Objective
Preimplantation factor (PIF) is a novel, 15 amino acid peptide, secreted by viable embryos. This study aims to elucidate PIF’s effects in human endometrial stromal cells (HESC) decidualized by estrogen and progestin, which mimics the preimplantation milieu, and in first-trimester decidua cultures (FTDC).
Study Design
HESC or FTDC were incubated with 100 nmol/L synthetic PIF or vehicle control. Global gene expression was analyzed using microarray and pathway analysis. Proteins were analyzed using quantitative mass spectrometry, and PIF binding by protein array.
Results
Gene and proteomic analysis demonstrate that PIF affects immune, adhesion, and apoptotic pathways. Significant up-regulation in HESC (fold change) include: nuclear factor-k-β activation via interleukin-1 receptor-associated kinase binding protein 1 (53); Toll-like receptor 5 (9); FK506 binding protein 15, 133kDa protein (2.3); and Down syndrome cell adhesion molecule like 1 (16). B-cell lymphoma protein 2 was down-regulated in HESC (21.1) and FTDC (27.1). Protein array demonstrates PIF interaction with intracellular targets insulin-degrading enzyme and beta-K + channels.
Conclusion
PIF displays essential multitargeted effects, of regulating immunity, promoting embryo-decidual adhesion, and regulating adaptive apoptotic processes.
The mammalian embryo/allograft may be considered a “perfect transplant.” The mother/host tolerates the invading embryo and subsequent fetoplacental unit while preserving maternal immunity, ability to fight infection, and ability to control autoimmune processes. Multiple factors have to act synergistically to allow such pregnancy-specific immune paradox. Maternal recognition of pregnancy occurs even before implantation, thus indicating that embryo-specific signaling is present before the embryo is in intimate contact with the endometrium.
Decidual cells play a central role in implantation, and they have been investigated extensively using a well-established in vitro culture system based upon induction of decidualization using ovarian steroids. This culture system serves as a valuable model to examine the role of different compounds during periimplantation, a time frame in human reproduction that poses challenges to investigation in vivo.
Preimplantation factor (PIF) (MVRIKPGSANKPSDD) is a novel peptide, secreted only by viable embryos, absent in nonviable ones, detected in the circulation of several species of pregnant mammals, and expressed in the placenta. Recently, we demonstrated that synthetic PIF replicates native PIF action, modulates peripheral immune cells, and creates a favorable immune environment shortly after fertilization. PIF regulates uterine natural killer cell toxicity in vivo, implying that PIF contributes to the maternal adaptation to pregnancy.
Herein, we used genomic and proteomic analysis to further define PIF’s precise mechanism of action in a well-characterized in vitro system of human endometrial stromal cells (HESC) decidualized by estrogen and progestin, which mimics the preimplantation milieu, and in first-trimester decidua cultures (FTDC).
Materials and Methods
Peptide synthesis
Synthetic PIF analog (MVRIKPGSANKPSDD) was produced using solid-phase peptide synthesis (Peptide Synthesizer; Applied Biosystems, Foster City, CA) employing Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Final purification was carried out by reversed-phase high-pressure liquid chromatography, and peptide identity was verified by mass spectrometry. Alexa Fluor 647-PIF conjugate was generated as well (Bio-Synthesis Inc, Lewisville, TX).
Endometrial cell cultures
Institutional review board approval was obtained for this study. Discarded endometrial tissue from premenopausal women undergoing hysterectomies due to benign indications were used. Decidual specimens from the first trimester were obtained from women undergoing elective terminations in weeks 6-12 of normal pregnancy.
Using our previously established cell culture method, we studied nonpregnant HESC and cells collected from healthy first trimester of pregnancy decidua. Endometrial and decidual cells were isolated and resuspended in Roswell Park Memorial Institute-1640, grown to confluence, and found to be leukocyte free (<1%). After reaching confluence, cells were decidualized using 10 –8 mol/L estradiol and 10 –7 mol/L synthetic progestin analog (R5020) (Du Pont/NEN, Boston, MA), in both cases for 7 days. Cells were switched to a serum-free medium containing insulin, transferrin, and selenium (Collaborative Research Inc, Waltham, MA); 5 μmol/L trace elements (GIBCO, Carlsbad, CA); and 50 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), and treated overnight with synthetic PIF (100 nmol/L) or vehicle control. The tissue was collected and frozen at –80°C.
Microarray analysis
Total RNA was extracted from each cell culture. Analysis of HESC or FTDC ± PIF 100 nmol/L (n = 3/group) was examined using Affymetrix (Santa Clara, CA) U133 Plus 2.0 Array (18,400 human genes), followed by fluorescence labeling and hybridization with Fluidics Station 450 and optical scanning with GeneChip Scanner 3000 (Affymetrix) at W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT.
Raw data were analyzed by GeneSpring software (Agilent, Santa Clara, CA), normalized for interchip and intrachip variation to eliminate false-positive results.
Mass spectrometry analysis
HESC protein lysates (n = 3/group) were homogenized in buffer (100 mmol/L Tris, 250 mmol/L NaCl, 0.1% Triton X-100, 0.1% SDS) and assayed for protein concentration. A total of 10 μg of each lysate was loaded in singlet onto a Novex 4-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (Invitrogen, Carlsbad, CA) followed by electrophoresis. Each lane was excised into 10 equal slices, digested with trypsin, and analyzed by nanoliquid chromatography/mass spectrometry on an LTQ-Orbitrap XL tandem mass spectrometer (Thermo Fisher Scientific, Inc., San Jose, CA). Data were searched against the Mascot concatenated forward-and-reversed v3.46 International Protein Index database (Matrix Science Ltd, London, UK) and collated into nonredundant lists using Scaffold software (Proteome Software Inc, Portland, OR). A dual-filtering strategy of target-decoy database searching and employing the Peptide and ProteinProphet algorithms provides high confidence in the proteins identified (false detection rate <0.5%).
Expression of Toll-like receptor-2 in FTDC
Western blotting for Toll-like receptor (TLR)-2 (diluted 1:1000) and β-actin (diluted 1:10,000) was conducted (n = 1) using established methods. Antibodies for TLR-2 and β-actin were obtained from R&D Systems (Minneapolis, MN) and Abcam Inc (Cambridge, MA), respectively. Densitometric analyses were carried out with the National Institutes of Health’s ImageJ software (available at http://rsb.info.nih.gov.easyaccess1.lib.cuhk.edu.hk/ij ; Sun Microsystems, Santa Clara, CA). Values were expressed as the ratio of TLR-2 to β-actin, correcting for loading efficiency.
Alexa-PIF interaction with human proteome array
ProtoArray Human Protein Microarrays v5.0 (Invitrogen) with 9000 possible targets were initially blocked followed by exposure to PIF-Alexa 647 (250 nmol/L and 2.5 μmol/L). Unbound probe was washed off, and arrays were assessed by a fluorescence microarray scanner. As negative control, an array was incubated with wash buffer while calmodulin kinase was used as positive control detected by Alexa Fluor 647-conjugated anti-V5 antibody. Three stringent conditions determined Alexa-PIF specificity of binding: signal value >200 relative fluorescent units, >6 time higher than the negative control, and Z-factor >5.
Statistical analysis
For gene experiments, data were first analyzed using Student t test, with P < .05 results, followed by fold-change testing, with a cutoff >2-fold only reported. Pathway analysis was performed using the Ingenuity Systems Inc (Redwood, CA) software, ranking by greatest number of genes in a given pathway. For proteome data, spectral counting was employed for relative quantitation and Student t test P < .05 was considered significant.
Results
Effect of PIF on HESC
PIF 100 nmol/L significantly changed >500 genes. The genes that were significantly changed (>10-fold) are featured in Table 1 . Notably, there was an 18.1-fold decrease in phosphodiesterase 4B, cyclic AMP-specific (phosphodiesterase, a gene encoding for a protein that specifically hydrolyzes cyclic AMP, a critical molecule promoting decidualization.
| Gene | Description | Fold PIF/control |
|---|---|---|
| IRAK1BP1 | IL-1 receptor-assoc kinase1 bind.Prot1 | 53 |
| CALD1 | Caldesmon 1 | 29.9 |
| STX3 | Syntaxin 3 | 22.8 |
| DSCAML1 | Down syndrome cell adhesion like 1 | 16 |
| FNDC3B | Fibronectin type III domain containing 3B | 14.7 |
| TLX2 | T-cell leukemia homeobox 2 | 13.8 |
| UNC5CL | Unc-5 homolog C (C. elegans)-like | 11.9 |
| RPE65 | Retinal pigment epithelium protein 65kDa | 11.8 |
| SLC22A1 | Solute carrier family 22 A1 | 11.6 |
| GABRG1 | GABA A receptor, gamma 1 | 11.6 |
| ZNF148 | Zinc finger protein 148 | –16.6 |
| GRM1 | Glutamate receptor, metabotropic 1 | –17.2 |
| PDE4B | Phosphodiesterase 4B, cAMP-specific, E4 | –18.1 |
| RYBP | RING1 and YY1 binding protein | –19.3 |
| TLK1 | Tousled-like kinase1 | –19.6 |
| BCL2 | B-cell CLL/lymphoma2 | –21.2 |
| COL8A1 | Collagen, type VIII, alpha1 | –24.9 |
| GBF1 | Golgi-specific brefeldin A resistance factor1 | –26.2 |
| VPS26A | Vacuolar protein sorting 26 homolog A | –29 |
| MCCC2 | Methylcrotonoyl-coenzyme A carboxylase2B | –33.1 |
We focused on 3 pathways that are relevant for implantation: (1) immune tolerance; (2) embryo adhesion; and (3) apoptosis/remodeling.
Effects on HESC gene expression
Immune pathway
PIF increased IRAK1BP1 (53-fold), which interacts with TLR5 ( Figure 1 ), a pathway involved in innate immune response to microbial agents. A decrease in IL12RB2 (9.1-fold), a receptor for a Th1 cytokine promoter of T-cell proliferation and interferon γ secretion, was observed. PIF modulates the calcineurin pathway by down-regulating FK506 binding protein 1A, 12kDa (2.8-fold). The IgG immunoglobulin genes SEMA3D and SEMA3C were down-regulated, 10.8- and 9.9-fold, respectively.
Adhesion pathway
PIF up-regulated Down syndrome cell adhesion molecule like 1 (16-fold) and sorbin and SH3 domain containing (SORBS)2 (2.6-fold), which interacts with actin cytoskeleton. Connexin 45, a gene encoding for a protein that facilitates cellular interaction, was up-regulated 2.1-fold.
Apoptotic pathway
Figure 2 demonstrates that PIF suppressed Bcl-2 (21.1-fold), and induced downstream effects in the Bcl-2 network. PIF increased MDM2 (5-fold), resulting in limiting cell proliferation via interaction with P53, thus favoring apoptosis. PIF up-regulated BCOR (4.2-fold), which acts as a Bcl6 corepressor.
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