Proteomic Identification of Betaig-h3 as a Lysophosphatidic Acid-Induced Secreted Protein of Human Mesenchymal Stem Cells: Paracrine Activation of A549 Lung Adenocarcinoma Cells by Betaig-h3*

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor1/3 inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA1. LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA1– Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

MSCs exist predominantly in the bone marrow, they are also distributed throughout many other tissues, where they are thought to be the local sources of tissue-resident stem cells (15). Moreover, bone marrow-derived MSCs are recruited into the stroma of developing tumors (16). MSCs constitute a large proportion of non-neoplastic stromal cells within the tumor microenvironment (3). Accumulating evidence suggests that MSCs could also have an adverse effect that favors tumor growth. Tumor cells mixed with MSCs, when transplanted subcutaneously, exhibited elevated capability of proliferation and rich angiogenesis in tumor tissues (17). MSCs stimulated the metastatic potency of breast carcinoma when they were co-injected with human breast carcinoma cells into a subcutaneous site by xenograft transplantation (18). Furthermore, MSCs exposed to tumor-conditioned medium have been reported to exhibit phenotypic and functional characteristics of CAFs, including sustained expression of stromal cell-derived factor-1 (SDF-1) and the ability to promote tumor cell growth in vitro and in an in vivo co-implantation model (19). These results suggest that tumorigenesis and metastasis of carcinoma cells are acquired by paracrine signals from MSCs within the tumor-associated stroma. However, the paracrine signaling mechanisms by which MSCs stimulate tumorigenesis are largely unknown.
Periostin and ␤ig-h3 are extracellular matrix proteins that are structurally homologous to the axon guidance protein fasciclin I (FAS1) (20). Both periostin and ␤ig-h3 contain four tandem repeats of FAS1 domains and an EMI protein-protein interaction domain, and they play a key role in a variety of cellular responses, including adhesion, migration, proliferation, angiogenesis, wound healing and tumorigenesis (21)(22)(23). We have reported that periostin is secreted from human adipose tissue-derived mesenchymal stem cells (hASCs) in response to LPA treatment, and the recombinant periostin protein stimulates the adhesion and migration of epithelial ovarian cancer cells (24). ␤ig-h3 (also known as transforming growth factor beta-induced protein ig-h3 or TGFBI) was originally identified as transforming growth factor-␤1 (TGF-␤1)induced protein in A549 human adenocarcinoma cells (25). ␤ig-h3 is normally expressed in fibroblasts, keratinocytes, and muscle cells (26 -28). The expression of ␤ig-h3 was increased or downregulated in various tumor cells, depending on tumor types (21). ␤ig-h3 promoted the adhesion and migration of human hepatoma by interacting with ␣3␤1 integrin (29). Elevated expression of ␤ig-h3 is associated with high-grade human colon cancers and ectopic expression of the ␤ig-h3 enhanced the aggressiveness and altered the metastatic potentials of colon cancer cells (30). Furthermore, ␤ig-h3 has been reported to regulate tumor angiogenesis by regulating endothelial cell adhesion and migration (31). Despite the various reports implicating ␤ig-h3 in tumorigenesis, it is still unclear whether ␤ig-h3 is expressed in cancer stroma and whether ␤ig-h3 is involved in the crosstalk between cancer cells and stromal cells.
Lysophosphatidic acid (LPA) is a small bioactive phospholipid produced by activated platelets, mesothelial cells, fibroblasts, adipocytes, and some cancer cells (32)(33)(34). Accumulating evidence suggests that LPA is relevant to the tumorigenesis and metastasis (33). We have previously reported that LPA treatment induced the migration of hASCs and stimulated the expression of ␣-SMA and SDF-1, which have been known as markers for CAFs in vitro (35,36). Furthermore, co-transplantation of A549 human lung adenocarcinoma cells and hASCs stimulated in vivo growth of A549 cells and tumor angiogenesis and elicited differentiation of hASCs to CAFs expressing ␣-SMA and vascular endothelial growth factor, an angiogenic cytokine, through an LPA receptor 1 (LPA 1 )-mediated mechanism. Conditioned medium from A549 lung adenocarcinoma cells induced expression of ␣-SMA and vascular endothelial growth factor in hASCs through an LPA 1 -dependent mechanism in vitro. These results suggest a pivotal role of the LPA-LPA 1 signaling axis in the differentiation of hASCs to CAFs and in the paracrine function of hASCs within tumor microenvironment. However, the mechanism by which LPA-activated hASCs can regulate tumorigenic potential of cancer cells is largely elusive.
In order to clarify the paracrine mechanisms involved in the crosstalk between cancer cells and hASCs, we characterized secreted proteins included in LPA-conditioned medium from hASCs, using a shotgun proteomic analysis. The present study demonstrates a pivotal role of ␤ig-h3 as an LPA-induced paracrine factor of hASCs on the adhesive and proliferative properties of A549 cells.
Cell Culture-Subcutaneous adipose tissue was obtained from elective surgeries with patient's consent, and this protocol was approved by the Institutional Review Board of Pusan National University Hospital. For isolation of hASCs, adipose tissues were washed at least three times with sterile phosphate-buffered saline (PBS) and treated with an equal volume of collagenase type I suspension (1 g/L of Hank's Balanced Salt Solution with 1% BSA) for 60 min at 37°C with intermittent shaking. The floating adipocytes were separated from the stromal-vascular fraction by centrifugation at 300 ϫ g for 5 min. The cell pellet was resuspended in ␣-minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin, and cells were plated in tissue culture dishes at 3500 cells/cm 2 . The primary hASCs were cultured for 4 -5 days until they reached confluence and were defined as passage "0." The passage number of hASCs used in these experiments was 3-10. The hASCs were positive for CD29, CD44, CD73, CD90, and CD105, whereas CD31, CD34, and CD45 were not expressed in hASCs (supplemental Fig. S1).
Preparation of Conditioned Medium-hASCs were seeded on 150-mm cell culture dishes and cultured in growth medium until reaching confluence. The cells were briefly rinsed twice with PBS and then incubated with 15 ml of ␣-minimum essential medium in the absence or presence of 10 M LPA for 48 h before collecting media. The conditioned medium was centrifuged at 1000 ϫ g for 10 min to remove cell debris, filtered using 0.45-m Millipore syringe filters (Millipore, Bedford, MA), and stored at Ϫ70°C for subsequent use.
Cell Adhesion Assay-Ninety-six-well microculture plates (Falcon, Becton-Dickinson, Mountain View, CA) were incubated with recombinant ␤ig-h3 proteins or conditioned medium from hASCs at 37°C for 1 h and then blocked with PBS containing 0.2% BSA for 1 h at 37°C. Cells were trypsinized and suspended in the culture media at a density of 2 ϫ 10 5 cells/ml, and 0.1 ml of the cell suspension was then added to each well of the plates. Cell attachment was analyzed as follows. After incubation for 1 h at 37°C, unattached cells were removed by rinsing twice with PBS. The number of attached cells was determined by counting the cells under microscopy at 100ϫ magnification after staining with hematoxylin and eosin.
Cell Proliferation-Proliferation was determined with a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay: MTT is metabolized by NAD-dependent dehydrogenase to form a colored reaction product (formazan), and the amount of dye formed directly correlates with the number of cells. To determine cell numbers, A549 cells were seeded in a 24-well culture plate at a density of 2 ϫ 10 4 cells/well, cultured for 48 h in normal growth medium, serum-starved for 24 h, and treated with various reagents for the indicated times. The cells were washed twice with PBS and incubated with 100 l of MTT (0.5 mg/ml) for 2 h at 37°C. The formazan granules generated by the cells were dissolved in 100 l of dimethyl sulfoxide, and the absorbance of the solution at 562 nm was determined by using a PowerWave x microplate spectrophotometer (Bio-Tek Instruments, Inc.; Winooski, VT) after dilution to a linear range.
Sample Preparation and Tryptic Digestion-hASCs were cultured in 150-mm diameter culture dishes until they reached subconfluence and were washed twice with Hank's balanced salt solution to remove the serum component. The cells were incubated in 20 ml nonsupplemented (no serum, phenol red, or antibiotics) ␣-minimum essential medium in the absence or presence of 10 M LPA for 48 h. Conditioned media were collected and centrifuged at 1000 ϫ g for 10 min using a MF 300 centrifuge (Hanil Science Industrial, Inchon, Korea) to remove cell debris, filtered through a 0.2-m filter, and concentrated using Amicon Ultracel-3K (3000 Da) molecular weight cutoff centrifugal filter device (Millipore). For tryptic digestion, each sample was heated at 90°C for 15 min and 5 l of 100 mM dithiothreitol was added and incubated in 56°C for 20 min. Then, 5 l of 200 mM iodoacetamide was added and incubated at room temperature in the dark for 15 min. To consume any unreacted iodoacetamide, an additional 10 l of 100 mM dithiothreitol was supplemented. Reduced and alkylated proteins were digested with 500 ng of trypsin (Promega, Madison, WI) for 12 h at 37°C.
LC-MS/MS-Mass spectrometry analysis was performed using nano-scaled liquid chromatography tandem mass spectrometry (LC-MS/MS) system consisting of an Agilent 1100 high-pressure liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA) and a QSTAR quadrupole-time-of-flight mass spectrometer (MDS SCIEX, Concord, Ontario, Canada) equipped with a nano-electrospray ionization source. To achieve high-resolution separation, a nanoscale reversed phase chromatography analytical column (ZORBAX C18, 0.1 mm, 0.075 mm i.d.; Agilent Technologies) was used. Mobile phase A consisted of HPLC-grade water containing 0.1% formic acid and mobile phase B consisted of 84% HPLC grade acetonitrile containing 0.1% formic acid. Separation was performed at a flow rate of 300 nL/min and the applied gradient was 0 -40% phase B over 60 min. For MS/MS analysis, each scan cycle consisted of 1 full scan mass spectrum (m/z 400 -1500), followed by three MS/MS events. Digested samples were run in duplicate, and representative LC-MS/MS data from three independent experiments are shown (supplemental Table S1).
Database Searching-LC-MS/MS results were transformed to MASCOT generic files using MASCOT Daemon (version 2.2.2; Matrix Science, Manchester, U.K.). The MASCOT generic files were searched against the concatenated database (153,194 sequence entries and their reverse sequences) combining Homo sapiens and Bos taurus databases from UniProt (release 2010_08) using in-house MASCOT software (version 2.2.04). The following parameters were used: Trypsin (cuts C-terminal side of KR unless next residue is P); 2 missed cuts; carbamidomethylation (C) as fixed modification; N-acetyl (Protein), oxidation (M), pyroglutamylation (N-term EQ) as variable modification; and charge states ϩ2, ϩ3, and ϩ4. Windows of mass accuracy of 100 ppm and 0.25 Da were used for precursor ions and MS/MS data respectively. Peptide identification and protein assembly were performed in multiple stages. Initial peptide filtering was used to determine an estimated 1% false discovery rate, which was calculated using the target-decoy method (37). Proteins supported by less than 2 spectral counts or no unique peptides were removed. Tandem mass spectra of each protein annotated by a single peptide are shown in the supplemental Fig. S2. Proteins identified with a higher MASCOT score in the bovine database than in the human database were considered serum contamination and removed.
Quantitative Analysis of MS Results-To determine the foldchanges in the amounts of identified proteins between experimental groups, we used a normalized spectral index based on fragment ion intensity measurement with modification (38). Briefly, we calculated the spectral index (SI) of each protein, which is the sum of fragment ion intensities for identified peptides (including all its spectra) that are comprised of a protein and is normalized (SI N ) by dividing the total SI for all identified proteins (SI T ). To avoid taking logarithms on values of zero, we set the values of SI as 0.349 and 0.538 if no peptide was identified in the control and LPA-treated group, respectively, which is half the smallest value among all SI. We compared the protein quantity between two groups using log2 ratios of SI N , Log2 (SI N_ LPA/ SI N_ Con).
Bioinformatic Analysis-Identified proteins were assessed to define "putative secretory proteins." Classical secretory proteins with signal peptide were predicted by SignalP 3.0 neural network (NN) scoring. Nonclassical secretory proteins without signal peptides were predicted using secretomeP 2.0 mammalian neural network scoring. DAVID 2008 was used for information mining and functional annotation analysis (http://david.abcc.ncifcrf.gov/).
Immunodepletion of ␤ig-h3 from Conditioned Medium-For immunoprecipitation of ␤ig-h3, aliquots (30 l) of a suspension (50% slurry) of protein A-agarose beads (Sigma-Aldrich) in PBS were mixed with 1 g of anti-␤ig-h3 and control rabbit antibodies at 4°C for 1 h with intermittent shaking. After recovery by centrifugation, beads were washed three times and used for immunodepletion of ␤ig-h3. LPA CM was incubated with protein A-agarose beads immobilized with anti-␤ig-h3 and control antibodies for 1 h at 4°C. Immune complexes absorbed to protein A-agarose beads were precipitated by centrifugation. Resultant supernatants were collected and immediately used for experiments.
siRNA-mediated Gene Silencing-Small interfering RNA (siRNA) duplexes were synthesized, desalted, and purified by Samchully Pharm. Co. Ltd. (Siheung, GyeongGi, Korea) as follows: LPA 1 , 5Ј-GGACUUGGAAUCACUGUUUUU-3Ј (sense) and 5Ј-AAACAGUGAU-UCCAAGUCCUU-3Ј (antisense). ␤ig-h3 siRNA (J-019370 -06-0005) and nonspecific control siRNA (D-001206 -13-05) were purchased from Dharmacon, Inc. (Chicago, IL). For siRNA experiments, hASCs were seeded on 60-mm dishes at 70% confluence, and they were then transfected with siRNAs by using the Lipofectamine plus TM reagent according to the manufacturer's instructions. Briefly, Lipofectamine plus TM reagent was incubated with serum-free medium for 15 min, and respective siRNAs were then added to the mixtures. After incubation for 15 min at room temperature, the mixtures were diluted with serum-free medium and added to each well. The final concentration of siRNAs in each well was 100 nM. After incubation of hASCs in serum-free medium containing siRNAs for 4 h, the cells were cultured in growth medium for 24 h, and the expression levels of LPA 1 and GAPDH were then determined by reverse transcription-polymerase chain reaction analysis.
Statistical Analysis-The results of multiple observations are presented as mean Ϯ S.D. Student's t test were used to analyze differences between two groups. For multivariate data analysis, group differences were assessed with two-way analysis of variance (ANOVA), followed by post hoc comparisons tested with Scheffe's method.

LPA-conditioned Medium from hASCs Stimulate Adhesion and Proliferation of A549 Human Lung Adenocarcinoma
Cells-To assess whether LPA-activated hASCs can regulate the tumorigenic potential of tumor cells, in the present study, we explored the effects of LPA-conditioned medium (LPA CM) from hASCs on the adhesion and proliferation of A549 human lung adenocarcinoma cells in vitro. To measure the effects of LPA CM on the adhesion of A549 cells, adhesive capacities of A549 cells on LPA CM-or control CM-coated dishes were measured. As shown in Fig. 1A, adhesion of A549 cells onto LPA CM-coated dishes was increased in a dosedependent manner with LPA CM. The adhesive activity of A549 cells on LPA CM-coated dishes was greater than on control CM-coated dishes. To explore the effect of LPA CM on the proliferation of A549 cells, the cells were exposed to different concentrations of LPA CM for 3 days, and the numbers of A549 cells were determined. LPA CM stimulated proliferation of A549 cells in a dose-dependent manner, with a maximal stimulation at 20% concentration (Fig. 1B). To exclude the possibility that exogenous LPA may be responsible for LPA CM-induced proliferation of A549 cells, we compared the effects of LPA CM, control CM, and LPA on cell proliferation. As shown in Fig. 1C, LPA itself had no significant impact on cell proliferation of A549 cells, in contrast to the potent stimulation of cell proliferation by LPA CM, suggesting that LPA stimulates secretion of mitogenic factors from hASCs. To explore whether protein factors are involved in the LPA CMinduced adhesion and proliferation of A549 cells, LPA CM was heated at 95°C for 5 min to denature protein factors. As shown in Fig. 1D, A549 cells did not adhere onto the culture dishes coated with heat-denatured LPA CM, in contrast to strong adherence of A549 cells onto LPA CM-coated dishes. Furthermore, LPA CM-induced proliferation of A549 cells was abrogated by heat denaturation of LPA CM (Fig. 1E). These results imply a key role of protein factors in LPA CM-induced adhesion and proliferation.
Proteomic Identification of ␤ig-h3 as an LPA-induced Secreted Protein in LPA CM-To identify the protein factors responsible for LPA CM-stimulated adhesion and proliferation, serum-starved hASCs were incubated with serum-free medium in the absence or presence of 10 M LPA for 2 days, and the conditioned media were subjected to LC-MS/MS analysis for protein identification. After single spectrummatched proteins were excluded, 146 proteins were identified from comparative proteomic analysis of hASC conditioned medium (supplemental Table S1). The identified proteins were analyzed for the possibility of secretion using SignalP and SecretomeP. In total, 130 (89%) proteins were considered as "putative secretory proteins"; 116 proteins were considered to be secreted through a classical pathway (endoplasmic reticulum/Golgi apparatus-dependent pathway), because a signal peptide was predicted by SignalP, whereas 14 proteins were secreted through a nonclassical pathway, predicted by SecretomeP.
For the comparative analysis of secretomes in response to LPA treatment, a label-free quantitative approach was adopted, and the relative abundance was based on the sum of total fragment ion intensities of peptides matched in control CM and LPA CM. The identities and relative abundance of the secretome are summarized in supplemental Table S1, and the subcellular localization and molecular function of the whole secretome are summarized in the supplemental Tables S2. Sequences of all peptides assigned are listed in the supplemental Table S3. We classified 16 features as LPAinduced proteins, of which Log2 ratio values are higher than 1. An abbreviated list of the LPA-induced proteins is presented in Table I. These proteins included extracellular matrix proteins (␤ig-h3 and periostin), proteases and protease inhibitors (matrix metalloproteinase-14, interstitial collagenase, plas-minogen activator inhibitor 1, and glia-derived nexin), and cytokine signaling (cytokine insulin-like growth factor-binding protein 3 and interleukin-8). Because periostin is secreted from hASCs in response to LPA treatment (24), these results suggest that ␤ig-h3 and periostin may be responsible for LPA CM-stimulated adhesion and migration of A549 cells. The  representative mass spectra of ␤ig-h3 and periostin are shown in supplemental Fig. S3. LPA Induces Expression of ␤ig-h3 in hASCs through LPA 1dependent Mechanism-To confirm the proteomic data that ␤ig-h3 is secreted from hASCs in response to LPA treatment, we determined the expression levels of ␤ig-h3 in hASCs by Western blotting. Because LPA treatment induced differentiation of hASCs to ␣-SMA-positive cells (35), we explored the effects of LPA on the expression levels of ␤ig-h3 and ␣-SMA. LPA treatment dose-dependently increased the expression levels of not only ␣-SMA but also ␤ig-h3 in hASCs ( Fig. 2A). Moreover, LPA treatment time-dependently increased the expression levels of ␤ig-h3 and ␣-SMA in hASCs (Fig. 2B). The expression levels of ␤ig-h3 in cell lysates were up-regulated significantly at 12 h after LPA treatment, in contrast to increased expression of ␣-SMA at 96 h after LPA treatment, suggesting that LPA-induced ␤ig-h3 expression occurred before ␣-SMA expression. Consistent with the LPA-induced expression of ␤ig-h3 in hASCs, the amounts of ␤ig-h3 protein in conditioned medium from hASCs were increased after a 48-h treatment with LPA (Fig. 2C).
We previously reported that LPA induced ␣-SMA expression through an LPA receptor 1 (LPA 1 )-dependent mechanism. To assess the involvement of LPA 1 in the LPA-induced expression of ␤ig-h3, we examined the effect of the LPA 1selective antagonist Ki16425 on the expression levels of ␤ig-h3 and ␣-SMA in hASCs. As shown in Fig. 2D, pretreatment of hASCs with Ki16425 completely abrogated the expression of ␤ig-h3 and ␣-SMA induced by LPA. Furthermore, depletion of endogenous LPA 1 expression using siRNA blocked the LPA-stimulated expression of ␤ig-h3 and ␣-SMA (Fig. 2E). These results indicate that LPA 1 plays a key role in the LPA-induced expression of ␤ig-h3 and ␣-SMA.
LPA-induced ␤ig-h3 Expression is Mediated by a Rho Kinase-dependent Pathway-We have previously reported that the LPA-induced secretion of TGF-␤1 from hASCs and the autocrine TGF-␤1-dependent pathway mediated LPA-induced ␣-SMA expression (35). Because ␤ig-h3 was originally identified as a TGF-␤1-induced protein (25), we examined the effects of TGF-␤1 on ␤ig-h3 expression in hASCs. As shown in Fig. 3A, TGF-␤1 dose-dependently increased expression levels of ␣-SMA and ␤ig-h3 in hASCs. To ascertain whether a TGF-␤1-dependent autocrine pathway is involved in the LPAinduced ␤ig-h3 expression, we examined the effects of SB431542, a TGF-␤ receptor I inhibitor, on the expression levels of ␤ig-h3 and ␣-SMA. As shown in Fig. 3B, the TGF-␤1-induced expression of ␤ig-h3 and ␣-SMA was completely abrogated by pretreatment of hASCs with SB431542. However, LPA-induced ␤ig-h3 expression was not affected by pretreatment of the cells with SB431542, in contrast to significant attenuation of LPA-induced ␣-SMA expression by SB431542 (Fig. 3C). These results suggest that the TGF-␤1dependent pathway is not involved in LPA-induced ␤ig-h3 expression.
RhoA plays a key role in LPA-induced cellular responses (40). To ascertain whether a Rho kinase is involved in the LPA-stimulated expression of ␤ig-h3, we examined the effect of the Rho kinase inhibitor Y27632 on the expression levels of ␤ig-h3 and ␣-SMA. As shown in Fig. 3D, pretreatment of hASCs with Y27632 completely abrogated the LPA-induced expression of ␤ig-h3 and ␣-SMA, implying a pivotal role of the Rho kinase in LPA-induced expression of ␤ig-h3 and ␣-SMA.

␤ig-h3 is Responsible for LPA CM-stimulated Adhesion and
Proliferation of A549 Cells-To explore the role of ␤ig-h3 on LPA CM-stimulated adhesion and proliferation of A549 cells, ␤ig-h3 was immunodepleted from LPA CM with anti-␤ig-h3 antibody (Fig. 4A). Adhesion of A549 cells onto culture dishes, which were coated with ␤ig-h3-depleted LPA CM, was markedly attenuated compared with that onto LPA CM-coated dishes (Fig. 4B). Furthermore, LPA CM-stimulated proliferation of A549 cells was attenuated by immunodepletion of ␤ig-h3 from LPA CM (Fig. 4C). These results suggest that ␤ig-h3 is responsible for the LPA CM-stimulated adhesion and proliferation of A549 cells.
To support these findings, ␤ig-h3 expression was depleted by transfection of hASCs with ␤ig-h3-specific siRNA. The LPA-induced secretion of ␤ig-h3 was blocked by transfecting the cells with ␤ig-h3-specific siRNA (Fig. 5A). Adhesion of A549 cells onto LPA CM-coated culture dishes was abrogated by siRNA-mediated knockdown of ␤ig-h3 in LPA CM (Fig. 5B). Furthermore, LPA CM-stimulated proliferation of A549 cells was inhibited by the siRNA-mediated knockdown of endogenous ␤ig-h3 in LPA CM. These results suggest a key role of ␤ig-h3 in LPA CM-induced adhesion and proliferation of A549 cells.
Overexpression of ␤ig-h3 in hASCs Potentiates the Paracrine Functions of hASCs on the Adhesion and Proliferation of A549 Cells-To further confirm the result that ␤ig-h3 secreted from hASCs stimulated the adhesion and proliferation of A549 cells, we overexpressed ␤ig-h3 in hASCs by using a retroviral system. As shown in Fig. 6A, the protein levels of ␤ig-h3 were increased in the cell lysates and conditioned medium from the hASCs infected with the ␤ig-h3 retrovirus. Conditioned medium from the ␤ig-h3-overexpressing hASCs can also stimulate the adhesion and proliferation of A549 cells (Figs. 6B and 6C). These results suggest that ␤ig-h3 secreted from hASCs accelerated the adhesion and proliferation of A549 cells. In order to clarify whether ␤ig-h3 acts as an adhesion molecule, we determined the adhesive capability of A549 cells onto recombinant ␤ig-h3-coated culture dishes. ␤ig-h3 dose-dependently stimulated the adhesion and proliferation of A549 cells, and treatment of A549 cells with purified ␤ig-h3 stimulated the proliferation of A549 cells (supplemental Fig. S4). Moreover, both ␤ig-h3 and LPA CM stimulated the adhesion and proliferation of not only A549 cells but also other cell types, including HeLa human cervical carcinoma cells and WI-38 human lung fibroblasts (supplemental Fig. S5). These results suggest that ␤ig-h3 acts as an extracellular adhesion molecule and stimulates the proliferation of cancer cells.  (41). In the present study, we identified ␤ig-h3 as an LPA-induced secreted protein in hASCs. LPA stimulated the expression of not only ␣-SMA but also ␤ig-h3 in hASCs by activating the LPA 1 receptor. ␤ig-h3 was originally identified as a TGF-␤-induced protein (25). High glucose induced the expression of ␤ig-h3 through the autocrine TGF-␤1-dependent signaling loop in human renal proximal tubule cells and vascular smooth muscle cells (42,43). LPA-induced ␣-SMA expression was mediated by an autocrine TGF-␤1-dependent signaling pathway (35), whereas the TGF-␤1-dependent pathway was not associated with the LPA-stimulated ␤ig-h3 expression. We demonstrated that the Rho kinase-dependent pathway plays a pivotal role in the LPA-induced expression of ␣-SMA and ␤ig-h3. These results suggest that LPA is a unique G protein-coupled receptor agonist stimulating expression of ␤ig-h3 through a TGF-␤1-independent but Rho kinase-dependent mechanism.
To our knowledge, this is the first study showing that ␤ig-h3 secreted from hASCs stimulated the adhesion and proliferation of A549 cells. Immunodepletion or siRNA-mediated silencing of endogenous ␤ig-h3 abrogated LPA CM-induced adhesion and proliferation of A549 cells, whereas conditioned medium from the ␤ig-h3-overexpressing hASCs stimulated the adhesion and proliferation of A549 cells. Accumulating evidence suggests a pivotal role of ␤ig-h3 as an adhesion molecule for various cell types, including dermal fibroblasts, corneal epithelial cells, keratinocytes, astrocytoma cells, and ovarian cancer cells (23, 26, 28, 44 -46). ␤ig-h3 secreted from peritoneal cells increased the adhesion of ovarian cancer cells to peritoneal cells and promoted the motility and invasion of ovarian cancer cells (47). Furthermore, ␤ig-h3 stimulated the proliferation of keratinocytes, smooth muscle cells, and renal FIG. 5. Effects of siRNA-mediated silencing of ␤ig-h3 expression on LPA CM-stimulated adhesion and proliferation of A549 cells. A, ASCs were transfected with si-control or si-␤ig-h3 and then treated with LPA or vehicles for 2 days. The protein levels of ␤ig-h3 in conditioned medium from hASCs were determined by Western blotting. B, 96-well culture dishes were coated with the conditioned medium (each 20%) from hASCs and adhesion of A549 cells onto the wells was determined. C, A549 cells were treated with the conditioned medium (each 20%) from hASCs for 3 days, and the number of cells was quantified. Data represent mean Ϯ S.D. (n ϭ 4). *, p Ͻ 0.01 by two-way ANOVA and Scheffe's post hoc test.
FIG. 6. Effects of conditioned medium from ␤ig-h3-overexpressed hASCs on the adhesion and proliferation of A549 cells. A, hASCs were infected with a retrovirus bearing ␤ig-h3 gene or control retrovirus and the protein levels of ␤ig-h3 and GAPDH in cell lysates and conditioned medium were determined by Western blotting. B, 96-well culture dishes were coated with conditioned medium (each 20%) from the retrovirus-infected hASCs, and adhesion of A549 cells onto the wells was determined. C, A549 cells were exposed to conditioned medium (each 20%) from the retrovirus-infected hASCs for 3 days and the number of A549 cells was quantified. Data represent mean Ϯ S.D. (n ϭ 4). *, p Ͻ 0.01 by two-way ANOVA and Scheffe's post hoc test.
proximal epithelial cells (26,42,48). ␤ig-h3 expression is elevated in high-grade human colon cancers, and ectopic expression of ␤ig-h3 enhances the aggressiveness and metastatic potential of colon cancer cells in vivo (30). In addition, increased expression of ␤ig-h3 has been reported in various tumor tissues, including lung cancer (23). However, there is conflicting data in the literature reporting the role of ␤ig-h3 in tumor progression. Transfection of ␤ig-h3 into lung cancer cells reduced tumor growth in a xenograft nude mouse model (49). Moreover, the loss of ␤ig-h3 predisposes mice to spontaneous tumor development (50), suggesting a role of ␤ig-h3 as a tumor suppressor. Contrary to the findings of the present study, conditioned medium from Chinese hamster ovary cells transfected with ␤ig-h3 cDNA inhibited the attachment of A549, HeLa, and WI-38 cells to plastic culture plates in vitro (51). The discrepancies in the effects of ␤ig-h3 on cell adhesion are likely because of different experimental conditions. Precoating the culture dishes with ␤ig-h3 stimulated the adhesion of various cell types onto culture dishes (23, 26, 28, 44 -46). Similarly, the adhesion of A549, WI-38, and HeLa cells onto culture plates was augmented by pre-coating the culture plates with recombinant ␤ig-h3 protein or LPA CM. However, coincubation of cancer cells with ␤ig-h3 protein or LPA CM in the culture medium during the adhesion of the cells onto culture plates inhibited cell adhesion (supplemental Fig. S5). These results suggest that pre-coated ␤ig-h3 stimulates cell adhesion as an extracellular matrix protein, whereas soluble ␤ig-h3 may interfere with the attachment of cells onto culture dishes. Taken together, these results support the notion that ␤ig-h3 secreted from hASCs acts as an adhesion molecule and stimulates the proliferation of cancer cells in vitro.
In addition to ␤ig-h3, using a shotgun proteomic analysis, we have identified a variety of extracellular proteins as LPAinduced secreted proteins, which include periostin, interleukin-8, insulin-like growth factor-binding protein 3/6, and proteases and protease inhibitors (matrix metalloproteinase-14, interstitial collagenase, plasminogen activator inhibitor 1, and glia-derived nexin/Serine protease inhibitor-E2). We have recently reported that LPA stimulated secretion of periostin, an extracellular protein structurally similar to ␤ig-h3, from hASCs through the LPA 1 -dependent mechanism and recombinant periostin protein augmented the adhesion and migration of epithelial ovarian cancer cells (24), suggesting that both ␤ig-h3 and periostin are involved in the regulation of adhesion, migration, and proliferation of tumor cells. In addition, LPA has been shown to stimulate IL-8 expression in ovarian cancer cells (52) and IL-8 mediated LPA-stimulated invasion of ovarian cancer cells (53). Insulin-like growth factor-binding proteins 3 and 6 bind to insulin-like growth factor, which is implicated in tumorigenesis. Taken together, these results suggest that LPA-induced extracellular protein factors, including extracellular matrix, proteases, and cytokines, play a key role in tumor growth by regulating tumor microenvironment.
CAFs play a key role in tumorigenesis and metastasis of various solid tumors by modulating tumor microenvironment (2)(3)(4)(5). Reports have suggested that bone marrow-derived MSCs contributed to 25% of the total myofibroblast population in the tumor stroma in a mouse pancreatic insulinoma model (54) and in a subcutaneous pancreatic xenograft tumor (55). Furthermore, lung cancer stromal fibroblasts have been reported to be derived from blood-borne progenitor cells of patients (56). Incubation of human bone marrow-derived MSCs to conditioned medium from human colorectal cancer cells increased the expression of ␣-SMA (57). Furthermore, tumor-conditioned medium induced differentiation of human bone marrow-derived MSCs into CAFs, which express ␣-SMA and SDF-1 (19). We have previously reported that cancerconditioned medium induced differentiation of hASCs to CAFs, which express ␣-SMA, SDF-1, and VEGF in vitro (35,58). Furthermore, co-transplanted hASCs were differentiated into ␣-SMA-positive CAFs and stimulated tumor angiogenesis through a VEGF-dependent mechanism in an A549 xenograft tumor model (41). Together with the findings that LPA-activated hASCs stimulated the adhesion and proliferation of A549 cells through a ␤ig-h3-dependent mechanism, these results suggest that hASCs promote tumor growth and angiogenesis within tumor microenvironment through secreting paracrine factors, including proteases, extracellular matrix proteins, and angiogenic cytokines.