Original Article
Periostin Enhances Migration, Invasion, and Adhesion of Human Endometrial Stromal Cells Through Integrin-Linked Kinase 1/Akt Signaling Pathway
Reproductive Sciences 1-9 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115572481 rs.sagepub.com
Xiaoxuan Xu, MD1, Qiaomei Zheng, PhD1, Zongzheng Zhang, MD2, Xiaolei Zhang, PhD1, Ruihan Liu, MD1 and Peishu Liu, PhD1
Abstract Although our previous study confirmed that periostin (PN) was overexpressed in the eutopic and ectopic endometrial stroma of women with endometriosis by immunohistochemitry, the role of PN in the pathophysiology of endometriosis remains unknown. Thus, we aimed to investigate the effects of PN on endometrial stromal cells (ESCs) migration, invasion, adhesion, and proliferation and to further study the mechanism under this process. Eutopic (EuSCs), ectopic (EcSCs), and normal ESCs (NSCs) were isolated and cultured. We evaluated the above-mentioned biology behaviors and the expression of PN, integrin-linked kinase 1 (ILK1), and phospho-Akt (p-Akt) in NSCs, EuSCs as well as EcSCs before and after receiving PN small-interfering RNA (siRNA). The protein and messenger RNA (mRNA) levels of PN were upregulated in EuSCs (P < .05; P ¼ .2261 in proliferative phase and P ¼ .3385 in secretory phase) and EcSCs (P < .001; P < .001 in proliferative phase and P < .05 in secretory phase) compared with NSCs, although there was no significant difference in PN mRNA between EuSCs and NSCs. In EcSCs, abilities of migration, invasion, and adhesion and the expressions of ILK1 and p-Akt were enhanced; and all of those were downregulated after PN siRNA interference. Thus, PN enhanced ESCs migration, invasion, and adhesion due to the ILK1/Akt signal pathway. As an agonist in the development and progression of endometriosis, PN may be a new clinical treatment target of endometriosis. Keywords periostin, ESCs, ILK1/Akt, migration, invasion, adhesion
Introduction Endometriosis is classified as the presence of endometrial glands and stroma beyond the uterine cavity. It is an estrogen-dependent chronic disease, which affects almost 10% of women in their reproductive period, and is related to dysmenorrhea, chronic pelvic pain, and infertility.1 The etiology of endometriosis remains unclear, but the theory of reflux menses Sampson proposed in 1921 is still the leading theory.2 In recent years, Lang proposed the determinant of uterine eutopic endometrium for the supplementary of Sampson theory, including ectopic adhesion, invasion, and proliferation of endometrial debris.3 Although endometriosis is a benign disease, it exhibits malignant-like biological behaviors, namely, adhesion, aggression, and angiogenesis (3-step procedure of pathogenesis),4 which leads to the development of endometriosis. Currently, there is no known cure for endometriosis and treatment aims at relieving pains, ameliorating infertility, eliminating lesions, and preventing recurrence. The available therapies are surgical excision and various medical treatments, which include gonadotropin-releasing hormone analogs, aromatase
inhibitors, and progestins,5 However, these medical therapies are not suitable long term due to their numerous side effects, such as massive hemorrhage, perimenopausal stage symptoms, and liver dysfunction.6-8 Moreover, the recurrence rate of endometriosis is still high following conservative surgery, unless patients accepted hysterectomy or reached menopause.7 Thus, it is necessary to continue seeking for an effective targeted therapy for endometriosis. Periostin (PN), a 90-kDa secreted protein, belongs to the Fasciclin I family.9 It is generally present at low level in most adult tissues but plays a crucial role in osteology,
1 Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, Shandong, China 2 Department of Orthopedics, Taishan Medical University, Tai’an, Shandong, China
Corresponding Author: Peishu Liu, Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, No. 107 Wenhua Xi Road, Lixia District, Jinan, Shandong, China. Email: [email protected]
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cardiovascular system, oncology, injury, or inflammation.10 High level of PN can promote cell migration, invasion, adhesion, fibrogenesis, proliferation, survival, and epithelial– mesenchymal transition, and downregulation of PN obviously decreased above-mentioned behaviors.9,11-13 In cancers, PN can enhance cell invasion, metastasis, angiogenesis, and survival through binding to integrins and activating Akt- and focal adhesion kinase (FAK)-mediate pathways.14 In myocardial infarction and injury, PN upregulates integrin receptors to activate phosphatidylinositol 3 kinase/Akt and mitogen-activated protein kinase pathways and facilitates tissue remodeling.15,16 Integrin-linked kinase 1 (ILK1), linking with integrinb cytoplasmic tails (CTs), is a member of the serine/threonine kinases.17 Primarily, ILK1 is a biological regulator of integrin pathway. The ILK- and FAK-mediated signal axes constitute 2 major signaling pathways of integrin. Growing evidence indicates that ILK1 expression is upregulated in cancers, including astrocytoma, ovarian cancer, breast cancer, and nonsmall cell lung cancer.18-20 In addition, ILK is directly associated with Akt (Ser-473) phosphorylation.21 The loss of the ILK gene markedly impairs cell adhesion and motility22 and inhibits the activation of Akt.23 However, little is known about the expression and function of ILK in ectopic stromal cells (EcSCs). In our previous work, we have confirmed that PN was highly expressed in the eutopic and ectopic endometrial stroma of women with endometriosis compared with normal endometrium by immunohistochemitry.24 But the mechanism PN induced in endometriosis, particularly in ESCs, has not yet been studied. Normally, retrograde endometrial debris could not survive. In view of previous studies, we hypothesized that PN may promote ESCs migration, invasion, adhesion, and proliferation by activating ILK1/Akt, which accelerates the formation of ectopic lesions. Therefore, we aimed to investigate the effects of PN on ESCs migration, invasion, adhesion, and proliferation and to further study its mechanism.
Materials and Methods Sample Collection and Cell Culture The eutopic and ectopic endometria were obtained from 32 women (25-45 years old; proliferative phase: n ¼ 22; secretory phase: n ¼ 10; no luteinizing hormone [LH] timing) who underwent laparoscopy for ovarian endometriosis. Control endometria were obtained from 25 normal women (22-46 years old; proliferative phase: n ¼ 15; secretory phase: n ¼ 10; and no LH timing), which involved in uterine malformation, teratoma, and ovary simple cyst. Then all samples were confirmed by pathological examination, and proliferative and secretory endometrium phases were distinguished by hematoxylin–eosin staining. All patients had regular menstruation and no hormonotherapy for at least 6 months. This study received an ethical support from the institutional review board of Qilu hospital affiliated to Shandong University, and all the participants knew the purpose of collecting samples and gave their written consent. 2
The ESCs were isolated following the digestion of type IV collagenase (5 mg/mL) and deoxyribonuclease I (15 U/mL). For removal of undigested tissues, cell suspension was filtrated through a sterile stainless steel wire mesh (100 mm). For purity of endometrial stromal cells (ESCs), epithelial cells were removed via passing a 40-mm sieve. After centrifugated at 1000 rpm for 7 minutes, the supernatant was removed. Cells were then cultured at 37 C and 5% carbon dioxide in Dulbecco modified Eagle medium F-12 (DMEM/F12; Sigma-Aldrich, St Louis, Missouri) containing 10% fetal bovine serum (FBS; Gibco, Australia) and 1% antibiotic. The purity of ESCs at passage 2 was evaluated by cell immunofluorescence using mouse antihuman cytokeratin (the phenotype of epithelial cells; 1:25; Cell Signaling Technology, Danvers, Massachusetts) and rabbit antihuman vimentin (the phenotype of stromal cells; 1:50; Cell Signaling Technology). The purity of ESCs was over 95%, which was shown as the proportion of stromal cells in 5 randomly selected pictures (20 magnification). These cells were used for the following experiments.
Western Blot Until reaching 80% to 90% confluence, cells were lysed in RIPA (Beyotime, Jiangsu, China) for Western blot analysis, as previous description.25 Protein samples (4 mg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Primary antibodies used for the assay included rabbit antibodies against PN (1:1000; Abcam, Cambridge, Massachusetts, cat no. ab14041), ILK1, phospho-Akt (p-Akt), Akt (1:1000; Cell Signaling Technology), and a mouse antibody against glyceraldehyde 3-phosphate dehydrogenase (1:2000; Santa Cruz Biotechnology, Dallas, Texas, cat no. sc47724). Secondary antibodies were peroxidase-conjugated goat antimouse immunoglobulin G (IgG) and antirabbit IgG (Beijing Zhongshan Biotech Company, Beijing, China). Blots were detected using the chemiluminescent reagent according to the manufacturer’s instructions. The gray value of bands was analyzed by Image J (National Institutes of Health, Bethesda, Maryland). The result is expressed as the protein expression level compared to that of normal stromal cells (NSCs).
Quantitative Real-Time Reverse TranscriptionPolymerase Chain Reaction Total RNA was isolated from ESCs with trizol (Invitrogen Life Technologies, Carlsbad, California), and RNA concentration was detected by Nanophotometer Pearl (Implen Company, Mu¨nchen, Germany). The complementary DNA was acquired from 500 ng RNA according to the reverse transcription specification (TaKaRa, Shiga, Japan). Polymerase chain reaction (PCR) amplification was performed in Roche LightCyclerR480 with SYBR-green (Toyobo, Osaka, Japan) in a 10mL reaction system. The threshold cycle (CT) method (2Dct) was utilized to calculate gene expression levels. The sequence of primers used are listed in Table 1.
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Table 1. Primers Used for Real-Time RT-PCR. Gene
Forward, 50 -30
Reverse, 50 -30
Periostin P-actin
TG CCC AGC AGT TTT GCC CAT CG GGA CCT GAC TGA CTA CCT
CG TTG CTC TCC AAA CCT CTA AA GCA TTT GCG GTG GA
Abbreviation: RT-PCR, reverse transcription-polymerase chain reaction.
Table 2. The Sequences of Periostin siRNA. Number
Sense (50 -30 )
Antisense (50 -30 )
Periostin-homo-355 Periostin-homo-876 Periostin-homo-1205 Negative control
GCC CUG GUU AUA UGA GAA UTT GCC AUC ACA UCG GAC AUA UTT GGU CCU AAU UCC UGA UUC UTT UUC UCC GAA CGU GUC ACG UTT
AUU CUC AUA UAA CCA GGG CTT AUA UGU CCG AUG UGA UGG CTT AGA AUC AGG AAU UAG GAC CTT ACG UGA C AC GUU CGG AGA ATT
Abbreviation: siRNA, small-interfering RNA.
Silencing of the PN Gene in EcSCs The small-interfering RNA (siRNA) sequences targeting human PN were designed by GenePharma Company (Shanghai, China). Cells were seeded in 6-well plate without antibiotics treated for 24 hours and transfected with blank sequence or PN siRNA (50 nmol/L) using lipofectamine 2000 (Invitrogen Life Technologies) when cell confluence gets 50% to 60%. After 48 hours of transfection, cells were digested for the ensuing cell experiments. Protein was extracted for Western blot analysis after 72 hours of transfection. The PN siRNA sequences are shown in Table 2.
for 4 hours before dimethyl sulfoxide treated. The ability of cell proliferation was evaluated by measuring the absorbance at 470 nm. Data are calculated as the ratio of optical density value compared to that of NSCs.
Statistical Analysis Data were analyzed by 1-way analysis of variance (SPSS13.0 software) followed by Student-Newman-Keuls (equal variances) or Dunnett T3 (nonequal variances) post hoc test. All data are presented as mean + standard error of the mean. P < .05 was considered statistically significant.
Migration/Invasion and Cell Adhesion Assay
Results
When the plates were already precoated with matrigel (1:7; 60 mL; BD Bioscience, San Diego, California) or fibronectin (40 mg/mL; 50 mL; Beyotime, Shanghai, China), cells were digested for migration/invasion and adhesion assays, as previously described.26 For cell adhesion assay, cells were, respectively, incubated for 15, 30, 60 minutes, and 2 hours at 37 C. For migration/invasion assay, cells were incubated for 24 hours. After fixation with methanol, the extent of migration, invasion, and adhesion was determined by counting the number of stained cells (crystal violet staining) with Image-Pro Plus in 3 randomly selected pictures. The results are described as the fold changes compared to NSCs.
The Purity of ESCs
Cell Proliferation Assay A total of 5 103 cells in DMEM/F12 supplemented with 10% FBS were distributed into each well of a 96-well flatbottomed microplate and incubated overnight. Then the medium was removed, and cells were incubated, respectively, for 24, 48, and 72 hours with PN siRNA. Thereafter, 10 mL of methyl thiazolyl tetrazolium (MTT; Sigma-Aldrich) solution was added to each well, and the cells were further incubated
The purity of stromal cells was 96.1% + 2.2% as confirmed by cell immunofluorescence, which was judged by red fluorescence for vimentin, green fluorescence for cytokeratin and blue fluorescence for cell nucleus (Figure 1).
The Expression of PN, ILK1, and p-Akt in NSCs, Eutopic Stromal Cells, and EcSCs To determine the expression of PN in NSCs, eutopic stromal cells (EuSCs), and EcSCs, Western blot and quantitative PCR (qPCR) were performed. The protein (Figure 2A) and messenger RNA (mRNA; Figure 2B) of PN were upregulated in EuSCs and EcSCs both in proliferative and secretory phase, while the mRNA level showed no significance between EuSCs and NSCs throughout the menstrual cycle (Figure 2B). Besides, the expression level of PN in proliferative phase was higher than that in secretory phase, but there was no significance. Simultaneously, Western blot was utilized to detect the expressions of ILK1 and p-Akt, which were higher in EuSCs and EcSCs when compared to NSCs (Figure 2C and D).
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Figure 1. Identification of purity of endometrial stromal cells. Representative staining of cell nucleus is shown in the first image; red immunofluorescence shows the expression of vimentin; lack of green fluorescence represents negative immunoreactivity for cytokeratin; first three images are merged together, as shown in the last picture. The purity of endometrial stromal cells (ESCs) used for our study was 96.1% + 2.2%, which was calculated as the proportion of red fluorescence. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
Inhibition of PN Decreased the Expression of ILK1 and p-Akt in ESCs After transfection of 3 PN siRNAs in EcSCs, the expression of PN mRNA obviously decreased, which was evaluated by qPCR (Figure 3A). Especially, the transfection of PN-homo-1205 had the strongest effect on the downregulation of PN. So PN-homo1205 was used in the remaining tests. To understand the effects of PN on ILK1 and p-Akt, we detected the expression of PN, ILK1, and p-Akt after transfection of PN siRNA by Western blot. The result illustrated that protein level of PN was reduced, and the loss of PN protein and mRNA significantly impaired the expression of ILK1 and p-Akt (Figure 3B–D).
Periostin Enhanced ESCs Migration, Invasion, and Adhesion Through Activating ILK1/Akt Signaling Pathway To study whether PN regulated malignant-like behaviors of ESCs, transwell and adhesion assays were performed to assess the ability of migration, invasion, and adhesion in NSCs, EuSCs, and EcSCs before and after transfection of PN siRNA. The result indicated that abilities of migration and invasion in EuSCs and EcSCs were stronger than NSCs (Figure 4). The strongest ability of adhesion was presented in EcSCs at 15 and 30 minutes; however, there is no significance between NSCs and EuSCs (Figure 5). Importantly, the downregulation of PN decreased above-mentioned abilities. Meanwhile, inhibition of PN led to the low level of ILK1 and p-Akt (Figure 3). Thus, we speculated that the changes PN induced was ascribed to ILK1/Akt signaling pathway.
Periostin Failed to Regulate ESCs Proliferation The influence of PN on ESCs proliferation was determined by MTT assay. The result showed no statistical significance before and after transfection of PN siRNA (see Supplemental Figure). 4
Discussion In this study, we isolated and cultured NSCs, EuSCs, and EcSCs. Their immunophenotype and purity were analyzed by cell immunofluorescence. We found that all cell types were positively stained for vimentin of mesenchymal origin, and the purity was over 95%. The result showed that the similarity of immunophenotype may be considered as a common origin of NSCs, EuSCs, and EcSCs, which is in line with the report of Dimitrov et al.27 Accumulating evidences suggest that PN is involved in various diseases.9-13 However, little is known about the role of PN in the pathophysiology of endometriosis. Our group previously indicated that PN was highly expressed in the stroma of endometriosis by immunochemistry.24 In this article, we first assessed the expression of PN in NSCs, EuSCs, and EcSCs. Indeed, PN protein and PN mRNA were upregulated in EuSCs and EcSCs compared with their expression in NSCs. Notably, regardless of no significant changes in PN mRNA, PN protein was highly existed in EuSCs but less than that in EcSCs. This indicates that PN expression in EuSCs is supposed to be affected at posttranscriptional level,28,29 which is an important explanation for the inherent different behavior compared to NSCs. In short, PN may be a key element in the theory of determinant of uterine eutopic endometrium. However, the potential mechanism of posttranscriptional level is required to be further investigated. The PN serves as dual roles: a regulator of cell invasion and metastasis and a cell adhesion molecule.30 Hence, PN is deemed to own 2 main function, that is, enhancing cell migration and invasion as well as fibrillogenesis.31 All the results of the present and previous studies support that PN can enhance cell migration, invasion, and adhesion and the inhibition of PN results in the drop of above-mentioned biological behaviors.9,11-13 Furthermore, a published research showed that PN could increase cell migration, invasion, and adhesion by 2- to 9-fold.13 In many diseases, PN regulates cell migration, invasion, angiogenesis, and fibrogenesis via Akt and FAK signaling pathways.11,13,32 Here, we report the molecular
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Figure 2. Comparison of periostin, ILK1, and p-Akt levels in NSCs, eutopic stromal cells (EuSCs), and EcSCs. Panel A, respectively, shows the PN protein level (target/glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) of proliferative (*P < .05; ***P < .001) and secretory phase (#P < .05; ###P < .001) compared to NSCs. Panel B shows that the highest level of PN mRNA was presented in EcSCs throughout the menstrual cycle (***P < .001; #P < .05 vs NSCs). The periostin expression shows no significant changes between proliferative and secretory phases whether in protein level or mRNA level. Panel C describes the expression of ILK1 (*P < .05 vs NSCs). The expression of p-Akt (p-AKT/t-Akt) is shown in panel D (*P < .05 vs NSCs). EcSCs indicates ectopic stromal cells; ILK1, integrin-linked kinase 1; mRNA, messenger RNA; NSCs, normal stromal cells; p-Akt, phospho-Akt; PN, periostin; total-Akt, t-Akt.
mechanism PN mediated in ESCs due to ILK1/AKT signaling pathway. Interestingly, in our investigation, PN was found to promote fibrogenesis of ESCs and the fiber-like substance was dissolved after the loss of PN (see Supplemental Figure).
Similar phenomenon has been reported in other tissues, such as bone, skin, and heart diseases.33-35 Collagen cross-links were defective in the periosteum from PN / femurs, PNdeficient mouse skin, and myocardial infarction.33-35
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Figure 3. Effects of periostin on ILK1 and p-Akt in human endometrial stromal cells. The downregulation level of PN after the transfection of 3 PN small-interfering RNAs (siRNAs) was measured by quantitative polymerase chain reaction (qPCR; A). Of the 3 sequences, periostin-homo1205 displayed the strongest influence (A). Therefore, periostin-homo-1205 was used to knockdown the PN gene for the following study. The expression of PN, ILK1, and p-Akt after periostin-homo-1205 was analyzed by Western blot. After the PN gene was silenced, the protein level of PN was reduced (B), and ILK1 (C) and p-Akt (D) levels were also lower than before (*P < .05 vs ectopic stromal cells [EcSCs] and NC). ILK1 indicates integrin-linked kinase 1; p-Akt, phospho-Akt; PN, periostin.
However, our study demonstrated PN failed to regulate ESCs proliferation, which was consistent with that of ovary cancers in vitro and bladder cancers in vivo.30,36 Moreover, an associated report demonstrated that the proliferative activity of ectopic lesions was markedly lower, but there was no significance in eutopic endometria between endometriotic and normal patients.37 6
At present, the molecular mechanism of PN action on ESCs is still unclear. Periostin is widely involved in integrin pathway, including activating the intracellular signal transduction and the linkage between integrin and cytoskeleton.11,15,38 Notably, the connection of integrin and actin is mediated by a group of 30 adaptor proteins,39 and one of them is ILK1, which plays an essential role in the biologically relevant regulation of
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Figure 4. The influence of periostin on the ability of migration and invasion of human endometrial stromal cells. The typical pictures (20 magnification) of migratory and invasive in NSCs and EuSCs, as well as EcSCs before and after PN siRNA transfection (A). Bars ¼ 20 mm. In the migration assay (B), the number of staining cells was more in EuSCs and EcSCs than those in NSCs (**P < .01) and fewest in cells transfected by PN siRNA (*P < .01 vs EcSCs). As well, the performance of invasive cells was the same to that of migration (C; ***P < .001 vs NSCs; **P < .01 vs EcSCs). EcSCs indicates ectopic stromal cells; EuSCs, eutopic stromal cells; NSCs, normal stromal cells; siRNA, small-interfering RNA. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
integrin signaling pathway.40 Once PN binds to integrin b, the CTs were exposed to link to a heterotrimer of PINCH, ILK, and parvin and immediately stimulate Akt phosphorylation at Ser 473.21 In addition, PN can also activate Akt independently to regulate cell invasion, metastasis, angiogenesis, and survival.17 Recently, associated studies reported PI3K/AKT signaling pathway was activated in endometriosis.41,42 Especially, pAKT (Ser473) was highly expressed in endometriotic stromal
cells, which led to the reduced decidualization and cell survival.41,43 Therefore, we evaluated the levels of PN, ILK1, and p-Akt with the hypothesis that PN overexpression may upregulate the expressions of ILK1 and p-Akt. Moreover, we verified that the expressions of ILK1 and p-Akt were inhibited by the PN siRNA. Taken together, we considered that the mechanism of PN conducted in endometriosis may owe to the activation of ILK1/Akt.
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Figure 5. The influence of periostin on the ability of adhesion of human endometrial stromal cells. Panel A shows the adhesive ability of NSCs, EuSCs, and EcSCs at 15, 30, 60, and 120 minutes. The EcSCs had the strongest ability of adhesion (*P < .05), but there was no significance between NSCs and EuSCs (P > .05). Panel B shows the alteration of cell adhesion after interference of PN siRNA. The adhesive ability of EcSCs transfected with siRNA was always in the plateau and reached the biggest gap at 60 minutes (*P < .05). All the data were compared with NSCs at 15minutes. EcSCs indicates ectopic stromal cells; EuSCs, eutopic stromal cells; NSCs, normal stromal cells; PN, periostin; siRNA, small-interfering RNA.
Periostin, as an agonist in the pathophysiological process of endometriosis, may be a promising target of therapeutical intervention for endometriosis in the future. Recently, some researchers have evaluated the therapeutic potential of PN, which proposes that serum PN can be an effective prognostic indicator and targeted therapy marker.44,45 In our findings, PN siRNA suppresses PN/ILK1/Akt signaling pathway and produces strong abilities of antimigration, anti-invasion, and antiadhesion. These results suggest that PN-targeting therapy should be considered for its clinical value against endometriosis. Acknowledgment We acknowledge and thank Mr Daoxin Ma of Haematology Laboratory of Qilu Hospital of Shandong University.
Authors’ Note Xiaoxuan Xu and Qiaomei Zheng contributed equally to this work. This study was completed in the laboratory under his technical help.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (Nos. 81370696 and 81101984) and the Science and Technology Development planning of Shandong (2013GGE27031).
Supplemental Material The online data supplements are available at http://rs.sagepub.com/ supplemental. 8
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alphavbeta5 and focal adhesion kinase (FAK) pathway. Atherosclerosis. 2010;208(2):358-365. Liu C, Huang SJ, Qin ZL. Inhibition of periostin gene expression via RNA interference suppressed the proliferation, apoptosis and invasion in U2OS cells. Chin Med J (Engl). 2010;123(24):3677-3683. Yan W, Shao R. Transduction of a mesenchyme-specific gene periostin into 293T cells induces cell invasive activity through epithelial–mesenchymal transformation. J Biol Chem. 2006; 281(28):19700-19708. Morra L, Moch H. Periostin expression and epithelial–mesenchymal transition in cancer: a review and an update. Virchows Arch. 2011;459(5):465-475. Cho YH, Cha MJ, Song BW, et al. Enhancement of MSC adhesion and therapeutic efficiency in ischemic heart using lentivirus delivery with periostin. Biomaterials. 2012;33(5):1376-1385. Ontsuka K, Kotobuki Y, Shiraishi H, et al. Periostin, a matricellular protein, accelerates cutaneous wound repair by activating dermal fibroblasts. Exp Dermatol. 2012;21(5):331-336. Li J, Zhang H, Wu J, et al. Prognostic significance of integrinlinked kinase1 overexpression in astrocytoma. Int J Cancer. 2010;126(6):1436-1444. Ahmed N, Oliva K, Rice GE, Quinn MA. Cell-free 59 kDa immunoreactive integrin-linked kinase: a novel marker for ovarian carcinoma. Clin Cancer Res. 2004;10(7):2415-2420. Okamura M, Yamaji S, Nagashima Y, et al. Prognostic value of integrin beta1-ILK-pAkt signaling pathway in non-small cell lung cancer. Hum Pathol. 2007;38(7):1081-1091. Yang HJ, Zheng YB, Ji T, et al. Overexpression of ILK1 in breast cancer associates with poor prognosis. Tumour Biol. 2013;34(6): 3933-3938. Hannigan G, Troussard AA, Dedhar S. Integrin-linked kinase: a cancer therapeutic target unique among its ILK. Nat Rev Cancer. 2005;5(1):51-63. Sakai T, Li S, Docheva D, et al. Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation. Genes Dev. 2003;17(7):926-940. Edwards LA, Thiessen B, Dragowska WH, Daynard T, Bally MB, Dedhar S. Inhibition of ILK in PTEN-mutant human glioblastomas inhibits PKB/Akt activation, induces apoptosis, and delays tumor growth. Oncogene. 2005;24(22):3596-3605. Shen L, Liu P, Zhang P, Zhang X, Cui J. Characterization of periostin expression in human endometrium and endometriotic lesions. Gynecol Endocrinol. 2012;28(10):815-818. Xi G, Hu X, Wu B, et al. Autophagy inhibition promotes paclitaxel-induced apoptosis in cancer cells. Cancer Lett. 2011; 307(2):141-148. Xu YL, Wang DB, Liu QF, Chen YH, Yang Z. Silencing of cofilin-1 gene attenuates biological behaviours of stromal cells derived from eutopic endometria of women with endometriosis. Hum Reprod. 2010;25(10):2480-2488. Dimitrov R, Timeva T, Kyurkchiev D, et al. Characterization of clonogenic stromal cells isolated from human endometrium. Reproduction. 2008;135(4):551-558. McBride S, Walsh D, Meleady P, Daly N, Clynes M. Bromodeoxyuridine induces keratin protein synthesis at a posttranscriptional level in human lung tumour cell lines. Differentiation. 1999;64(3):185-193.
29. Berger A, Stierkorb E, Nickenig G. The role of the AUUUUA hexamer for the posttranscriptional regulation of the AT1 receptor mRNA stability. Biochem Biophys Res Commun. 2005;330(3): 805-812. 30. Kim CJ, Yoshioka N, Tambe Y, Kushima R, Okada Y, Inoue H. Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells. Int J Cancer. 2005;117(1):51-58. 31. Kudo A. Periostin in fibrillogenesis for tissue regeneration: periostin actions inside and outside the cell. Cell Mol Life Sci. 2011; 68(19):3201-3207. 32. Shao R, Bao S, Bai X, et al. Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through upregulation of vascular endothelial growth factor receptor 2 expression. Mol Cell Biol. 2004;24(9):3992-4003. 33. Kii I, Nishiyama T, Li M, et al. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture. J Biol Chem. 2010;285(3):2028-2039. 34. Norris RA, Damon B, Mironov V, et al. Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues. J Cell Biochem. 2007;101(3):695-711. 35. Shimazaki M, Nakamura K, Kii I, et al. Periostin is essential for cardiac healing after acute myocardial infarction. J Exp Med. 2008;205(2):295-303. 36. Zhu M, Fejzo MS, Anderson L, et al. Periostin promotes ovarian cancer angiogenesis and metastasis. Gynecol Oncol. 2010;119(2): 337-344. 37. Beliard A, Noel A, Foidart JM. Reduction of apoptosis and proliferation in endometriosis. Fertil Steril. 2004;82(1):80-85. 38. Borg TK, Markwald R. Periostin: more than just an adhesion molecule. Circ Res. 2007;101(3):230-231. 39. Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002;110(5):599-511. 40. Boulter E, Van Obberghen-Schilling E. Integrin-linked kinase and its partners: a modular platform regulating cell-matrix adhesion dynamics and cytoskeletal organization. Eur J Cell Biol. 2006; 85(3-4):255-263. 41. Yin X, Pavone ME, Lu Z, Wei J, Kim JJ. Increased activation of the PI3K/AKT pathway compromises decidualization of stromal cells from endometriosis. J Clin Endocrinol Metab. 2012;97(1): E35-43. 42. Samartzis EP, Noske A, Dedes KJ, Fink D, Imesch P. ARID1A mutations and PI3K/AKT pathway alterations in endometriosis and endometriosis-associated ovarian carcinomas. Int J Mol Sci. 2013;14(9):18824-18849. 43. Franco-Murillo Y, Miranda-Rodriguez JA, Rendon-Huerta E, et al. Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3beta [published online September 6, 2014.]. Reprod Sci. 2014. 44. Castronovo V, Waltregny D, Kischel P, et al. A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer. Mol Cell Proteomics. 2006;5(11):2083-2091. 45. Kudo Y, Siriwardena BS, Hatano H, Ogawa I, Takata T. Periostin: novel diagnostic and therapeutic target for cancer. Histol Histopathol. 2007;22(10):1167-1174.
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Periostin Enhances Migration, Invasion, and Adhesion of Human Endometrial Stromal Cells Through Integrin-Linked Kinase 1/Akt Signaling Pathway
Reproductive Sciences 1-9 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115572481 rs.sagepub.com
Xiaoxuan Xu, MD1, Qiaomei Zheng, PhD1, Zongzheng Zhang, MD2, Xiaolei Zhang, PhD1, Ruihan Liu, MD1 and Peishu Liu, PhD1
Abstract Although our previous study confirmed that periostin (PN) was overexpressed in the eutopic and ectopic endometrial stroma of women with endometriosis by immunohistochemitry, the role of PN in the pathophysiology of endometriosis remains unknown. Thus, we aimed to investigate the effects of PN on endometrial stromal cells (ESCs) migration, invasion, adhesion, and proliferation and to further study the mechanism under this process. Eutopic (EuSCs), ectopic (EcSCs), and normal ESCs (NSCs) were isolated and cultured. We evaluated the above-mentioned biology behaviors and the expression of PN, integrin-linked kinase 1 (ILK1), and phospho-Akt (p-Akt) in NSCs, EuSCs as well as EcSCs before and after receiving PN small-interfering RNA (siRNA). The protein and messenger RNA (mRNA) levels of PN were upregulated in EuSCs (P < .05; P ¼ .2261 in proliferative phase and P ¼ .3385 in secretory phase) and EcSCs (P < .001; P < .001 in proliferative phase and P < .05 in secretory phase) compared with NSCs, although there was no significant difference in PN mRNA between EuSCs and NSCs. In EcSCs, abilities of migration, invasion, and adhesion and the expressions of ILK1 and p-Akt were enhanced; and all of those were downregulated after PN siRNA interference. Thus, PN enhanced ESCs migration, invasion, and adhesion due to the ILK1/Akt signal pathway. As an agonist in the development and progression of endometriosis, PN may be a new clinical treatment target of endometriosis. Keywords periostin, ESCs, ILK1/Akt, migration, invasion, adhesion
Introduction Endometriosis is classified as the presence of endometrial glands and stroma beyond the uterine cavity. It is an estrogen-dependent chronic disease, which affects almost 10% of women in their reproductive period, and is related to dysmenorrhea, chronic pelvic pain, and infertility.1 The etiology of endometriosis remains unclear, but the theory of reflux menses Sampson proposed in 1921 is still the leading theory.2 In recent years, Lang proposed the determinant of uterine eutopic endometrium for the supplementary of Sampson theory, including ectopic adhesion, invasion, and proliferation of endometrial debris.3 Although endometriosis is a benign disease, it exhibits malignant-like biological behaviors, namely, adhesion, aggression, and angiogenesis (3-step procedure of pathogenesis),4 which leads to the development of endometriosis. Currently, there is no known cure for endometriosis and treatment aims at relieving pains, ameliorating infertility, eliminating lesions, and preventing recurrence. The available therapies are surgical excision and various medical treatments, which include gonadotropin-releasing hormone analogs, aromatase
inhibitors, and progestins,5 However, these medical therapies are not suitable long term due to their numerous side effects, such as massive hemorrhage, perimenopausal stage symptoms, and liver dysfunction.6-8 Moreover, the recurrence rate of endometriosis is still high following conservative surgery, unless patients accepted hysterectomy or reached menopause.7 Thus, it is necessary to continue seeking for an effective targeted therapy for endometriosis. Periostin (PN), a 90-kDa secreted protein, belongs to the Fasciclin I family.9 It is generally present at low level in most adult tissues but plays a crucial role in osteology,
1 Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, Shandong, China 2 Department of Orthopedics, Taishan Medical University, Tai’an, Shandong, China
Corresponding Author: Peishu Liu, Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, No. 107 Wenhua Xi Road, Lixia District, Jinan, Shandong, China. Email: [email protected]
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cardiovascular system, oncology, injury, or inflammation.10 High level of PN can promote cell migration, invasion, adhesion, fibrogenesis, proliferation, survival, and epithelial– mesenchymal transition, and downregulation of PN obviously decreased above-mentioned behaviors.9,11-13 In cancers, PN can enhance cell invasion, metastasis, angiogenesis, and survival through binding to integrins and activating Akt- and focal adhesion kinase (FAK)-mediate pathways.14 In myocardial infarction and injury, PN upregulates integrin receptors to activate phosphatidylinositol 3 kinase/Akt and mitogen-activated protein kinase pathways and facilitates tissue remodeling.15,16 Integrin-linked kinase 1 (ILK1), linking with integrinb cytoplasmic tails (CTs), is a member of the serine/threonine kinases.17 Primarily, ILK1 is a biological regulator of integrin pathway. The ILK- and FAK-mediated signal axes constitute 2 major signaling pathways of integrin. Growing evidence indicates that ILK1 expression is upregulated in cancers, including astrocytoma, ovarian cancer, breast cancer, and nonsmall cell lung cancer.18-20 In addition, ILK is directly associated with Akt (Ser-473) phosphorylation.21 The loss of the ILK gene markedly impairs cell adhesion and motility22 and inhibits the activation of Akt.23 However, little is known about the expression and function of ILK in ectopic stromal cells (EcSCs). In our previous work, we have confirmed that PN was highly expressed in the eutopic and ectopic endometrial stroma of women with endometriosis compared with normal endometrium by immunohistochemitry.24 But the mechanism PN induced in endometriosis, particularly in ESCs, has not yet been studied. Normally, retrograde endometrial debris could not survive. In view of previous studies, we hypothesized that PN may promote ESCs migration, invasion, adhesion, and proliferation by activating ILK1/Akt, which accelerates the formation of ectopic lesions. Therefore, we aimed to investigate the effects of PN on ESCs migration, invasion, adhesion, and proliferation and to further study its mechanism.
Materials and Methods Sample Collection and Cell Culture The eutopic and ectopic endometria were obtained from 32 women (25-45 years old; proliferative phase: n ¼ 22; secretory phase: n ¼ 10; no luteinizing hormone [LH] timing) who underwent laparoscopy for ovarian endometriosis. Control endometria were obtained from 25 normal women (22-46 years old; proliferative phase: n ¼ 15; secretory phase: n ¼ 10; and no LH timing), which involved in uterine malformation, teratoma, and ovary simple cyst. Then all samples were confirmed by pathological examination, and proliferative and secretory endometrium phases were distinguished by hematoxylin–eosin staining. All patients had regular menstruation and no hormonotherapy for at least 6 months. This study received an ethical support from the institutional review board of Qilu hospital affiliated to Shandong University, and all the participants knew the purpose of collecting samples and gave their written consent. 2
The ESCs were isolated following the digestion of type IV collagenase (5 mg/mL) and deoxyribonuclease I (15 U/mL). For removal of undigested tissues, cell suspension was filtrated through a sterile stainless steel wire mesh (100 mm). For purity of endometrial stromal cells (ESCs), epithelial cells were removed via passing a 40-mm sieve. After centrifugated at 1000 rpm for 7 minutes, the supernatant was removed. Cells were then cultured at 37 C and 5% carbon dioxide in Dulbecco modified Eagle medium F-12 (DMEM/F12; Sigma-Aldrich, St Louis, Missouri) containing 10% fetal bovine serum (FBS; Gibco, Australia) and 1% antibiotic. The purity of ESCs at passage 2 was evaluated by cell immunofluorescence using mouse antihuman cytokeratin (the phenotype of epithelial cells; 1:25; Cell Signaling Technology, Danvers, Massachusetts) and rabbit antihuman vimentin (the phenotype of stromal cells; 1:50; Cell Signaling Technology). The purity of ESCs was over 95%, which was shown as the proportion of stromal cells in 5 randomly selected pictures (20 magnification). These cells were used for the following experiments.
Western Blot Until reaching 80% to 90% confluence, cells were lysed in RIPA (Beyotime, Jiangsu, China) for Western blot analysis, as previous description.25 Protein samples (4 mg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Primary antibodies used for the assay included rabbit antibodies against PN (1:1000; Abcam, Cambridge, Massachusetts, cat no. ab14041), ILK1, phospho-Akt (p-Akt), Akt (1:1000; Cell Signaling Technology), and a mouse antibody against glyceraldehyde 3-phosphate dehydrogenase (1:2000; Santa Cruz Biotechnology, Dallas, Texas, cat no. sc47724). Secondary antibodies were peroxidase-conjugated goat antimouse immunoglobulin G (IgG) and antirabbit IgG (Beijing Zhongshan Biotech Company, Beijing, China). Blots were detected using the chemiluminescent reagent according to the manufacturer’s instructions. The gray value of bands was analyzed by Image J (National Institutes of Health, Bethesda, Maryland). The result is expressed as the protein expression level compared to that of normal stromal cells (NSCs).
Quantitative Real-Time Reverse TranscriptionPolymerase Chain Reaction Total RNA was isolated from ESCs with trizol (Invitrogen Life Technologies, Carlsbad, California), and RNA concentration was detected by Nanophotometer Pearl (Implen Company, Mu¨nchen, Germany). The complementary DNA was acquired from 500 ng RNA according to the reverse transcription specification (TaKaRa, Shiga, Japan). Polymerase chain reaction (PCR) amplification was performed in Roche LightCyclerR480 with SYBR-green (Toyobo, Osaka, Japan) in a 10mL reaction system. The threshold cycle (CT) method (2Dct) was utilized to calculate gene expression levels. The sequence of primers used are listed in Table 1.
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Table 1. Primers Used for Real-Time RT-PCR. Gene
Forward, 50 -30
Reverse, 50 -30
Periostin P-actin
TG CCC AGC AGT TTT GCC CAT CG GGA CCT GAC TGA CTA CCT
CG TTG CTC TCC AAA CCT CTA AA GCA TTT GCG GTG GA
Abbreviation: RT-PCR, reverse transcription-polymerase chain reaction.
Table 2. The Sequences of Periostin siRNA. Number
Sense (50 -30 )
Antisense (50 -30 )
Periostin-homo-355 Periostin-homo-876 Periostin-homo-1205 Negative control
GCC CUG GUU AUA UGA GAA UTT GCC AUC ACA UCG GAC AUA UTT GGU CCU AAU UCC UGA UUC UTT UUC UCC GAA CGU GUC ACG UTT
AUU CUC AUA UAA CCA GGG CTT AUA UGU CCG AUG UGA UGG CTT AGA AUC AGG AAU UAG GAC CTT ACG UGA C AC GUU CGG AGA ATT
Abbreviation: siRNA, small-interfering RNA.
Silencing of the PN Gene in EcSCs The small-interfering RNA (siRNA) sequences targeting human PN were designed by GenePharma Company (Shanghai, China). Cells were seeded in 6-well plate without antibiotics treated for 24 hours and transfected with blank sequence or PN siRNA (50 nmol/L) using lipofectamine 2000 (Invitrogen Life Technologies) when cell confluence gets 50% to 60%. After 48 hours of transfection, cells were digested for the ensuing cell experiments. Protein was extracted for Western blot analysis after 72 hours of transfection. The PN siRNA sequences are shown in Table 2.
for 4 hours before dimethyl sulfoxide treated. The ability of cell proliferation was evaluated by measuring the absorbance at 470 nm. Data are calculated as the ratio of optical density value compared to that of NSCs.
Statistical Analysis Data were analyzed by 1-way analysis of variance (SPSS13.0 software) followed by Student-Newman-Keuls (equal variances) or Dunnett T3 (nonequal variances) post hoc test. All data are presented as mean + standard error of the mean. P < .05 was considered statistically significant.
Migration/Invasion and Cell Adhesion Assay
Results
When the plates were already precoated with matrigel (1:7; 60 mL; BD Bioscience, San Diego, California) or fibronectin (40 mg/mL; 50 mL; Beyotime, Shanghai, China), cells were digested for migration/invasion and adhesion assays, as previously described.26 For cell adhesion assay, cells were, respectively, incubated for 15, 30, 60 minutes, and 2 hours at 37 C. For migration/invasion assay, cells were incubated for 24 hours. After fixation with methanol, the extent of migration, invasion, and adhesion was determined by counting the number of stained cells (crystal violet staining) with Image-Pro Plus in 3 randomly selected pictures. The results are described as the fold changes compared to NSCs.
The Purity of ESCs
Cell Proliferation Assay A total of 5 103 cells in DMEM/F12 supplemented with 10% FBS were distributed into each well of a 96-well flatbottomed microplate and incubated overnight. Then the medium was removed, and cells were incubated, respectively, for 24, 48, and 72 hours with PN siRNA. Thereafter, 10 mL of methyl thiazolyl tetrazolium (MTT; Sigma-Aldrich) solution was added to each well, and the cells were further incubated
The purity of stromal cells was 96.1% + 2.2% as confirmed by cell immunofluorescence, which was judged by red fluorescence for vimentin, green fluorescence for cytokeratin and blue fluorescence for cell nucleus (Figure 1).
The Expression of PN, ILK1, and p-Akt in NSCs, Eutopic Stromal Cells, and EcSCs To determine the expression of PN in NSCs, eutopic stromal cells (EuSCs), and EcSCs, Western blot and quantitative PCR (qPCR) were performed. The protein (Figure 2A) and messenger RNA (mRNA; Figure 2B) of PN were upregulated in EuSCs and EcSCs both in proliferative and secretory phase, while the mRNA level showed no significance between EuSCs and NSCs throughout the menstrual cycle (Figure 2B). Besides, the expression level of PN in proliferative phase was higher than that in secretory phase, but there was no significance. Simultaneously, Western blot was utilized to detect the expressions of ILK1 and p-Akt, which were higher in EuSCs and EcSCs when compared to NSCs (Figure 2C and D).
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Figure 1. Identification of purity of endometrial stromal cells. Representative staining of cell nucleus is shown in the first image; red immunofluorescence shows the expression of vimentin; lack of green fluorescence represents negative immunoreactivity for cytokeratin; first three images are merged together, as shown in the last picture. The purity of endometrial stromal cells (ESCs) used for our study was 96.1% + 2.2%, which was calculated as the proportion of red fluorescence. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
Inhibition of PN Decreased the Expression of ILK1 and p-Akt in ESCs After transfection of 3 PN siRNAs in EcSCs, the expression of PN mRNA obviously decreased, which was evaluated by qPCR (Figure 3A). Especially, the transfection of PN-homo-1205 had the strongest effect on the downregulation of PN. So PN-homo1205 was used in the remaining tests. To understand the effects of PN on ILK1 and p-Akt, we detected the expression of PN, ILK1, and p-Akt after transfection of PN siRNA by Western blot. The result illustrated that protein level of PN was reduced, and the loss of PN protein and mRNA significantly impaired the expression of ILK1 and p-Akt (Figure 3B–D).
Periostin Enhanced ESCs Migration, Invasion, and Adhesion Through Activating ILK1/Akt Signaling Pathway To study whether PN regulated malignant-like behaviors of ESCs, transwell and adhesion assays were performed to assess the ability of migration, invasion, and adhesion in NSCs, EuSCs, and EcSCs before and after transfection of PN siRNA. The result indicated that abilities of migration and invasion in EuSCs and EcSCs were stronger than NSCs (Figure 4). The strongest ability of adhesion was presented in EcSCs at 15 and 30 minutes; however, there is no significance between NSCs and EuSCs (Figure 5). Importantly, the downregulation of PN decreased above-mentioned abilities. Meanwhile, inhibition of PN led to the low level of ILK1 and p-Akt (Figure 3). Thus, we speculated that the changes PN induced was ascribed to ILK1/Akt signaling pathway.
Periostin Failed to Regulate ESCs Proliferation The influence of PN on ESCs proliferation was determined by MTT assay. The result showed no statistical significance before and after transfection of PN siRNA (see Supplemental Figure). 4
Discussion In this study, we isolated and cultured NSCs, EuSCs, and EcSCs. Their immunophenotype and purity were analyzed by cell immunofluorescence. We found that all cell types were positively stained for vimentin of mesenchymal origin, and the purity was over 95%. The result showed that the similarity of immunophenotype may be considered as a common origin of NSCs, EuSCs, and EcSCs, which is in line with the report of Dimitrov et al.27 Accumulating evidences suggest that PN is involved in various diseases.9-13 However, little is known about the role of PN in the pathophysiology of endometriosis. Our group previously indicated that PN was highly expressed in the stroma of endometriosis by immunochemistry.24 In this article, we first assessed the expression of PN in NSCs, EuSCs, and EcSCs. Indeed, PN protein and PN mRNA were upregulated in EuSCs and EcSCs compared with their expression in NSCs. Notably, regardless of no significant changes in PN mRNA, PN protein was highly existed in EuSCs but less than that in EcSCs. This indicates that PN expression in EuSCs is supposed to be affected at posttranscriptional level,28,29 which is an important explanation for the inherent different behavior compared to NSCs. In short, PN may be a key element in the theory of determinant of uterine eutopic endometrium. However, the potential mechanism of posttranscriptional level is required to be further investigated. The PN serves as dual roles: a regulator of cell invasion and metastasis and a cell adhesion molecule.30 Hence, PN is deemed to own 2 main function, that is, enhancing cell migration and invasion as well as fibrillogenesis.31 All the results of the present and previous studies support that PN can enhance cell migration, invasion, and adhesion and the inhibition of PN results in the drop of above-mentioned biological behaviors.9,11-13 Furthermore, a published research showed that PN could increase cell migration, invasion, and adhesion by 2- to 9-fold.13 In many diseases, PN regulates cell migration, invasion, angiogenesis, and fibrogenesis via Akt and FAK signaling pathways.11,13,32 Here, we report the molecular
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Figure 2. Comparison of periostin, ILK1, and p-Akt levels in NSCs, eutopic stromal cells (EuSCs), and EcSCs. Panel A, respectively, shows the PN protein level (target/glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) of proliferative (*P < .05; ***P < .001) and secretory phase (#P < .05; ###P < .001) compared to NSCs. Panel B shows that the highest level of PN mRNA was presented in EcSCs throughout the menstrual cycle (***P < .001; #P < .05 vs NSCs). The periostin expression shows no significant changes between proliferative and secretory phases whether in protein level or mRNA level. Panel C describes the expression of ILK1 (*P < .05 vs NSCs). The expression of p-Akt (p-AKT/t-Akt) is shown in panel D (*P < .05 vs NSCs). EcSCs indicates ectopic stromal cells; ILK1, integrin-linked kinase 1; mRNA, messenger RNA; NSCs, normal stromal cells; p-Akt, phospho-Akt; PN, periostin; total-Akt, t-Akt.
mechanism PN mediated in ESCs due to ILK1/AKT signaling pathway. Interestingly, in our investigation, PN was found to promote fibrogenesis of ESCs and the fiber-like substance was dissolved after the loss of PN (see Supplemental Figure).
Similar phenomenon has been reported in other tissues, such as bone, skin, and heart diseases.33-35 Collagen cross-links were defective in the periosteum from PN / femurs, PNdeficient mouse skin, and myocardial infarction.33-35
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Figure 3. Effects of periostin on ILK1 and p-Akt in human endometrial stromal cells. The downregulation level of PN after the transfection of 3 PN small-interfering RNAs (siRNAs) was measured by quantitative polymerase chain reaction (qPCR; A). Of the 3 sequences, periostin-homo1205 displayed the strongest influence (A). Therefore, periostin-homo-1205 was used to knockdown the PN gene for the following study. The expression of PN, ILK1, and p-Akt after periostin-homo-1205 was analyzed by Western blot. After the PN gene was silenced, the protein level of PN was reduced (B), and ILK1 (C) and p-Akt (D) levels were also lower than before (*P < .05 vs ectopic stromal cells [EcSCs] and NC). ILK1 indicates integrin-linked kinase 1; p-Akt, phospho-Akt; PN, periostin.
However, our study demonstrated PN failed to regulate ESCs proliferation, which was consistent with that of ovary cancers in vitro and bladder cancers in vivo.30,36 Moreover, an associated report demonstrated that the proliferative activity of ectopic lesions was markedly lower, but there was no significance in eutopic endometria between endometriotic and normal patients.37 6
At present, the molecular mechanism of PN action on ESCs is still unclear. Periostin is widely involved in integrin pathway, including activating the intracellular signal transduction and the linkage between integrin and cytoskeleton.11,15,38 Notably, the connection of integrin and actin is mediated by a group of 30 adaptor proteins,39 and one of them is ILK1, which plays an essential role in the biologically relevant regulation of
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Figure 4. The influence of periostin on the ability of migration and invasion of human endometrial stromal cells. The typical pictures (20 magnification) of migratory and invasive in NSCs and EuSCs, as well as EcSCs before and after PN siRNA transfection (A). Bars ¼ 20 mm. In the migration assay (B), the number of staining cells was more in EuSCs and EcSCs than those in NSCs (**P < .01) and fewest in cells transfected by PN siRNA (*P < .01 vs EcSCs). As well, the performance of invasive cells was the same to that of migration (C; ***P < .001 vs NSCs; **P < .01 vs EcSCs). EcSCs indicates ectopic stromal cells; EuSCs, eutopic stromal cells; NSCs, normal stromal cells; siRNA, small-interfering RNA. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
integrin signaling pathway.40 Once PN binds to integrin b, the CTs were exposed to link to a heterotrimer of PINCH, ILK, and parvin and immediately stimulate Akt phosphorylation at Ser 473.21 In addition, PN can also activate Akt independently to regulate cell invasion, metastasis, angiogenesis, and survival.17 Recently, associated studies reported PI3K/AKT signaling pathway was activated in endometriosis.41,42 Especially, pAKT (Ser473) was highly expressed in endometriotic stromal
cells, which led to the reduced decidualization and cell survival.41,43 Therefore, we evaluated the levels of PN, ILK1, and p-Akt with the hypothesis that PN overexpression may upregulate the expressions of ILK1 and p-Akt. Moreover, we verified that the expressions of ILK1 and p-Akt were inhibited by the PN siRNA. Taken together, we considered that the mechanism of PN conducted in endometriosis may owe to the activation of ILK1/Akt.
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Figure 5. The influence of periostin on the ability of adhesion of human endometrial stromal cells. Panel A shows the adhesive ability of NSCs, EuSCs, and EcSCs at 15, 30, 60, and 120 minutes. The EcSCs had the strongest ability of adhesion (*P < .05), but there was no significance between NSCs and EuSCs (P > .05). Panel B shows the alteration of cell adhesion after interference of PN siRNA. The adhesive ability of EcSCs transfected with siRNA was always in the plateau and reached the biggest gap at 60 minutes (*P < .05). All the data were compared with NSCs at 15minutes. EcSCs indicates ectopic stromal cells; EuSCs, eutopic stromal cells; NSCs, normal stromal cells; PN, periostin; siRNA, small-interfering RNA.
Periostin, as an agonist in the pathophysiological process of endometriosis, may be a promising target of therapeutical intervention for endometriosis in the future. Recently, some researchers have evaluated the therapeutic potential of PN, which proposes that serum PN can be an effective prognostic indicator and targeted therapy marker.44,45 In our findings, PN siRNA suppresses PN/ILK1/Akt signaling pathway and produces strong abilities of antimigration, anti-invasion, and antiadhesion. These results suggest that PN-targeting therapy should be considered for its clinical value against endometriosis. Acknowledgment We acknowledge and thank Mr Daoxin Ma of Haematology Laboratory of Qilu Hospital of Shandong University.
Authors’ Note Xiaoxuan Xu and Qiaomei Zheng contributed equally to this work. This study was completed in the laboratory under his technical help.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (Nos. 81370696 and 81101984) and the Science and Technology Development planning of Shandong (2013GGE27031).
Supplemental Material The online data supplements are available at http://rs.sagepub.com/ supplemental. 8
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