gi|4504165 gi|47115317 gi|57209813 gi|62414289 gi|87196339 gi|62414289 gi|62414289 gi|14250401 gi|62897409 gi|4507651 gi|4826659

-1.21 1.04 2.62 1.36 1.62 -1.08 1.22 -1.46 1.16 1.35 -1.56 1.41 -1.22

-1.80 1.16 1.22 1.80 1.86 -1.31 1.67 -1.47 1.26 2.02 -1.62 4.24 -1.49

-2.8 1.48 1.08 1.97 3.03 -1.5 1.73 -2.28 1.41 2.14 -4.25 5.20 -2.93

-1.72 1.48 1.07 1.38 1.89 -3.85 1.30 -2.27 2.26 1.98 -1.69 4.14 -3.05

1.40 1.12 1.77 1.57 2.15 -1.78 1.53 -1.94 -1.31 -1.78

1.57 1.79 2.46 1.92 2.35 -1.88 2.61 -2.57 -1.77 -2.5

1.24 1.55 3.46 -1.29 1.21 -1.72 2.07 2.01 -2.24 -2.36

-1.10 1.28 1.20 -1.14 -1.10 1.78

-1.39 2.79 1.80 -2.10 -1.53 2.42

-2.37 3.24 2.27 -3.40 -1.94 2.77

-4.56 2.50 1.70 -1.48 -6.40 1.78

1.88 -1.29 -1.38

2.64 -1.50 -1.56

3.03 -1.57 -3.18

1.57 -1.36 -1.62

Production gi|119594451 gi|21411235

-1.04 1.15

-1.30 1.33

-2.20 3.10

-1.64 3.54

gi|19923748 gi|515634

1.12 -1.13

1.26 -1.25

2.28 -1.42

1.02 -1.23

gi|89574029

-1.07

-1.19

-1.96

-2.52

1.06 -1.40 1.65 2.22 -1.11

1.20 -1.31 1.94 2.34 -1.64

1.50 -1.08 1.94 2.68 -2.00

2.86 2.64 2.13 1.57 1.92

1.30 1.61 1.25 1.72 1.51

1.61 2.21 3.46 2.25 1.52

1.43 1.78 -1.06 -1.43 1.82

1.

1

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

2 8 9 23 24 25 26 32 33 34 35 38 43

15. 16. 17 18. 19. 20. 21. 22. 23. 24.

5 7 12 13 16 17 20 21 22 27

25. 26. 27. 28. 29. 30.

28 44 45 46 47 48

Stress Response, Protein Tumor rejection antigen (gp96) 1 Heat shock protein 90 kDa beta, member 1 GRP78 precursor GRP78 precursor Heat shock 70 kDa protein 8 isoform 1 Heat shock 70 kDa protein 9B precursor Mitochondrial heat shock 60 kDa protein 1 Mitochondrial heat shock 60 kDa protein 1 Chaperonin containing TCP1, subunit 8 (theta) Protein disulfide isomerase family A, member 3, isofrom cra_a T-complex polypeptide 1 Heat shock protein beta-1 Heat shock protein beta-1 Peroxiredoxin 2 isoform a Peroxiredoxin 3, isoform CRA_c Chain A, Crystal Structure Of Human DJ-1

31. 32. 33.

37 40 42

Annexin A5 Annexin A5 Annexin A4

34. 35.

3 11

36. 37.

29 30

38.

31

39. 40. 41. 42. 43.

4 6 10 39 41

44. 45. 46. 47. 48.

14 15 18 19 36

Folding, and Protein Turnover gi|61656607 1.36 gi|4507677 1.10 gi|386758 1.56 gi|386758 1.16 gi|5729877 1.70 gi|62897075 -1.15 gi|189502784 1.05 gi|189502784 -1.03 gi|62896539 -1.10 gi|119597640 -1.20 gi|36796 gi|4504517 gi|4504517 gi|32189392 gi|119569783 gi|42543006

Immunity gi|49168528 gi|49168528 gi|39645467

Energy Glucosidase, alpha; neutral AB, isoform CRA_a NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa Dihydrolipoamide S-succinyltransferase Ubiquinol-cytochrome c reductase core I protein Mitochondrial ATP synthase, H+ transporting F1 complex beta subunit Major vault protein Valosin-containing protein Motor protein 14-3-3 Protein Epsilon Guanine nucleotide binding protein (G protein), beta polypeptide

stage II (sec)

Structural Proteins gi|115527062

Signal Transduction gi|194389978 gi|111305821 gi|516764 gi|67464424 gi|66347285

RNA Biogenesis, Protein Biosynthesis, and Nuclear Proteins Lamin B1 gi|15126742 1.02 Lamin B1 gi|15126742 1.12 Lamin B2 gi|27436951 1.05 Lamin B2 gi|27436951 1.38 B23 nucleophosmin (280 AA) gi|825671 1.09

a Spot code as in Figure 1. b Accession numbers are according to NCBI 2009 database. c A positive fold change indicates that protein expression was upregulated in endometriosis. A negative fold change indicates that protein expression was downregulated in endometriosis. Sec and pro indicate secretory and proliferative phase, respectively.

software were used to detect and quantify protein spots as well as to match the profiles across a gel series. Protein spots in the gels were identified after normalization based on the local regression model (LOESS). The protein spots from all the gels in the same group were matched for reproducibility analysis in our test system and the scatter plot tool was used to show the correlation coefficient among the gels in each group. For gel comparison, a statistical approach was applied when

determining differentially expressed proteins using the PDQuest software. Mann-Whitney Unpaired 2-sample test was performed with 95% significance level to determine which proteins were differentially expressed between the eutopic endometrium from women having endometriosis and control gels. A minimum of 1.2-fold change was considered for the upregulated/ downregulated proteins. Comparisons between multiple groups were performed using one-way analysis of variance. A BonferJournal of Proteome Research • Vol. 9, No. 9, 2010 4409

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Figure 2. Immunoblot analysis of eutopic endometrium tissue protein lysates from stage IV endometriosis and controls in proliferative phase. Panels A-H show representative immunoblots of DJ-1, HSP27, HSP60, HSP70, GRP78, HSP90 beta, MVP, and ERp57, respectively. Panels A′-H′ show the graphical representation of the immunoblots A-H, which was obtained by densitometry analysis. The semiquantitative data of A-H immunoblots was normalized by relative intensity of Gapdh. Asterisk (*) indicates significant difference (p < 0.05, as determined by Student’s t test) between cases and controls. Experiments were performed with endometrial tissues from 12 different individuals representing six each from stage IV endometriosis and controls, respectively.

roni correction was applied to the significance levels obtained in order to determine whether the observed significant differences in protein spot expression between the study groups may have occurred due to multiple analyses. In-Gel Digestion and Protein Identification by Mass Spectrometry. The protein spots were manually cut out from gels and processed for MALDI. The excised protein spots were destained for 1 h using 50% (v/v) acetonitrile (ACN) and 100 mM NH4HCO3 and then briefly washed with 100% ACN, vacuum-dried in a SpeedVac concentrator (Labconco Corporation, Kansas City, MO) and then incubated in 10 µL of trypsin solution (10 µg/mL trypsin in 10 mM NH4HCO3) for 16 h at 37 °C. The tryptic peptides were spotted on the MALDI plate and dried prior to the addition of 1 µL of 5 mg/mL of CHCA in 50% ACN. Protein spots were then identified by MALDI MS and/or MS/MS using MALDI TOF/ TOF 4800 Proteomics Analyzer (Applied Biosystems, Foster city, CA). Peptide mass calibration was performed with external mass standard (Calmix 5; Applied Biosystems) and measured over a mass range of 850-4000 Da. Monoisotopic spectra were analyzed using in-house GPS Explorer software, version 3.5 with integrated MASCOT version 2. The search parameters used were fixed carbamidomethyl cysteine and variable methionine oxidation, mass tolerance of (50 ppm, minimum sound to noise ratio 10, and one missed cleavage. Database used was Homo sapiens, NCBI 2009. For protein identification based on MS/MS, the five most intense pep4410


tides detected in MS were selected for MS/MS analysis. The search parameters were the same as for MS analysis mentioned above, with MS/MS fragment tolerance of (0.25 Da. Proteins that were identified by MALDI MS (protein score > 67, p < 0.05) and/or MS/MS (individual ion score > 37, p < 0.05) have been tabulated. The results were also manually validated based on protein pI and molecular weight. Immunoblotting. Endometrium proteins from cases and controls were resolved by 1-D PAGE and then electrotransferred onto an NC membrane at 100 V for 1 h using the wet transfer system (Hoefer Scientific Instruments, San Francisco, CA). Subsequently, the membrane was stained with 0.1% ponceau S to check for equal loading of the proteins. Membranes were then blocked with 5% (w/v) nonfat milk in TBST (Tween 0.1% (v/v) in TBS) for 1 h at room temperature, washed, and incubated with the primary antibody prepared in TBST containing BSA 1% (w/v) for 1 h. Primary antibodies were diluted as follows: (i) HSP 27 (Stressgen, Ann Arbor, MI), 1:1000; (ii) DJ-1 (MBL International, Woburn, MA), 1:1000; (iiii) GRP 78 (Abcam, Cambridge, MA), 1:1000; (iv) HSP 90 beta (Abcam), 1:1000; (v) HSP 70 (Abcam), 1:1000; (vi) HSP 60 (Millipore, Billerica, MA), 1:1000; (vii) major vault protein 37 (MVP-37) (generous gift of George L. Scheffer, VU Medical Center, Amsterdam, The Netherlands), 1:1000; (viii) ERp57 (Millipore), 1:1000; and (ix) glyceraldehyde phosphate dehydrogenase (Gapdh), loading control (Abcam), 1:5000. After the incubation period, the


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Figure 3. Immunoblot analysis of eutopic endometrium tissue protein lysates from stage II endometriosis and controls in secretory phase. Panels A-H show representative immunoblots of DJ-1, HSP27, HSP60, HSP70, GRP78, HSP90 beta, MVP, and ERp57, respectively. Panels A′-H′ show the graphical representation of the immunoblots A-H, which was obtained by densitometry analysis. The semiquantitative data of A-H immunoblots was normalized by relative intensity of Gapdh. Asterisk (*) indicates significant difference (p < 0.05, as determined by Student’s t test) between cases and controls. Experiments were performed with endometrial tissues from 12 different individuals representing six each from stage II endometriosis and controls, respectively.

membranes were washed three times (10 min each wash with TBST) and incubated in the appropriate secondary antibody prepared in TBST containing BSA 1% (w/v) for 1 h at room temperature. The secondary antibody used was conjugated to horseradish peroxidase (HRP) (Sigma) and was used at a concentration of 1:10 000. The blots were developed using the ECL kit (Millipore, Billerica, MA). Exposed films were scanned and bands of interest were quantified using GeneTools version 3.06.04 from SynGene (Cambridge, U.K.). Immunohistochemistry. Tissue samples from controls and cases were thawed and rinsed with PBS to remove blood. The tissue was then immediately immersed in freshly prepared 10% neutral buffered formalin and left for 1-2 days at room temperature. After fixing, the tissue pieces were rinsed in distilled water for 1 h. They were then transferred to 70% alcohol for 1 h and then through a series of graded alcohols (80%, 95%, and absolute alcohol) after every 1 h. The tissues were then transferred to xylene for 1 h, twice, and were finally embedded in paraffin wax. Molds were prepared with paraffin wax and 5 µm thick sections were prepared. The sections were first deparaffinized by incubation in xylene for 3 min, twice, and then in xylene 1:1 with absolute alcohol for 3 min. They were further transferred to absolute alcohol for 3 min (× 2) and then through a series of graded alcohol (95%, 70%, and 50%) after every 3 min. The slides were incubated in antigen retrieval buffer (10 mM Sodium citrate and 0.05% Tween 20, pH 6.0) at 98 °C for 20 min. After 20 min, the slides were washed with running cold

water for 10 min. The slides were then washed for 2 × 5 min with TBS plus 0.025% Triton X-100 with gentle agitation. Blocking was done in 10% normal serum with 1% BSA in TBS for 2 h at room temperature. After blocking, the slides were drained, wiped around the section, and primary antibody diluted in TBS with 1% BSA was added for overnight incubation at 4 °C. Primary antibodies were diluted as follows: (i) HSP 27 (Stressgen, Ann Arbor, MI), 1:200; (ii) DJ-1 (MBL International, Woburn, MA), 1:200; (iiii) GRP 78 (Abcam, Cambridge, MA), 1:200; (iv) HSP 90 beta (Abcam), 1:200; (v) HSP 70 (Abcam), 1:200; (vi) HSP 60 (Millipore, Billerica, MA), 1:1200; (vii) LRP-56 for major vault protein (generous gift of George L. Scheffer, VU Medical Center, Amsterdam, The Netherlands), 1:200 and (viii) ERp57 (Millipore), 1:250. This was followed by washing, 2 × 5 min with TBS plus 0.025% Triton X-100 with gentle agitation. Fluorophore-conjugated secondary antibodies diluted in TBS with 1% BSA were then applied and incubated for 1 h at room temperature. After incubation period, the slides were rinsed with TBS (3 × 5 min) and mounted using Vectashield (Vector Laboratories, CA) as the mounting medium and viewed under an Axioplan2 epifluorescence microscope (Carl Zeiss, Inc., Jena, Germany). All individual tissue samples were stained at one time for consistency. Staining intensity was scored for each tissue and each antibody in glandular epithelium and stroma using Image J software. Journal of Proteome Research • Vol. 9, No. 9, 2010 4411

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Figure 4. Immunoblot analysis of eutopic endometrium tissue protein lysates from stage III endometriosis and controls in secretory phase. Panels A-H show representative immunoblots of DJ-1, HSP27, HSP60, HSP70, GRP78, HSP90 beta, MVP, and ERp57, respectively. Panels A′-H′ show the graphical representation of the immunoblots A-H, which was obtained by densitometry analysis. The semiquantitative data of A-H immunoblots was normalized by relative intensity of Gapdh. Asterisk (*) indicates significant difference (p < 0.05, as determined by Student’s t test) between cases and controls. Experiments were performed with endometrial tissues from 12 different individuals representing six each from stage III endometriosis and controls, respectively.

Results Comparison of Protein Expression in Proliferative Phase Eutopic Endometrium from Women with and without Endometriosis. The eutopic endometrium tissues from women having stage IV (n ) 6; replicates) endometriosis in proliferative phase of menstrual cycle were compared with control (n ) 6; replicates) to gain an insight into the aberrant protein expression during the proliferative phase of menstrual cycle in endometriosis. The replicate 2D gels of stage IV endometriosis tissue samples were compared with control using PDQuest software. The reproducibility of 2D gels was analyzed by the scatter plots generated in the same software. The correlation coefficient of >0.8 in all the groups indicated good reproducibility of the gels in each group. These gels were then used to synthesize a master gel. The synthesized master gel contained 215 discovered spots and was chosen for comparison with the proteomes of eutopic endometrium from women with stage IV endometriosis and with the corresponding control samples. A total of 100 protein spots out of 215 marked spots were altered during the proliferative phase of the menstrual cycle in eutopic endometrium of women with endometriosis compared to women without the disease. Out of the 100 protein spots, that were altered in intensity in the endometrium from women with endometriosis, 51 spots showed a significant increase (p < 0.05), 41 showed a significant decrease (p < 0.05), and 8 were unique. A subset of 48 protein spots with statistically significant changes in expression levels (Figure 1) were identi4412


fied by MALDI MS and/or MS/MS. Table 1 shows list of identified proteins and fold differences in endometriosis. The identity of the differentially expressed proteins and characteristics such as its pI, mass, peptides matched, and sequence coverage have been tabulated in Supplementary Tables 1 and 2. Comparison of Protein Expression in Secretory Phase Eutopic Endometrium from Women with and without Endometriosis. To identify proteins associated with different stages of endometriosis in secretory phase, the 2D proteome of eutopic endometrium tissue from 18 women having endometriosis (rAFS II-6; III-6; IV-6; replicates) and control women (n ) 18; replicates), in the molecular weight range of 10-110 kDa and pI range of 4-7 were compared and analyzed using PDQuest software. The reproducibility of 2D gels was analyzed by the scatter plots generated in the same software. The correlation coefficient of >0.8 in all the groups indicated good reproducibility of the gels in each group. Those protein spots that showed significant changes of more than (1.2-fold (p < 0.05) across all individuals in the group were considered as differentially expressed. When the proteome of secretory phase eutopic endometrium in stage II, III, and IV endometriosis, respectively, was compared with the corresponding control, 72, 91, and 116 protein spots were identified as differentially expressed. Among these differentially expressed protein spots a total of 42, 47, and 73 protein spots showed a significant increase, whereas 29, 43, and 42 protein spots showed a


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Figure 5. Immunoblot analysis of eutopic endometrium tissue protein lysates from stage IV endometriosis and controls in secretory phase. Panels A-H show representative immunoblots of DJ-1, HSP27, HSP60, HSP70, GRP78, HSP90 beta, MVP, and ERp57, respectively. Panels A′-H′ show the graphical representation of the immunoblots A-H, which was obtained by densitometry analysis. The semiquantitative data of A-H immunoblots was normalized by relative intensity of Gapdh. Asterisk (*) indicates significant difference (p < 0.05, as determined by Student’s t test) between cases and controls. Experiments were performed with endometrial tissues from 12 different individuals representing six each from stage IV endometriosis and controls, respectively.

significant decrease in expression in stage II, III, and IV endometriosis, respectively. One protein spot was unique to endometriosis in all the three stages. Out of these differential protein spots, 48 protein spots were successfully identified by MALDI MS and/or MS/MS (Figure 1). Table 1 gives the identity of the proteins which are consistently differentially expressed from stage II to stage IV endometriosis. Interestingly, several of the identified proteins were the same or isoforms of the same protein as those identified in proliferative phase, in particular actin and vimentin. However, it was interesting to observe that expression of one of the isoforms of GRP 78 and Lamin B2 was found to be increased while valosin containing protein is found to be downregulated during the secretory phase when compared to proliferative phase of menstrual cycle in endometriosis. Validations of Differential Proteins by Immunoblotting. To validate the differential protein expression in either proliferative or secretory phase of the menstrual cycle in eutopic endometrium of women with endometriosis compared to women without the disease as identified by PDQuest analysis, the proteins were separated by 1-D SDS-PAGE and immunoblotting was performed with the respective antibody. Immunoblot analysis was performed using a total of 48 samples representing 24 from case (rAFS II-6, rAFS III-6, rAFS IV-6 in secretory phase and rAFS IV-6 in proliferative) and 24 from control group (n ) 18; secretory phase and n ) 6; proliferative phase). Our study is restricted to analysis of only stage IV endometriosis in proliferative phase, due to constraints in procuring eutopic endometrium from stage II and III endometriosis as during this

phase of cycle the endometrium lining is very thin20 and gynecologists prefer to intervene surgically only at a later stage. Figure 2 shows the results of immunoblot analysis and quantitation results of major vault protein 37 (MVP), HSP90 beta, GRP78, HSP70, HSP60, HSP27 (Heat shock protein beta-1), DJ1, and ERp57 (Protein disulfide isomerase family A, member 3) of three individual samples in stage IV endometriosis with controls during proliferative phase. Figures 3, 4, and 5 show immunoblot and quantitation results of three individual samples in each group from stage II, III, and IV and control in secretory phase, respectively. The results from the remaining six samples were also identical (data not shown). Gapdh was used as an internal loading control. The band intensities were normalized with internal control and were compared as case versus control. The level of significance for the differences in expression was determined by the Student’s t test (p < 0.05). The results of DJ-1, HSP27, HSP60, HSP70, GRP78, HSP90 beta, and MVP revealed that there were about 1.2- to 3-fold upregulation in various stages of endometriosis affected endometrium tissue compared to the normal, while ERp57 was about 1.2- to 2.5fold downregulated in the various stages of endometriosis. These results were consistent with the results of the eight differentially expressed proteins as obtained in PDQuest analysis. Validations of Differential Proteins by Immunohistochemistry. To further validate the observed changes in protein expression in either proliferative or secretory phase of the menstrual cycle in eutopic endometrium of women with endometriosis compared to women without the disease, imJournal of Proteome Research • Vol. 9, No. 9, 2010 4413

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Figure 6. Changes in DJ-1, HSP27, HSP60, HSP70, and MVP in stage IV endometriosis and controls in the proliferative phase using immunohistochemical analyses. Panels A-E show representative endometrial tissue sections from controls and A′-E′ from cases, that were stained for DJ-1, HSP27, HSP60, HSP70, and MVP, respectively. Panels A′′-E′′ and A′′′-E′′′, show the quantitative analysis of staining intensity in the glandular epithelium and stroma, respectively. Asterisk (*) indicates significant difference (p < 0.05, as determined by Mann-Whitney Unpaired 2-sample test) between cases and controls.

munohistochemical staining of the endometrial tissue samples was performed using the respective antibodies. Immunohistochemical analysis was performed using a total of 9 samples from cases [rAFS IV secretory phase (n ) 4) and rAFS IV proliferative phase (n ) 5)] and 11 from control group [secretory phase (n ) 6) and proliferative phase (n ) 5)]. Figures 6 and 7 show the results of immunohistochemical analysis and quantitation of the results for the DJ-1, HSP27 (Heat shock protein beta-1), HSP60, HSP70, major vault protein (MVP), GRP78, HSP90 beta, and ERp57 (Protein disulfide isomerase family A, member 3) of representative tissue sections in stage IV endometriosis with controls during proliferative phase, respectively. Figures 8 and 9 show immunohistochemical and quantitation of the results of representative tissue sections in stage IV endometriosis with control in secretory phase. The level of significance for the differences in expression was determined by Mann-Whitney Unpaired 2-sample test (p < 0.05). Immunohistochemical analysis of endometrial tissue section from stage IV endometriosis and controls in proliferative phase revealed strong staining for DJ-1, HSP 27, HSP 70, HSP 90 beta, and MVP, both in the epithelial glands and stroma of endome4414


trial tissue in cases compared to controls (Figures 6 and 7). Interestingly, HSP 60 showed more intense staining in the epithelial glands (p ) 0.0009) and it was more in cases, but no significant difference in staining intensity was observed in the stroma (Figure 6C-C′′′) between cases and controls. Immunostaining of tissue sections revealed that GRP 78 staining was intense in epithelial glands but no significant difference was observed in the intensity between control and cases. Surprisingly, in cases the stromal region showed significant increase in intensity as compared to controls (Figure 7A-A′′). ERp57 staining was observed both in epithelial glands and stroma, but the total staining was significantly lower in the glands of endometrium from women with endometriosis compared to controls (Figure 7C-C′′). In case of immunohistochemical analysis of secretory phase endometrial tissue sections from stage IV endometriosis and controls, it was observed that intensity of staining for DJ-1, HSP 27, HSP 60, HSP 70, and MVP was significantly higher in epithelial glands in cases than controls (Figure 8). No significant difference in intensity was observed in the stroma of endometrium tissue section between cases and controls. On the contrary, GRP 78 and HSP 90 beta staining was significantly


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Figure 7. Changes in GRP78, HSP90 beta, and ERp57 in stage IV endometriosis and controls in proliferative phase using immunohistochemical analyses. Panels A-C show representative endometrial tissue sections from controls and A′-C′ from cases, that were stained for GRP78, HSP90 beta, and ERp57, respectively. Panel A′′ shows the quantitative analysis of staining intensity of GRP78 in the stroma, whereas panels B′′ and C′′ show the quantitative analysis of staining intensity of HSP90 beta and ERp57 in glandular epithelium, respectively. Asterisk (*) indicates significant difference (p < 0.05, as determined by Mann-Whitney Unpaired 2-sample test) between cases and controls.

stronger in both epithelial glands and stroma of endometrium tissue section from cases than controls (Figure 9A-A′′ and B-B′′). Immunostaining of ERp57 showed staining both in glands and stroma, but epithelial glands showed more intense staining in controls than in cases (Figure 9C-C′′).

Discussion The identification of molecular differences in the endometrium of women with endometriosis compared to normal women is an important step toward understanding the pathogenesis of endometriosis and toward developing novel strategies for the treatment of associated infertility and pain. In the present study using comparative proteomics approach and mass spectrometry, the differential protein expression of eutopic endometrium in the proliferative phase of menstrual cycle from women having stage IV endometriosis was compared with women without endometriosis. Further, the eutopic endometrium in the secretory phase of menstrual cycle from women having stage II, III, and IV endometriosis was compared with women without endometriosis. We chose eutopic endometrium for our study since earlier reports suggested that large number of proteins are differentially expressed in the eutopic endometrium of women having endometriosis.12-14 The proteome analysis revealed molecular dysregulation of many proteins between endometriosis-affected endometrium and normal endometrium. In the present study, more than 70 proteins were found to be differentially expressed in stage IV in proliferative phase and

stage II, III, and IV in secretory phase of endometriosis compared to control samples based on PDQuest analysis. The changes in the expression levels of seven upregualted proteins DJ-1, HSP27, HSP60, HSP70, GRP78, HSP90 beta, and MVP and one downregulated protein ERp57 were further confirmed by immunoblot analysis (Figures 2-5) and immunohistochemical analysis (Figures 6-9). Among the 48 differential protein spots identified in this study, six proteins (Vimentin, β-actin, Heat shock protein 90 kDa beta, HSP70, Protein disulfide-isomerase, and peroxiredoxin-2) were consistent with the earlier three reports,12-14 thus, implying the identification of 42 additional differentially expressed proteins that have previously escaped identification. Our report also supports the finding of Fowler et al.14 who had shown that, in endometriosis, protein expression profiles also change during the proliferative phase in contrast to gene array data8-11 which indicated that most of the changes in expression profiles are likely to occur during the secretory phase. Some of the proteins like HSP60,21 HSP70,22 HSP90 beta, and peroxiredoxin-2,14 which were identified during the course of this study, have either been previously associated with endometriosis and/or endometrial functioning.15,23 Proteins which are differentially expressed in different stages of endometriosis in secretory phase of menstrual cycle as well as in proliferative phase of the menstrual cycle cover most of the functional groups and they could be novel candidates for further research on endometrium, especially with reference to endometriosis. Journal of Proteome Research • Vol. 9, No. 9, 2010 4415

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Figure 8. Changes in DJ-1, HSP27, HSP60, HSP70, and MVP in stage IV endometriosis and controls in secretory phase using immunohistochemical analyses. Panels A-E show representative endometrial tissue sections from controls and A′-E′ from cases, that were stained for DJ-1, HSP27, HSP60, HSP70, and MVP, respectively. Panels A′′-E′′ show the quantitative analysis of staining intensity in the glandular epithelium. Asterisk (*) indicates significant difference (p < 0.05, as determined by Mann-Whitney Unpaired 2-sample test) between cases and controls.

A majority of the proteins which are found to be differentially expressed in the proliferative and secretory phase of endometriosis are structural proteins. Vimentin and isoforms of actin are the persistent themes in the investigation of endometriosis related proteins12-14 and genes.8-11 Both these proteins are found to be differentially expressed in women with endometriosis. It is of interest to note that the expression of one of the isoforms of actin-binding protein, gelsolin isoform a, decreases from stage II to stage IV endometriosis. This decreased gelsolin expression was assumed to result in deregulation of cellular differentiation and motility, thus, facilitating malignant transformation, invasion, and metastasis.24,25 On the contrary, the expression of gelsolin isoform b increases from stage II to stage IV. Among other proteins which are found to be upregulated are tubulin, and isoforms of tropomysoin. Two proteins which are downregulated in endometriosis include vinculin, and F-actin capping protein beta subunit. Vinculin acts as a regulator of cellular mechanical functions26 and its expression is decreased in various cancers,27 suggesting that the suppression of vinculin is closely related to malignant progression. Since the cytoskeleton has crucial roles in a wide 4416


range of cellular functions, including proliferation, apoptosis, motility, differentiation, and second messenger pathways,28-30 it is highly likely that these differences could play a role in endometriosis. Within the ‘stress response, protein folding, and proteinturnover’ category, we have identified several members of HSPs including HSP90 beta, HSP70, HSP60, and HSP27 and the HSP homologues, glucose regulated proteins, GRP96, and GRP78, to be upregulated in various stages of endometriosis. However, the expression of few isoforms of HSP70 and 60, protein disulfide isomerase family A, member 3, and T-complex polypeptide 1 was decreased in endometriosis. Since HSPs are known to play a pivotal role in progression of a wide range of human cancers,31 deregulation of HSPs could be involved in the pathogenesis of endometriosis. The role of HSPs as molecularchaperones,andtheirinteractionwithsteroidreceptors32,33 and contribution to proliferation, and antiapoptosis23 further supports their role in endometriosis. Compelling evidence suggests that oxidative stress is increased in women with pelvic endometriosis. DJ-1, a cancer and Parkinson’s disease (PD)associated protein that protects cells from toxic stresses,34 is


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Figure 9. Changes in GRP78, HSP90 beta, and ERp57 in stage IV endometriosis and controls in secretory phase using immunohistochemical analyses. Panels A-C show representative endometrial tissue sections from controls and A′-C′ from cases, that were stained for GRP78, HSP90 beta, and ERp57, respectively. Panels A′′-C′′ show the quantitative analysis of staining intensity in the glandular epithelium. Asterisk (*) indicates significant difference (p < 0.05, as determined by Mann-Whitney Unpaired 2-sample test) between cases and controls.

found to be upregulated in endometriosis. Also, it is a positive regulator of the androgen receptor35 and found to be related to infertility.36 Peroxiredoxin 2 isoform ‘a’ and Peroxiredoxin 3 are the two proteins, which are related with oxidative stress and are downregulated in endometriosis. Our result confirms the report of Fowler et al.14 where they have reported downregulation of Peroxiredoxin 2 in endometriosis. Proteins having role in immunity such as Annexin A4, and Annexin A5 have also been found to be downregulated in endometriosis. But, interestingly, the expression of another isoform of Annexin A5 is increased in endometriosis. Karube et al.37 found a decrease of Annexin A5 mRNA and protein in carcinomas of the uterine cervix and endometrium when compared to normal tissues. Annexin A5 has been shown to exert an inhibitory action on protein kinase C;38 therefore, the decreased levels of Annexin A5 might lead to a dysregulated activation of protein kinase C, which plays a major role in tumor progression. Annexin A4 is known as a Ca2+ and lipidbinding protein. It is also involved in promoting vesicle aggregation, vesicle trafficking, and exocytosis.39 Several annexins have been implicated in the pathogenesis of benign and malignant neoplasms of different origins.40 The differential expression of annexins at various stages of endometriosis points toward a functional role of annexins in the pathogenesis of this disease. Proteins which are involved in energy production are also seen to be altered in endometriosis. Glucosidase alpha, neutral AB, mitochondrial ATP synthase, H+ transporting F1 complex beta subunit, and ubiquinol-cytochrome c reductase core I

protein are the proteins which are found to be downregulated, whereas NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa, and dihydrolipoamide S-succinyltransferase (DLST) are the proteins which are upregulated. Although the exact function with respect to endometrium and therefore with endometriosis is not known, it is likely that these proteins, apart from their role in glucose metabolism, are also involved in oxygen sensing, apoptosis, cell cycle regulation, and immune response and recognition.41 Major vault protein and 14-3-3 protein epsilon, which are involved in cell cycle, cell proliferation, and antiapoptosis are found to be upregulated in endometriosis.42-45 This is relevant to endometriosis as this disease is marked by the proliferation of heterogeneous endometrial cells and their invasion into ectopic sites. Valosin containing protein, which is known to be involved in the ubiquitin-dependent proteasome degradation pathway,46 and works in both the up-regulation of cell proliferation and the down-regulation of cell death in human cancer cells,47 is seen to be downregulated in all the stages of endometriosis during the secretory phase. But, interestingly, it is observed that the decrease in expression is more profound in stage II endometriosis. In contrast, in proliferative phase of stage IV endometriosis, this protein is found to be upregulated, suggesting that it might have a role in later stages of endometriosis. Guanine nucleotide binding protein (G protein), beta polypeptide is one of the other proteins in this category which is found to be downregulated. Proteins related to RNA biogenesis, protein biosynthesis, and nuclear proteins are also found to be altered. B23 nuJournal of Proteome Research • Vol. 9, No. 9, 2010 4417

research articles cleophosmin is found to be upregulated in endometriosis. Most importantly, it is linked to a variety of cellular processes including cell survival, cell cycle regulation, differentiation, and proliferation48 and are regulated by estrogen,49,50 suggesting their importance in estrogen driven disease endometriosis. Lamin B1 and B2, which play an essential role in maintaining nuclear architecture, DNA replication, and regulation of gene expression and which are important for cell proliferation,51,52 were also found to be upregulated.

Conclusions In the present study, we have applied proteomic techniques to study protein expression profiling between different stages of endometriosis using eutopic endometrium from women with and without endometriosis. This study has enabled us to identify 48 proteins that showed significant changes in expression levels in stage IV endometriosis during the proliferative phase and in different stages of endometriosis in secretory phase of the menstrual cycle. The upregulation of MVP, HSP90 beta, GRP78, HSP70, HSP60, HSP27, and DJ-1 and downregulation of ERp57 in different stages/phases of endometriosis were further confirmed by immunoblotting and immunohistochemistry. In this study, we have identified an additional 42 endometriosis related proteins that had previously escaped identification which might give us some insight into the biochemical pathways which are involved in the pathogenesis of this disease. However, the scope of this study is limited to only analysis of eutopic endometrium from different stages of endometriosis in secretory phase and analysis of only stage IV endometriosis in proliferative phase due to constraints in procuring eutopic endometrial tissue in proliferative phase from women having endometriosis. This study reinforces the validity of the proteomic approach in carrying out comparative analysis between normal and endometriotic women.

Acknowledgment. Priyanka Rai and Venkatesh Kota are the recipients of CSIR fellowship from Government of India. We thank Dr. Archana B. Siva, Dr. Curam Sreenivasacharlu Sundaram, Y. Kameshwari, Nandini Rangaraj and Avinash for their valuable suggestions and technical help. We sincerely thank Dr. George L. Scheffer for the anti-MVP-37 and anti-LRP-56 antibody. We are indebted to the patients who donated their tissues for this study and to the staff of IIRC, Hyderabad for tissue collection. Supporting Information Available: Tables listing the identity of the differentially expressed proteins and characteristics such as its pI, mass, peptides matched, and sequence coverage. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Giudice, L. C.; Kao, L. C. Endometriosis. Lancet 2004, 364, 1789– 1799. (2) Donnez, J.; Van Langendonckt, A.; Casanas-Roux, F.; Van Gossum, J. P.; Pirard, C.; Jadoul, P.; Squifflet, J.; Smets, M. Current thinking on the pathogenesis of endometriosis. Gynecol. Obstet. Invest. 2002, 54, 52–58. (3) Kitawaki, J.; Kado, N.; Ishihara, H.; Koshiba, H.; Kitaoka, Y.; Honjo, H. Endometriosis: the pathophysiology as an estrogen-dependent disease. J. Steroid Biochem. Mol. Biol. 2002, 83, 149–155. (4) Bulun, S. E. Endometriosis. N. Engl. J. Med. 2009, 360, 268–279. (5) Girling, J. E.; Rogers, P. A. Recent advances in endometrial angiogenesis research. Angiogenesis 2005, 8, 89–99. (6) Sampson, J. A. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity. Am. J. Obstet. Gynecol. 1927, 14, 442–469.

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