Proteomic Approach Identifies Alterations in Cytoskeletal Remodelling

Sep 17, 2010 - Remodelling Proteins during Decidualization of Human Endometrial ... In this study we isolated primary human endometrial stromal cells ...
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Proteomic Approach Identifies Alterations in Cytoskeletal Remodelling Proteins during Decidualization of Human Endometrial Stromal Cells Sarah G. Paule,* Lynette M. Airey,† Ying Li,† Andrew N. Stephens,† and Guiying Nie† Prince Henry’s Institute of Medical Research, 246 Clayton Road, Clayton, 3168, Australia Received May 25, 2010

Decidualization is a tissue remodelling process within the uterus in preparation for embryo implantation and pregnancy. In this study we isolated primary human endometrial stromal cells and stimulated decidualization with cAMP. We then used 2D- differential in-gel electrophoresis (DIGE) to identify proteins induced by decidualization. Eighty-eight out of 2714 spots were differentially regulated, 18 of which were assigned clear identities by mass spectrometry. Many of these are proteins known to be associated with cell structure and cytoskeletal remodelling. We validated five of these proteins by Western blot and immunohistochemistry on human endometrial tissue. The validated proteins are caldesmon 1, src substrate contactin 8, tropomyosin alpha-4 chain, protein disulfide isomerase 1A, and LIM and SH3 domain protein. With the exception of caldesmon 1, none of the identified proteins have previously been associated with decidualization. This study provides insight into our understanding of decidualization, which is important for successful embryo implantation and establishment of pregnancy. Keywords: decidualization • human endometrial stromal cells • src substrate cortactin 8

Introduction The endometrium undergoes extensive tissue remodelling during each menstrual cycle in preparation for embryo implantation and establishment of pregnancy. Following ovulation, during the secretory phase, considerable changes occur in the secretory profile of the endometrial epithelial cells. In the stromal compartment, late in the cycle, the important changes are in the vascular remodelling, accumulation of endometrial natural killer cells. In particular, differentiation of stromal fibroblast into a highly specialized cell type, the decidualized stromal cells. In a conception cycle, these changes continue, to provide maternal components of the placenta, the decidua.1 While the entire process is called decidualization, the term is also widely used to define just the stromal cell differentiation. During decidualization, the stromal cells surrounding the spiral arterioles become rounded, polygonal, and myofibroblastic in morphology.2,3 The decidualized stromal cells also become highly secretory, and secreted products such as prolactin (PRL) and insulin-like growth factor are often used as markers of decidualization in vitro.4-6 In addition to morphological changes, the decidualized stromal cells acquire the unique ability to regulate trophoblast invasion, resist inflammatory and oxidative insults, and diminish local maternal immune responses to support pregnancy. * To whom correspondence should be addressed. Address: Prince Henry’s Institute of Medical Research, P.O. Box 5152 Victoria, Australia 3168. Phone: +61 3 9594 4627. Fax: +61 3 9594 6125. E-mail: [email protected]. † E-mail addresses: (L.M.A) [email protected]; (Y.L.) Ying.Li@ princehenrys.org; (A.N.S.) [email protected]; (G.N.) [email protected]. 10.1021/pr100525a

 2010 American Chemical Society

The process of decidualization prior to embryo implantation is therefore of upmost importance and poor decidualization leads to pregnancy and infertility related disorders.7-11 Hence the understanding of the regulatory proteins involved during decidualization is critically important. Despite the fact that decidualization is important for embryo implantation, little is understood about the proteins involved in this process. The addition of exogenous cAMP is widely used to stimulate decidualization in vitro in baboon and human endometrial stromal fibroblasts.6,12 Microarray analysis of human decidual cells following cAMP treatment demonstrates a striking reprogramming of genes associated with regulated extracellular matrix, tissue strength, and tissue integrity.13,14 In this study, we decidualized primary human endometrial stromal cells (HESC) with cAMP and used a proteomic approach to identify proteins differentially regulated during this process.

Experimental Methods Patients and Human Endometrial Tissue Collection. Ethics approval for all tissue collections was obtained from the Human Ethics Committee at Monash Medical Centre, Melbourne, Australia. Prior to tissue collection, written and informed consent was obtained from all patients. Human endometrial biopsies from days 8-24 of the menstrual cycle were obtained from healthy fertile women undergoing curettage for benign conditions. A total of seven samples were collected in Dulbecco’s modified Eagle’s medium with Ham’s F12 medium (v/v; DMEM/F12; Sigma, St Louis, MO) and processed within 24 h of cell isolation. Journal of Proteome Research 2010, 9, 5739–5747 5739 Published on Web 09/17/2010

research articles Isolation and Culture of HESC. HESC were isolated from tissue by enzymatic digestion and filtration as previously described.8 In brief, tissues were finely chopped, washed with PBS, and digested with PBS containing 0.03% collagenase type 3 (Worthington, Lakewood, NJ), 0.02% deoxyribonuclease type I (Roche, Castle Hill, NSW, Australia) in a 37 °C shaking water bath for 40 min. The digest was diluted with serum-free DMEM/F12 and subjected to consecutive filtration through 44 and 11 µm nylon mesh. Stromal cells were harvested from the filtrate, plated into T75 cm2 tissue culture flasks and cultured in DMEM/F12 medium supplemented with 10% charcoal stripped fetal calf serum (CS-FCS; Thermo Electron Corporation, Maple Plain, MN), 2 mM L-glutamine (Sigma), 100 µg/ mL streptomycin, and 100 IU/ml penicillin (Gibco, Mulgrave, Victoria, Australia). Decidualization of HESC. Once 70-80% confluent, HESC were trypsinized, replated into two T75 cm2 tissue culture flasks and again cultured to 80% confluency. Cells were then treated with 5 × 10-4 M cAMP (Sigma) for 72 h in DMEM/F12 containing 2% CS-FCS at 37 °C. HESC treated with vehicle served as controls (control). At the end of the experiment, culture media were collected and assayed for prolactin (a marker of successful decidualization) by ELISA (Bioclone Australia Pty Ltd., NSW, Australia). Control media (HESC without cAMP treatment) were included in every assay. Protein Extraction in Preparation for Differential In-Gel Electrophoresis (DIGE). Cells were washed three times with ice-cold PBS and lysed with solubilization buffer (40 mM TrisHCl (pH7.4), 7 M urea, 2 M thiourea, 1% (w/v) C7BzO) (n ) 4). Following centrifugation at 16 000 × g for 5 min at 4 °C, the supernatant was transferred to a fresh tube and the pellet reextracted as above. The supernatants were pooled and the proteins recovered by precipitation with acetone (10 × volume for 90 min) followed by centrifugation for 30 min at 10 000 × g at room temperature. The protein pellet was air-dried and redissolved with CyDye labeling buffer (30 mM Tris-HCl (pH8.1), 7 M urea, 2 M thiourea, 1% (w/v) C7BzO). Protein concentration was determined using a modified Bradford method15 and adjusted to 1.75 mg/mL with CyDye labeling buffer. The samples were snap frozen on dry ice and stored at -80 °C until required. DIGE. Fluorescent labeling of proteins was performed by minimal CyDye labeling methodology as per manufacturer’s instructions (GE Healthcare Bioscience, Uppsala, Sweden). In brief, 50 µg of proteins from samples treated with or without cAMP were labeled with 400 pmol of Cy3 or Cy5, respectively. In addition, 25 µg from each sample were pooled and labeled with 400 pmol Cy2 for use as the internal standard. One microliter of 10 mM lysine was added to stop the CyDye labeling process. In each case, proteins were precipitated as described above, resuspended in 100 µL of CyDye labeling buffer, snap-frozen and stored at -80 °C. Experiments were repeated with four independent pairs of cell isolations, and reciprocal dye labeling experiments were performed to eliminate artifactual labeling effects between the dyes. Samples were passively rehydrated overnight into 24 cm Immobilin DryStrip pH 4-7 linear pH gradient (IPG) (BioRad, Hercules, CA). First-dimension separation of protein by isoelectric focusing (IEF) was performed using the following conditions: current limited to 60 µA per strip, 100 V for 9 min, 300 V for 90 min, 500 V for 3 h, gradient to 1000 V for 4 h, gradient to 8000 V for 3 h, and then at constant 8000 V to a total of 60 000 Vh. On completion, the strips were equilibrated 5740

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Paule et al. in 10 mL of equilibration buffer (50 mM Tris/HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) for 20 min on a shaking platform at 80 rpm. Second dimension separation was carried out in a 4-20% acrylamide gradient overnight at a constant 50 V using a BioRad Dodeca electrophoresis tank. Differential expression analysis based on normalized spot volumes was carried out using PG240 SameSpots software (Nonlinear Dynamics, Newcastle-upon-Tyne, U.K.). Image Capture. Two-dimensional gels were scanned using a Fuji FLA5100 laser scanner (FujiFilm, Tokyo, Japan) at the Cy2, Cy3, and Cy5 excitable wavelengths of 473, 535, and 635 nm, respectively. Image alignment, spot detection, background removal and expression analysis were performed using Progenesis SameSpot software (Nonlinear Dynamics, Newcastle Upon Tyne, U.K.). Fold changes were calculated based on normalized spot volumes and all statistical analyses were carried out automatically by the software. Protein Identification by MALDI-TOF/TOF MS or LCMS/MS. Protein spots of interest were excised from the gel using a ProPicII robotic spot picker (Genome Solutions, MI), based on X-Y coordinates exported directly from PG240 SameSpots. Protein identification by MALDI TOF MS was also as described.16 Monoisotopic peak masses were automatically extracted using GPS Explorer software (v 3.0 build 311; Applied Biosystems, CA) and peak lists searched against the nonredundant UniProtKB/Swiss-Prot database (version 57.2, H. sapiens, 462764 sequences entries; http://ww.expasy.org) using the MASCOT search engine (version 2.2; http://www.matrixscience. com.) Species was restricted to Homo sapiens, carbonylamidecysteine (CAM - fixed modification) and oxidation of methionine (variable modification) were taken into account, a parent ion mass tolerance of 60 ppm, and 1 missed cleavage (trypsin) were allowed. Up to 15 of the most intense peptides detected in each MS scan and peak lists were extracted using Data Analysis software version 3.4 (Bruker Diagnostics, Germany). The parameters used to create the peak lists were as follows: mass range 100-3000 Da; signal-to-noise threshold of 5; minimum compounds length of 10 spectra. Peptide mass (MS) data were searched using in-house MASCOT search engine (version 2.2, Matrix Science) against the UniProtKB/Swiss-Prot database as above with fragment mass tolerance of 0.1 Da. Protein identities were assigned using the following criteria to evaluate the search; statistically significant identity as indicated by MOWSE score (>43), minimum number of peptides matched (>4); and direct correlation between the identified protein and its estimated molecular mass and pI were determined from the 2D gel. Proteins that were not identified by MALDI TOF MS were submitted for further analysis by LC-MS/MS. The peptide mixture was then fractionated by nanoflow reversed-phase liquid chromatography using a 1200 series Capillary HPLC (Agilent Technologies, Santa Clara, CA) online equipped with a nanoAcquity C18 150 mm × 0.15 mm i.d. column (Waters, U.S.A.). Fractionation was performed at a flow rate of 0.5 µL/ min at 45 °C in a linear 60 min gradient from 100% solvent A (0.1% formic acid) to 100% solvent B (0.1% formic acid, 60% acetonitrile). Mass spectra were acquired using an LTQ mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) for automated MS/MS. Data-dependent MS analysis was performed by acquiring one FTMS scan followed by MS2 on the top five most intense ions. Dynamic exclusion was enabled at repeat count 1, exclusion

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Decidualized Proteins in Human Endometrial Stromal Cells list size 700, exclusion duration 180 s, and exclusion mass width (1.5 m/z. Collision-induced dissociation was performed by setting the ion isolation width at 2 m/z, normalized collision energy at 35%, activation Q at 0.25, and an activation time at 30 ms. Spectra were exported in mascot generic file format (.mgf) and analyzed using the Mascot search engine as detailed above. Standard search parameters included a peptide mass tolerance of 1.5 Da, peptide fragment tolerance of 0.8 Da, peptide charge of +2 or +3, and up to 1 missed cleavage was allowed. Western Blot Analysis. Decidualized and control cells were washed twice with ice cold PBS, lysed with a lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EGTA, 2 mM EDTA) containing protease inhibitors cocktail (1 mM AEBSF-HCl, 0.8 µM Aprotinin, 50 µM Bestatin, 15 µM E-64, 50 µM EDTA, 20 µM Leupeptin, 10 µM Pepstatin; Roche, Castle Hill, NSW, Australia) and centrifuged at 14 000 rpm at 4 °C for 10 min (n ) 3). The supernatants were transferred to fresh tubes and protein concentration was determined by Bradford protein assay as above. The prepared samples were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, NSW, Australia), and then probed with appropriate antibodies. The antibodies included caldesmon antibody (CALD; monoclonal mouse C6542, Sigma; 1:500), antitropomyosin 4 (TPM4, polyclonal rabbit, AB5449, Millipore, NSW, Australia; 1:1000), protein disulfide isomerase1A (PDIA1; monoclonal mouse, NB300-517, Novus Biologicals, Littleton, CO; 1:2000), LIM and SH3 protein1 (LASP1; clone 8C6, MAB8991, Millipore; 1:1000), and Src substrate cortactin (SRC8; clone 4F11, 05-150, Millipore; 1:2000) and β-tubulin (polyclonal rabbit, 2146, Cell Signaling Technology, Danver, MA; 1:2000). Proteins of interest were detected with horseradish peroxidise-conjugated anti rabbit or mouse IgG (Dako Botany, NSW, Australia) and visualized by enhanced chemiluminescent detection reagents (GE Healthcare) on hypersensitive film (GE Healthcare). Immunohistochemistry (IHC). Endometrial biopsies from mid proliferative (day 8-10, n ) 5-6) and late-secretory phase (day 25-28, n ) 5-6) of the menstrual cycle represented the control and decidualized stroma, respectively. Biopsies were embedded in paraffin, sectioned (5 µm), dewaxed in Histosol (Sigma), and rehydrated. Sections stained for caldesmon (monoclonal mouse, C-0297, Sigma) and LASP1 required an antigen retrival process. Sections were microwaved at high power (700W) in 0.01 mol/L sodium citrate buffer (pH 6.0) for 5 min, cooled, and treated with 3% H2O2 in methanol to black endogenous peroxide activity. Sections were blocked for 10 min in 15% horse serum in high salt TBS-0.1%Tween (0.3 M NaCl, in 50 mM Tris, pH 7.6, 0.1% Tween20) for caldesmon or 20% goat serum in high salt TBS-001% Tween for LASP1, respectively, to prevent nonspecific binding. Primary antibodies to caldesmon (1:1000) or LASP1 (1:2500) were applied to sections and incubated at 37 °C for 30 min. For sections probed incubated with caldesmon antibody, biotinylated horse antimouse IgG (1:200, Millipore, NSW, Australia) was then applied for 30 min at room temperature, and the signal was amplified and visualized with StreptABC/HRP (DAKO) and diaminobenzidine (DAKO). LASP1 was visualized with the Envision system (see below; DAKO). For the detection of PDIA1, TPM4, and SRC8, sections were blocked with goat serum for 10 min and antibodies were applied: 1:2500 PDI1A; 1:5000 anti-TPM 4; and 1:2500 SRC8. The application of Envision system and then peroxidise substrate 3,3′ diaminobenzidine (DAB) (Dako)

Figure 1. A representative 2D DIGE gel of control and decidualized human endometrial stromal cells. The gel shows the separation across a pH 4-10 gradient and separation on 8-16% polyacrylamide gradient gel (A). A box within panel A was enlarged to highlight spots of interest (B). Differentially expressed proteins identified by MALDI-TOF/TOF MS or LC-MS/MS are numbered and indicated by circles (Table 1).

produced a brown precipitate. A negative control was included for each section in which nonimmunized mouse or rabbit IgG (Dako) was appropriately replaced by the primary antibody. All sections were counterstained in Harris hematoxylin (Sigma; 1:10). Biological Profiling and Pathway Analysis. Differentially expressed proteins identified by MALDI-TOF/TOF MS and LC-MS/MS were first investigated for their roles in biological processing. The ontology was built and assigned by PANTHER (protein analysis through evolutionary relationships) classification systems [www.pantherdb.org]. Proteins identified were used as focus proteins to generate a biological network by Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood city, CA).

Results Proteins Identified by 2D-DIGE as Changed Following Decidudalization of Endometrial Stromal Cells. Primary HESC (n ) 4) were decidualized with cAMP and decidualization success was confirmed by significant increase in prolactin secretion (not shown) as previously described.8 Cell lysates from decidualized and control cells from the same women (n ) 4) were paired for CyDye labeling; one was labeled with Cy5 and the other with Cy3. A representative 2D-DIGE gel is shown in Figure 1. A total of 2714 protein spots were detected with 88 of those with fold changes of greater than 1.5 were significantly different between control and decidualized cells. From the 18 spots, 13 proteins were unambiguously identified by MALDITOF/TOF MS or LC-MS/MS (Table 1). The remaining 74 spots could not be identified, presumably due to their low abundance, and were not examined further. Journal of Proteome Research • Vol. 9, No. 11, 2010 5741

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Table 1. Assigned Protein Identification Using MALDI-TOF/TOF MS or LC-MS/MS spot no.c

name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Src substrate cortactin tropomyosin alpha-4 chain caldesmon caldesmon Src substrate cortactin protein disulfide-isomerase tubulin alpha-1C chain keratin, type II cytoskeletal 1 actin, cytoplasmic 2 keratin, type II cytoskeletal 1 Src substrate cortactin protein disulfide-isomerase drebrin-like protein elongation factor 1-delta LIM and SH3 domain protein 1 elongation factor 1-beta nuclear migration protein nudC UV excision repair protein RAD23 homologue B

18 a

Analysis by MALDI-TOF/TOF MS.

b

Swiss-prot accession ID

threshold

P value

pI

MW (Da)

peptide count

MOWSE

SRC8 TPM4 CALD1 CALD1 SRC8 PDIA1 TBA1C K2C1 ACTG K2C1 SRC8 PDIA1 DBNL EF1D LASP1 EF1B NUDC

1.6 1.7 1.5 1.6 1.8 1.7 1.7 1.6 1.8 1.5 1.7 1.5 2 2 -2.6 1.5 1.7

0.027 0.012 0.034 0.003 0.005 0.018 0.04 0.021 0.014 0.008 0.021 0.004 0.011 0.004 0.015 0.002 0.014

5.24 4.67 5.63 5.63 5.24 4.76 4.94 8.16 5.31

61598.5 28504.4 93194.5 93194.5 61598.5 57080.7 50103.6 65978 41765.8 65978 61598.5 57487 48468 31219 29698.2 24922 38277

14/16 14/16 21/16 15/16 15/16 17/16 7/16 14/16 14/16 10 16 6 11 9 15 9 14

102 197 224 145 76 196 82 229 227 58 72 104 77 116 243 201 117

a a a a a a a a a b b b b b b b b

28 55.3 32.7 21.4 20.8 42.9 16.8 29.6 49.7 21.2 14.7 14.6 18.5 19.6 37.9 20 41.1

RD23B

1.9

0.027

12

304

b

35.5

43203

% peptide coverage

c

Analysis by LC-MS/MS. Spots numbering from Figure 1.

Figure 3. Classification of identified proteins according to biological function. Differentially expressed proteins in decidualized human endometrial stromal cells were classified according to protein ontology groupings using the PANTHER classification system [www.pantherdb.com].

Figure 2. Histograms of the mean normalized volumes ( standard deviation (SD) of each protein numbered in Figure 1 as altered with decidualization (solid bar) and undecidualized controls (empty bar). The average normalized volume of (A) spots 1-5 and (B) spots 6-15 was from four individual experiments.

Of the 18 spots identified, 17 were significantly increased and one decreased in abundance following decidualization (Figure 2). CALD1 (Spots 3 and 4), PDI1A (Spots 3 and 12), and SRC8 (Spots 1, 5, and 11) were identified more than once with each isoform significantly elevated in decidualized stromal cells (Figure 2A,B). TPM4 (Spot 2) and LASP-1 (Spot 15) only appeared as individual spots. Spots 3 and 4, which are the same MW (93 kDa) but differ in pI, were both identified as CALD1. This was also observed for spots 1 and 5 which was SRC8. By contrast, another form of SRC8 (Spot 11) and two forms of PDI1A (Spot 6 and 12) 5742

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varied in both pI and MW suggesting different isoforms of the one protein. Interestingly, this form of SRC8 appeared at a greater MW and pI than proposed by Swiss-Prot database suggesting either a new, possibly tissue-specific isoform and/ or that SRC8 has undergone dramatic post-translational modifications during decidualization. Biological Function Analysis of Identified Proteins. The potential biological functions of the 13 proteins identified by 2D-DIGE as differing between control and decidualized stromal cells was assessed by PANTHER Classification System. Most of the proteins identified had multiple functional roles and this was taken into account. For example, the suggested functional roles of ACTG (Table 2; Spot 9) are in exocytosis, endocytosis, transport, cytokinesis, and cell structure. The major biological functions for the assigned identified proteins were associated with changes in cell structure and motility (41%), endocytosis/ exocytosis (9%), protein biosynthesis (9%), mitosis (9%), and DNA repair (9%). The functions of transport, cytokinesis, protein disulfide isomerize reaction, intracellular traffic, and chromosome segregation had a lesser contribution (4.6%) to decidualization (Figure 3). Validation by Western Blot Analysis. Five of the identified proteins (CALD1, SRC8, TPM4, LASP1, and PDI1A) were

Decidualized Proteins in Human Endometrial Stromal Cells selected for validation for the following reasons. CALD1, SRC8, and PDI1A were each identified more than once. TPM4 was selected due to its known functional role in cell structure while LASP1 was the only identified protein to be down-regulated during decidualization. Proteins were validated by two means, immunohistochemistry and Western blot analysis. Western blot analysis was performed on lysates of cells treated similarly to those subjected to 2D-DIGE. CALD1 in decidualized and control cell lysates appeared as multiple isoforms (Figure 4A). In control lysates, CALD1 appears at approximately 70, 77, and 95 kDa, whereas in decidualized cell lysates, only two bands were apparent for CALD1 at 77 and 95 kDa with both bands being increased with decidualization. The 2D-DIGE identified two isoforms of CALD1 at 93.2 kDa with different pI, and it may be that both are differentially regulated and other isoforms of caldesmon may also exist in the pool of 74 differentially proteins that were not identified. The lower MW form of caldesmon (∼70 kDa) detected by Western blot suggests either proteolytic processing or down regulation during decidualization. Expression of TPM4 (35 kDa) (Figure 4B), SRC8 at 80 and 85 kDa (Figure 4C) and PDIA1 at 61 kDa (Figure 4D) were all confirmed as upregulated with decidualization as demonstrated by 2D-DIGE. However, expression of LASP1 (at 35 kDa) decreased with decidualization, also validating 2D-DIGE results. Confirmation That CALD1, TPM4, SRC8, and PDI1A Are Present while LASP1 Is Absent in Decidualized Stromal Cells in Vivo in Human Uterus. To further validate that our proteomic findings reflect the in vivo situation in women, IHC was performed on endometrial tissue sections from two distinct phases of the menstrual cycle: midproliferative and latesecretory phases. Stromal cells spontaneously decidualize in the late secretory phase, attaining a distinct morphology that is clearly identified on histological sections. During the proliferative phase, CALD1 was absent in the stromal but punctate staining was evident in some epithelial glands (Figure 5A) whereas in the late secretory phase, it was clearly localized within the decidualized cells especially those surrounding the spiral arteries (Figure 5B). This staining pattern confirmed our proteomic findings that CALD1 is upregulated with decidualization and our previous study17 confirming the proteomic data. The blood vessel walls were also positive for CALD1 and some epithelial cells in the midproliferative but not late-secretory phase. Immunoreactive TPM4 was absent from stroma in the midproliferative phase although faint staining was observed in the glandular epithelium. However, in the late-secretory phase TPM4 was present only in decidual cells and absent from the glandular epithelium (Figure 5D). SRC8 was undetectable in both the stromal compartment and the glandular epithelium in the midproliferative phase (Figure 5E). In the late-secretory phase, both decidual cells and glandular epithelial, SRC8 staining were apparent (Figure 5F). Stromal PDI1A was dectectable in the decidualized cells during the late-secretory phase (Figure 5H) while PDIA1 was present in the glandular epithelium in both midproliferative and late-secretory phases (Figure 5G,H). Thus the IHC results confirm the proteomic findings that TPM4, SRC8, and PDI1A are upregulated during decidualization and are localized in the decidualized stroma during the late secretory phase of the menstrual cycle. In the 2D-DIGE analysis, LASP1 protein expression was lower in decidualized than nondecidualized stromal cells. The IHC results displayed punctate LASP1 staining in some but not all

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Figure 4. Representative Western blot of control and decidualized endometrial stromal lysates. Lysates were collected from three individual experiments and membranes were probed with antibodies against (A) CALD1, (B) TPM4, (C) SRC8, (D) PDIA1, and (E) LASP1. Equal loading was verified by β-actin.

cells in the endometrial stromal compartment during both the midproliferative and late-secretory phases of menstrual cycle (Figure 5I,J, respectively) and in the cytoplasm of the glandular epithelium during the midproliferative phase (Figure 5I). The stained cells in the stroma were more abundant in the midproliferative than in the late-secretory phase. Protein Networks Implicated In Decidualized Stromal Cells. We performed Ingenuity Pathways Analysis to investigate whether the identified proteins regulated with decidualization Journal of Proteome Research • Vol. 9, No. 11, 2010 5743

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Paule et al. cellular movement, assembly, and organization. From the validated proteins, the interactions are indirectly associated with PI3K and directly linked to CTTN (Figure 6B), which also have strong association with cellular movement, assembly, and organization, highlighting these as important functions in decidualized stromal cells.

Discussion

Figure 5. Representative immunohistochemical staining on human endometrium taken during the proliferative and late secretory phase of the menstrual cycle. Sections were stained for (A,B) CALD1, (C,D) TPM4, (E,F) SRC8, (G,H) PDIA1, and (I,J) LASP1. The insets are negative controls. GE, glandular epithelium; ST, stroma; Dec, decidual cells, SA; spiral arteriole. Closed arrows point to blood vessels and open arrows are directed to the LASP1 stains. Images were taken 100× magnification.

are involved in relevant networks and pathways. Each focus protein from our list was mapped onto the Ingenuity Pathways Knowledge Base, which contains important curated information on interactions between genes, proteins, and other biological molecules. Two pathways were generated from proteins either identified by MS (Figure 6A) or those identified and validated by Western blot analysis and IHC (Figure 6B). This analysis assessed the interactive networks important during the process of decidualization. Highly interconnected networks are likely to represent significant biological function. From the identified proteins, there are complex interactions indirectly associated with transforming growth factior β1 (TGFβ1), phosphoinositide-3 kinase (PI-3K), and SRC8 (or CTTN from IPA) (Figure 6A). The functions associated with this network include 5744

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The aim of this study was to identify proteins altered during decidualization of endometrial stromal cells using 2D-DIGE. The proteins identified were proteins known to be associated with cell structure and cytoskeletal remodelling in other cell lines.18-23 We not only confirmed the previous finding that CALD1 is upregulated17 but identified and validated SRC8, TPM4, PDI1A, and LASP1 as abundant proteins involved in decidualization of endometrial stromal cells. Decidualization of endometrial stromal cells requires major morphological, structural, and functional changes that are closely associated with cytoskeletal reorganization.24-26 Initiation of decidualization is accompanied by a significant downregulation of R-smooth muscle actin24 while disruption of actin filaments promotes decidualization.12 LASP1 was the only identified protein identified at reduced levels in decidualized stroma. LASP1 is a member of the LIM protein subfamily characterized by a LIM motif and a Src homology region 3. Upregulation of LASP1 has been implicated in the cell growth of human breast and hepatocellular carcinomas.21,22,27 LASP1 may therefore play an important role during the proliferative phase during endometrial restoration following menses. However during the secretory phase, stromal cells cease to proliferate and LASP1 would not be required for this function. LASP1 also functions as an F-actin-binding protein.28,29 Since reorganization and destabilization of actin cytoskeleton is necessary for decidualization, decreased LASP1 expression may induce destabilization of F-actin.29,30 The cytoskeleton provides necessary scaffolding to support and reorganize the cellular components. The ability of the cytoskeleton to deform and reform is critical for cellular differentiation. CALD1, TPM4, and SRC8 are proteins involved in cytoskeletal remodelling. Caldesmon is a calmodulin- and actin-binding protein that is essential for the regulation of smooth muscle and nonmuscle contraction.31,32 Isoforms of caldesmon exist through alternative splicing of a single gene. High (h) molecular weight caldesmon corresponds to isoform 1 (793 aa) and low (l) molecular weight caldesmon has isoforms 2-5 (532-564 aa) all of which are present in the decidualized stromal cells. Both forms of caldesmon have N-and C-termini and the main difference between them is that l-caldesmon lacks a central a R-helical domain (residues 201-447).33 Our results demonstrate the presence of 3 isoforms of CALD1 in control conditions and confirmed previous findings that two isoforms of CALD1 are increased with decidualization.17 The N-terminus of caldesmon interacts with tropomyosin and myosin while the C-terminus binds with actin, Ca2+-binding protein, myosin, phospholipids, and tropomyosin.34-36 Since CALD1 has several binding sites for tropomyosin, it is not surprising that TPM4 was also identified by 2D-DIGE in this study. To date nothing is known of TPM4 in decidualized stromal cells and its function may be tissue specific. In skeletal muscle, TPM4 is a marker of growth and repair/regeneration in response to injury, disease state, and stress.23 There are 40 isoforms of tropomyosin (TPM) resulting from alternative splicing.37 TPM co-operatively interacts with caldesmon and

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Figure 6. Functional biological networks of (A) identified and (B) validated differential proteins that were altered during decidualization of human stromal cells using Ingenuity program. Shaded proteins were those identifed by proteomics; unshaded proteins were associated with the network based on current literature. Direct interactions appear as solid lines whereas indirect interactions appear as dotted lines. (CALD1, caldesmon 1; CD2AP, CD2-associated protein; CHI3L1, Chitinase 3-like 1 (cartilage glycoprotein-39); CSF2, colony stimulating factor 2 (granulocyte-macrophage); CTNND1, catenin (cadherin-associated protein), delta 1; CTTN, cortactin; DNM2, dynamin 2; F-Actin (filamentous actin); HGF, hepatocyte growth factor (hepapoietin A; scatter factor); IL5, interleukin 5 (colony-stimulating factor, eosinophil); LASP1 (LIM and SH3 protein 1); MYCBPAP (includes EG:84073), MYCBP-associated protein; P4HB, protein disulfide isomerase 1A; PAK1, p21 protein (Cdc42/Rac)-activated kinase 1; PI3K, phosphatidylinositol 3-kinase; PKN2, protein kinase N2; PRKD1, protein kinase D1; PXN, paxillin; SRC, v-src sarcoma (Schmidt-Ruppin A-2); SYK, spleen tyrosine kinase; TCERG1, transcription elongation regulator 1; TPM4, tropomyosin 4; TPO, thyroid peroxidase.

non-cooperatively inhibits F-actin interaction with caldesmon (at the C-terminus). The binding of TPM to F-actin reduces TPM turnover rates and stabilizes actin microfilament bundles.38 The complex interaction between CALD1 and TPM4 is necessary to stabilize actin filaments and to resist F-actin

severing and depolymerization in both smooth muscle and nonmuscle cells.39,40 Whether this is also the case in decidualized endometrial stromal cells remains to be established. SRC8 is an adapter protein that also interacts closely with actin. SRC8 comprises an N-terminal acidic (NTA) domain Journal of Proteome Research • Vol. 9, No. 11, 2010 5745

research articles followed by 6.5 37-amino acid repeats that are proline rich and has an SH3 domain at the carboxy terminus.41 The amino terminal and the fourth repeat of SRC8 bind to F-actin.41 The NTA domain and the F-actin binding repeat domain are required for Arp2/3 complex activation,41 which facilitates actin network formation. Thus it is likely that CALD1, TPM4, and SRC8 closely interact with actin to promote cytoskeletal remodelling during decidualization. PDI1A is a multifunctional protein that catalyzes the formation, breakage, and rearrangement of disulfide bonds. The localization of PDI1A is important as it determines its functional role.42 To date it is known that when present at the cell surface, PDI1A acts as a reductase, cleaving disulfide bonds of proteins attached to the cell.43,44 Intracellular, PDI1A rearranges disulfide bonds of nascent proteins.45 At high concentrations, PDI1A functions as a chaperone that inhibits aggregation of misfolded proteins, while at low concentration PDI1A facilitates aggregation (antichaperone activity).46 PDI1A is upregulated in decidualized stroma and in particular is present in the cytoplasm of decidualized stromal cells. Since PDI1A catalyzes formation, breakage, and rearrangement of disulfide bonds, which are important in cytoskeletal remodelling,47,48 it was reassuring to find and validate that PDI1A increased with decidualization. TGFβ1 and PI-3K have previously been associated with stimulating growth factors, cytokines, sex steroids, and endometrial remodelling, all of which are important in decidualization and the establishment of pregnancy.49-55 Among the 18 identified spots proteins, such as SRC8 (CTTN) and RAD23B (Figure 6A), these two may be pivotal to decidualization. Currently there are no studies regarding the role of SRC8 and RAD23B in decidualized stromal cells. Here we have demonstrated the power of proteomics to identify novel proteins and unravel important functional pathways associated with decidualization. In particular, we have identified cytoskeletal changes, particularly those related to actin reorganization as one piece of the puzzle in understanding this unique process.

Acknowledgment. This work was supported by the National Health of Medical Research Council of Australia [Project Grants 41117 and 611804 and Fellowship No. 494808 (to G.N.)] by the Victorian Government Operational Infrastructure Support Program, and CONRAD CICCR, U.S.A. A.N.S. was supported by an Ovarian Cancer Research Foundation fellowship. We thank Dr. Lois Salamonsen for proofreading this manuscript. Supporting Information Available: The proteins identified by MALDI-TOF/TOF MS (Table S1) and LC-MS/MS (Table S2) from 2D-DIGE are sorted according to spot number and identified by Swiss-Prot accession numbers. The numbers listed in rows indicate the number of distinct peptides identified by mass spectrometry in human endometrial stromal cells. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Norwitz, E. R.; Schust, D. J.; Fisher, S. J. Implantation and the survival of early pregnancy. N. Engl. J. Med. 2001, 345 (19), 1400– 8. (2) Dunn, C. L.; Kelly, R. W.; Critchley, H. O. Decidualization of the human endometrial stromal cell: an enigmatic transformation. Reprod. Biomed. Online 2003, 7 (2), 151–61. (3) Salamonsen, L. A. The Menstrual and Estrous Cycles. In The Endometrium: molecular, cellular and clinical perspectives, 2nd ed.; Informa U.K. Ltd.: London, 2007; pp 25-45.

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