15-Deoxy-Δ<sup>12,14</sup>-Prostaglandin J<sub>2</sub> Inhibits Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury via Redox Pathway

Liu, Xin; Jia, Shuangshuang; Li, Weiyang; Yang, Le; Yang, Lin; Wang, Lin; Li, Liying

doi:https://doi.org/10.1155/2015/876160

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Research Article | Open Access

Volume 2015 | Article ID 876160 | https://doi.org/10.1155/2015/876160

15-Deoxy-Δ^12,14-Prostaglandin J₂ Inhibits Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury via Redox Pathway

Xin Liu,¹Shuangshuang Jia,¹Weiyang Li,¹Le Yang,¹Lin Yang,¹Lin Wang,¹and Liying Li¹

Academic Editor: Marcelo H. Napimoga

Received03 Jul 2015

Accepted27 Aug 2015

Published20 Sept 2015

Abstract

It has been reported that bone marrow-derived mesenchymal stem cells (BMSCs) have capacity to migrate to the damaged liver and contribute to fibrogenesis in chronic liver diseases. 15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), an endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), is considered a new inhibitor of cell migration. However, the actions of 15d-PGJ₂ on BMSC migration remain unknown. In this study, we investigated the effects of 15d-PGJ₂ on the migration of BMSCs using a mouse model of chronic liver fibrosis and primary mouse BMSCs. Our results demonstrated that in vivo, 15d-PGJ₂ administration inhibited the homing of BMSCs to injured liver by flow cytometric analysis and, in vitro, 15d-PGJ₂ suppressed primary BMSC migration in a dose-dependent manner determined by Boyden chamber assay. Furthermore, the repressive effect of 15d-PGJ₂ was blocked by reactive oxygen species (ROS) inhibitor, but not PPARγ antagonist, and action of 15d-PGJ₂ was not reproduced by PPARγ synthetic ligands. In addition, 15d-PGJ₂ triggered a significant ROS production and cytoskeletal remodeling in BMSCs. In conclusion, our results suggest that 15d-PGJ₂ plays a crucial role in homing of BMSCs to the injured liver dependent on ROS production, independently of PPARγ, which may represent a new strategy in the treatment of liver fibrosis.

1. Introduction

Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent nonhaematopoietic cells with the ability to differentiate toward a variety of cell types [1, 2]. They have received a great deal of attention as the therapeutic potential for wound healing process [3, 4]. However, it is noticeable that BMSCs have potential to differentiate toward myofibroblasts to exaggerate organ damage. It has been reported that after liver injury, BMSCs could migrate to the damaged liver and become the major origin of hepatic myofibroblasts to promote liver fibrosis by generating extracellular matrix components [5]. Although activated hepatic stellate cells (HSC), residential fibroblasts, circulating fibrocytes, and epithelial-mesenchymal transition (EMT) are proportions of hepatic myofibroblasts, BMSC-derived hepatic myofibroblasts are the overwhelming majority [6]. Considering the importance of BMSCs in liver fibrosis, identification of the molecular mechanism underlying BMSC migration may represent an effective strategy for the treatment of fibrotic liver disease.

BMSC migration can be regulated by a variety of molecules, such as platelet derived growth factor (PDGF), stromal-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) [7–10]. In addition, fibroblast activation protein (FAP), Sry-related high-mobility group box 11 (Sox11), and activin B and vitamin C transporter were also involved in the migration of BMSCs [11–14]. Furthermore, our previous results indicated that sphingosine 1-phosphate (S1P) gradient between liver and bone marrow induced migration of BMSCs to the damaged liver [15]. Although much work has already been done to elucidate the mechanistic basis underlying the migration of BMSCs, the potential effects of other endogenous molecules on BMSC migration is still undefined.

15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), an endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), has been considered a pleiotropic regulator in cell apoptosis, proliferation, and inflammation [16]. In addition, several reports have demonstrated that 15d-PGJ₂ could inhibit cell migration in vivo and in vitro. In mouse model of chronic eosinophilia, 15d-PGJ₂ suppressed eosinophil migration into the peritoneal cavity [17]. 15d-PGJ₂ also decreased neutrophil migration to the inflammatory site in experimental acute peritonitis [18]. In vitro, airway smooth muscle cell and mammary cancer cell migration are reduced by 15d-PGJ₂ treatment [19, 20]. More recently, the function of 15d-PGJ₂ in the liver fibrosis has garnered much interest, and our previous report has confirmed that 15d-PGJ₂ administration reduced bone marrow-derived monocyte/macrophage (BMM) migration to the damaged liver and ameliorated liver fibrosis in mouse models [21]. Even though BMSCs are another cell type derived from bone marrow and closely related to liver fibrogenesis, little is known about the effect and the underlying mechanism of 15d-PGJ₂ on the migration of BMSCs.

The cytoskeleton is a system of intracellular filaments which is crucial for cell shape, division, and other functions in the cell [22]. After tissue injury, a large component of cellular responses is related to the cytoskeleton [23]. For instance, it has been demonstrated that cytoskeletal remodeling plays an important role in cell migration in response to stimulation, including BMSCs [24, 25]. There are reports indicating that 15d-PGJ₂ could alter cytoskeletal structure of several cell types in which it reduces cell migration [18, 20]. Nevertheless, it is still not clear whether the potential function of 15d-PGJ₂ in the BMSC migration is linked to the cytoskeleton regulation.

The present study aims to investigate the effect of 15d-PGJ₂ on BMSC migration triggered by chronic liver injury. Here, we found that, in vivo, 15d-PGJ₂ administration reduced the homing of BMSCs to the injured liver and, in vitro, 15d-PGJ₂ inhibited primary mouse BMSC migration via production of ROS, independently of PPARγ. In addition, the effect of 15d-PGJ₂ in BMSCs was associated with cytoskeletal remodeling. These results suggest that 15d-PGJ₂ holds great promise in the treatment of liver fibrosis.

2. Materials and Methods

2.1. Materials

α-MEM was from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone/Thermo Scientific (Victoria, Australia). Anti-CD105 and anti-CD166 antibodies used for flow cytometry analysis were from eBioscience (San Diego, CA). Anti-PPARγ antibody used for immunofluorescence was from Santa Cruz Biotechnology (CA, USA). PCR reagents were from Applied Biosystems (Foster City, CA). 15d-PGJ₂ was from Cayman Chemical (Ann Arbor, MI). 2′,7′-Dichlorohydrofluorescein diacetate (DCFH-DA) was from Molecular Probes (Interchim, France). Troglitazone and ciglitazone were from Biomol (Tebu, France). GW9662, N-acetylcysteine (NAC), and other common reagents were from Sigma (St. Louis, MO).

2.2. BMSCs Preparation

Bone marrow (BM) cells were isolated from BM of ICR mice (closed colony mice) aged 3 weeks by flushing the tibias and femurs (Laboratory Animal Center, Capital Medical University) with a 25-gauge needle. Then, the cells were passed through 70 mm nylon mesh and washed with PBS containing 2% FBS for three times. BMSCs were cultured as described previously [5]. In brief, BM cells were cultured with α-MEM containing 20% FBS at 37°C in 5% CO₂ for 1 week. The culture medium was replaced twice a week to remove the nonadherent cells. After the first subculture, α-MEM containing 15% FBS was used to culture BMSCs. BMSCs were characterized by flow cytometry analysis, and passage 3 to passage 5 were used in the experiments. All animal work was performed under the ethical guidelines of the Ethics Committee of Capital Medical University.

2.3. Mouse Models

ICR mice aged 6 weeks received intraperitoneal injections of 1 μL/g body weight of CCl₄/olive oil (OO) mixture, 1 : 9 v/v, or OO singly, twice per week. 15d-PGJ₂ (0.3 mg/kg body weight) or saline firstly was administrated the day before CCl₄ or OO treatment and then twice per week before CCl₄ or OO treatment for 4 weeks ( per group).

Another group of ICR mice received lethal irradiation (8 Grays) and then immediately received transplantation by a tail-vein injection of 1.5 × 10⁷ whole BM cells obtained from 3-week-old enhanced green fluorescent protein (EGFP) transgenic mice. 4 weeks later, mice received intraperitoneal injections of CCl₄ or OO twice per week for 4 weeks. 15d-PGJ₂ (0.3 mg/kg body weight) or saline firstly was administered the day before CCl₄ or OO treatment and then twice per week before CCl₄ or OO treatment for 4 weeks ( per group).

2.4. Immunofluorescence and High Content Analysis

Cultured BMSCs with or without treatments were fixed in 4% paraformaldehyde in PBS for 30 minutes. Then cells were washed twice with PBS, permeabilized in 0.5% TritonX-100 in PBS for 15 minutes, blocked with 2% BSA for 1 hour, and then incubated with anti-PPARγ antibody (1 : 100), followed by incubation of secondary antibody conjugated with Cy3 (1 : 100; Jackson ImmunoResearch Laboratories, West Grove, PA). Filamentous actin (F-actin) was stained with FITC-conjugated phalloidin (1 : 80, Molecular Probes, Eugene, OR) for 20 minutes. The nuclei were stained with DAPI and 50 μL PBS was left in each well. The plates were imaged on a Thermo Scientific CellInsight personal cell imaging (PCI) platform (Cellomics, Inc., Thermo Fisher Scientific Inc., Waltham, MA), with a ×10 objective using the Thermo Scientific Cellomics iDEV software. Thirty-six fields were automatically acquired by the software, corresponding to at least 3,000 cells. The total Cy3 or FITC fluorescence intensity of each well was analyzed by Cellomics Cell Health Profiling BioApplication software.

2.5. Fluorescent Measurement of Intracellular Reactive Oxygen Species

BMSCs were plated in the wells of 96-well plates (Corning, NY) and allowed to attach overnight in α-MEM. Cells were then loaded for 15 minutes at 37°C with 5 μM DCFH-DA in α-MEM without FBS. After two washings with PBS, BMSCs were treated with 15d-PGJ₂ (1, 2, or 5 μM) or vehicle, and the fluorescence intensity of each well was determined after 5, 10, 15, 20, and 30 minutes by high content analysis.

2.6. Flow Cytometric Analysis

Nonparenchymal cells (NPCs) of mouse liver were isolated as described by Han et al. [21]. Cultured BMSCs were prepared to achieve single cell suspensions. The cells were resuspended at 1.5 × 10⁶ cells/100 μL in PBS and then incubated with PE-CD105 (1 : 40), PE-CD166 (1 : 80), or their isotype-matched negative control antibodies. After incubation in the dark for 15 minutes, the cells were washed with PBS and subjected to flow cytometric analysis. Flow cytometric analysis was performed on a FACS Aria and analyzed with FACS Diva4.1 (BD Biosciences).

2.7. Cell Migration Assay

BMSC migration was determined by Boyden chambers as described previously by Liu et al. [26]. Briefly, BMSCs were serum starved for 24 hours and then exposed to 15d-PGJ₂, troglitazone, ciglitazone, or vehicle for 12 hours. Then 4 × 10⁴ BMSCs were seeded to the upper chamber. Cell migration was allowed to proceed for 4 hours at 37°C in 5% CO₂. BMSCs migrating to the lower face of the porous membrane were fixed with cold methanol for 30 minutes and stained with hematoxylin for 1 hour. BMSCs on the upper membrane surface were removed with cotton swabs. Migrated BMSCs were photographed in at least six random fields per filter and quantified by cell counting.

2.8. Real-Time RT-PCR

Total RNA was extracted from frozen liver specimens or cultured BMSCs with or without treatments, using an RNeasy kit (Qiagen, Hilden, Germany). Real-time RT-PCR was performed with an ABI Prism 7300 sequence-detecting system (Life Technologies, Foster City, CA), as described previously [15]. Primers (MWG Biotech, Ebersberg, Germany) used for real-time RT-PCR were as follows: 18 s rRNA, sense, 5′-GTA ACC CGT TGA ACC CCA TT-3′, and antisense, 5′-CCA TCC AAT CGG TAG TAG CG-3′; PPARγ: sense, 5′-GCC CAC CAA CTT CGG AAT C-3′, and antisense, 5′-TGC GAG TGG TCT TCC ATC AC-3′.

2.9. Statistical Analysis

All results were confirmed at least by three independent experiments. The results are expressed as mean ± SEM. Statistical significance was determined by Student’s -test or ANOVA. Statistical significance was defined as .

3. Results

3.1. 15d-PGJ₂ Inhibits Homing of BMSCs to the Injured Liver

We previously have confirmed that 15d-PGJ₂ could inhibit homing of BMM to the damaged liver tissue in mouse model of chronic liver injury [21]. Although BMSCs are also known to migrate to the injured liver in this process, whether it could be regulated by 15d-PGJ₂ has not been elucidated. To investigate the effect of 15d-PGJ₂, we first used CCl₄ injection to induce mouse liver fibrosis. Four weeks later, NPCs in liver tissues were analyzed by flow cytometric analysis, and total MSCs were characterized as positive for markers CD166⁺ or CD105⁺. The results showed that 15d-PGJ₂ administration significantly decreased the proportion of total MSCs (CD166⁺ or CD105⁺ cells) in liver NPCs compared with that in the liver without 15d-PGJ₂ treatment (Figures 1(a) and 1(b)).

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(b)

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Figure 1

15d-PGJ₂ inhibits the migration of BMSCs toward injured liver. ((a) and (b)) 4 weeks of CCl₄ were used to induce mouse liver fibrosis with or without 15d-PGJ₂ administration ( per group). Total MSCs were isolated from the NPCs in the liver by flow cytometry analysis based on CD166 and CD105. Representative histograms and proportions of CD166⁺ (a) and CD105⁺ (b) in NPCs. ((c)–(f)) Mice were lethally irradiated and received whole BM transplants from EGFP transgenic mice, followed by CCl₄ injection for 4 weeks in the presence or absence of 15d-PGJ₂ treatment. BMSCs in the liver NPCs were identified as CD166⁺/EGFP⁺ or CD105⁺/EGFP⁺. ((c) and (e)) Representative histograms of BMSCs. ((d) and (f)) The proportions of CD166⁺/EGFP⁺ BMSCs and CD166⁺/EGFP⁻ resident MSCs (d) and CD105⁺/EGFP⁺ BMSCs and CD105⁺/EGFP⁻ resident MSCs in NPCs of liver tissues. , compared with olive oil (OO) group. , compared with CCl₄ group without 15d-PGJ₂ ().

MSCs are multipotential nonhematopoietic progenitor cells that can be obtained from several tissues, including the bone marrow (BMSCs) and the liver tissue (L-MSCs). We next want to examine whether these decreased MSCs by 15d-PGJ₂ are bone marrow derived or resident MSCs. For this purpose, we reconstituted BM in the irradiated mice by transplantation of the genetic EGFP-labeled BM cells. Liver fibrosis was also induced by CCl₄ administration for 4 weeks with or without 15d-PGJ₂ treatment. BMSCs in the liver were isolated and counted as double positive for CD166/EGFP and CD105/EGFP, respectively. The results indicated that, in liver NPCs, there was no significant difference in the proportions of resident MSCs (CD166⁺/EGFP⁻ or CD105⁺/EGFP⁻) in the 15d-PGJ₂-treated mice compared with 15d-PGJ₂ nontreatment group (Figures 1(c)–1(f)). However, the proportions of CD166⁺/EGFP⁺ and CD105⁺/EGFP⁺ BMSCs in the damaged liver were markedly decreased by 15d-PGJ₂ administration (Figures 1(c)–1(f)). These results suggested that 15d-PGJ₂ inhibited migration of BMSCs to the damaged liver tissue but had no influence on liver resident MSCs in the model of CCl₄-induced liver injury.

3.2. 15d-PGJ₂ Inhibits Migration of BMSCs In Vitro

To further investigate the effect of 15d-PGJ₂ on BMSC migration in vitro, we first performed flow cytometric analysis to identify the purity of BMSCs isolated from mouse BM. The results showed that these cells were predominantly positive for CD166 and CD105 (Figures 2(a) and 2(b)). Herein, these primary BMSCs were performed in the subsequent studies. Transwell migration assay indicated that 15d-PGJ₂ (1–5 μM) caused a powerful dose-dependent decrease in the migration of BMSCs (Figures 2(d) and 2(e)). In that studies have proved PDGF possess promigration property in liver diseases [27–29], we utilized PDGF to investigate BMSC migration in response to pathological stimuli. The results indicated that PDGF could augment migration of BMSCs, which was also inhibited by 15d-PGJ₂ in a concentration-dependent manner (Figures 2(d) and 2(f)). Given that 15d-PGJ₂ possesses proapoptotic and growth inhibitory potential, we determined the effect of 15d-PGJ₂ on BMSC proliferation using the Cell Counting Kit-8. As shown in Figure 2(c), 15d-PGJ₂ did not alter the number of living BMSCs at concentrations ranging from 1 to 5 μM. These observations suggested that 15d-PGJ₂ suppressed BMSC migration not only under normal condition but also in the context of pathological condition.

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Figure 2

Effects of 15d-PGJ₂ on BMSCs migration and proliferation. ((a) and (b)) Flow cytometry analysis was performed to identify BMSCs based on the specific marker of CD166 (a) and CD105 (b). (c) BMSCs were exposed to varying concentrations of 15d-PGJ₂ for 24 hours. Cell Counting Kit-8 was used to assess cell proliferation. ((d)–(f)) Serum-starved BMSCs were preincubated with indicated doses of 15d-PGJ₂ for 12 hours and then allowed to migrate for 4 hours with or without PDGF (30 ng/mL) administration. Representative images of migration of BMSCs (d). Quantitative analysis of BMSC migration in the presence (f) or absence (e) of PDGF. , compared with control (). Scale bars, 50 μm.

3.3. 15d-PGJ₂ Inhibits BMSCs Migration via ROS-Dependent Pathway, Independently of PPARγ

It is well revealed that 15d-PGJ₂ can act through PPARγ pathway [30–32]. Next, we evaluate whether the suppressive effects of 15d-PGJ₂ on BMSC migration were mediated by PPARγ. The results showed that application of synthetic ligands of PPARγ (troglitazone or ciglitazone) had no effects on BMSC migration (Figure 3(a)). In addition, pretreatment with GW9662 (an irreversible PPARγ antagonist) did not influence the inhibitory effect of 15d-PGJ₂ (Figure 3(a)). Furthermore, 15d-PGJ₂ had no influence on PPARγ mRNA in BMSCs (Figure 3(b)). Immunofluorescence was also performed to study the effect of 15d-PGJ₂ on the protein expression of PPARγ. The results indicated that there was comparable extent of immunoreactivities for PPARγ in the vehicle- or 15d-PGJ₂-treated BMSCs (Figure 3(d)). High content analysis showed that the fluorescence intensities of PPARγ did not achieve statistical significance among the two groups (Figure 3(c)).

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Figure 3

15d-PGJ₂ inhibits BMSCs migration independently of PPARγ. (a) Serum-starved BMSCs were pretreated with or without 10 μM GW9662 for 1 hour and then exposed to 5 μM 15d-PGJ₂ or 10 μM troglitazone (Trog) or ciglitazone (Cig) for 12 hours and were subsequently allowed to migrate for 4 hours. ((b)–(d)) Serum-starved BMSCs were incubated with 15d-PGJ₂ (5 μM) for 12 hours. Real-time RT-PCR was performed to determine mRNA expression of PPARγ in BMSCs (b). PPARγ protein expression was detected by immunofluorescence analysis (green) (d), and total fluorescence intensity of PPARγ was counted with high content analysis (c). , compared with control (). Scale bars, 50 μm.

Recent studies indicate that besides activation of PPARγ ROS production is another mechanism by which 15d-PGJ₂ elicits its effects [21, 33, 34]. Then we used antioxidant NAC to assess the role of ROS in the inhibitory action caused by 15d-PGJ₂. We found that preincubation with NAC eliminated the suppressive effect of 15d-PGJ₂ on BMSC migration (Figure 4(a)). The production of intracellular ROS in response to 15d-PGJ₂ was further confirmed by employing the peroxide-sensitive probe DCFH-DA prior to the addition of 15d-PGJ₂. As shown in Figure 4(c), application of 15d-PGJ₂-induced a significant rapid and transient increase in ROS production, which was in a dose-dependent fashion counted with high content analysis (Figure 4(b)). These results emphasized the importance of ROS in the inhibitory function of 15d-PGJ₂ on BMSC migration, but not PPARγ.

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Figure 4

The suppressive effect of 15d-PGJ₂ on BMSCs migration occurs via ROS production. (a) Serum-starved BMSCs were incubated with 5 μM 15d-PGJ₂ with or without 2.5 mM NAC (inhibitor of ROS) for 1 hour before treatment and then determined the effect of 15d-PGJ₂ on BMSCs migration. ((b) and (c)) BMSCs were loaded with DCFH-DA and further treated with 15d-PGJ₂ at different dose levels and durations. Representative images of fluorescence for ROS (green) in BMSCs (c). Fluorescence was measured using high content analysis (b). , compared with control. , compared with 15d-PGJ₂ group without NAC (). Scale bars, 50 μm.

3.4. 15d-PGJ₂ Inhibiting Migration of BMSCs Requires F-Actin Remodeling

In response to external stimulation, intracellular signals induce a dynamic remodeling of actin cytoskeleton, which results in changing cell shape and affecting cell motility [35]. It has been demonstrated that the number of actin fibers constitutes a principal profile related to migratory capacity [36, 37]. In addition, actin alignment in cell that represents order extent of fibers alters under different conditions. Herein, we further investigated the change of F-actin with FITC-conjugated phalloidin in BMSCs in the presence or absence of 15d-PGJ₂. As shown in Figure 5(a), under normal condition, BMSCs showed a migratory phenotype with abundant actin fibers and focal adhesion-like structures could be observed on the edge of cell membrane. Application of 15d-PGJ₂-induced a static phenotype with fewer actin fibers (Figure 5(b)). In particular, focal adhesion-like structures were disassembled in 15d-PGJ₂-treated BMSCs (Figure 5(b)). Furthermore, high content analysis was used to determine the amount and distribution of actin fibers in BMSC. The results showed that 15d-PGJ₂ administration time-dependently decreased number of fibers (Figure 5(c)) and fiber alignment (Figure 5(d)) in BMSCs. These data indicated that 15d-PGJ₂ could induce F-actin remodeling which were possibly associated with inhibitory action of 15d-PGJ₂ on BMSC migration.

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4. Discussion

Earlier reports have documented that 15d-PGJ₂ exhibits inhibitory effect on migration of several cells; however, it is still under investigation whether 15d-PGJ₂ plays an important role in migration of BMSCs, the main origin of hepatic myofibroblasts. In the current study, we investigated the effects of 15d-PGJ₂ on mouse BMSC migration in vivo and in vitro and the underlying mechanisms. We found that 15d-PGJ₂ could reduce homing of BMSC triggered by chronic liver injury. In addition, 15d-PGJ₂ inhibits migration of BMSCs in vitro, which is mediated by ROS production, independently of PPARγ. Meanwhile, the suppressive effect of 15d-PGJ₂ on BMSC migration was associated with cytoskeletal remodeling.

There are two main mechanisms by which 15d-PGJ₂ carries out its functions. Earlier report has shown that, as an endogenous ligand for PPARγ, 15d-PGJ₂ could activate PPARγ to regulate target genes expression [38]. In addition, 15d-PGJ₂ can change cellular redox status, such as production of ROS by forming covalent adducts with cysteine thiols via Michael addition due to its electrophilic α, β-unsaturated carbonyl group [39]. The effects of oxidative stress on cell migration remain controversial. Although considerable evidence points that ROS promotes migration of several cell types through direct or indirect interactions with migration-related molecules, including focal adhesion kinase (FAK), Rho GTPases, and mitogen activated protein kinase (MAPK) family of signaling pathways, some studies found oxidative stress also could exert suppressive function in cell migration [13, 40–45]. Here, ROS mediated the inhibitory effect of 15d-PGJ₂ on BMSC migration, eliminated by antioxidant NAC. 15d-PGJ₂ reduced another bone marrow-derived cell, BMM migration also via formation of ROS in our previous report [21]. Furthermore, in mammary cancer cells, a redox signaling pathway was involved in 15d-PGJ₂-induced focal adhesion disassembly and F-actin cytoskeletal changes in which 15d-PGJ₂ attenuated migration [20].

Evidence suggests that PPARγ also participates in cell migration. For instance, 15d-PGJ₂ suppresses eosinophil migration by activating PPARγ [17]. In addition, PPARγ is involved in 15d-PGJ₂-induced inhibition of migration in human airway smooth muscle cell and neutrophil [18, 19]. However, in the present study, PPARγ does not mediate the repressive function of 15d-PGJ₂ in BMSC migration. The effect of 15d-PGJ₂ was neither reproduced by PPARγ synthetic agonists nor blocked by PPARγ antagonist. In addition, 15d-PGJ₂ did not affect PPARγ content in the BMSCs. These results suggest that the underlying molecular mechanism of 15d-PGJ₂ on cell migration is complex and highly cell type specific.

High content analysis is a novel method, which could provide precise statistics according to cells selected for analysis [46]. In addition to detecting PPARγ protein and ROS production in BMSCs on the basis of labeled fluorescence, high content analysis also can measure F-actin remolding in BMSCs. It has been reported that cytoskeletal rearrangement of actin plays a central role in cell migration [24]. In the present study, 15d-PGJ₂ treatment caused a decrease in the number of fibers and fiber alignment. These findings suggest that 15d-PGJ₂-mediated actin remodeling is possibly involved in the inhibition of BMSC migration. Similar to our results, cytoskeletal organization is altered in 15d-PGJ₂ stimulated breast cancer cells (MCF-7) mediated through a mechanism unrelated to PPARγ transcriptional activation [47]. In addition, disruption of F-actin reorganization resulted in the reduction of migration in human MSCs [48]. Furthermore, inhibition of actin polymerization markedly suppressed migration of ovarian cancer cells [49]. In particular, we found 15d-PGJ₂ treatment disassembled focal adhesion-like structures in BMSCs. Although it is known that 15d-PGJ₂-induced focal adhesion disassembles via a redox pathway in mammary cancer cells [20], the underlying mechanisms for the regulatory effects of 15d-PGJ₂ on adhesion-like structures in BMSCs still require further investigation.

5. Conclusions

In summary, our results indicate that 15d-PGJ₂ inhibits homing of BMSCs toward injured liver, and 15d-PGJ₂ reduces BMSC migration through ROS production and cytoskeletal remodeling, independently of PPARγ. Therefore, we provide a novel regulatory mechanism of BMSC migration and suggest that 15d-PGJ₂ may be used as an antifibrotic agent during liver fibrosis.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Xin Liu and Shuangshuang Jia contributed equally to this work.

Acknowledgments

This work was supported by grants from the National Natural and Science Foundation of China (81430013 and 81300335) and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality (IDHT20150502).

References

A. I. Caplan, “Mesenchymal stem cells,” Journal of Orthopaedic Research, vol. 9, no. 5, pp. 641–650, 1991.
View at: Publisher Site | Google Scholar
M. F. Pittenger, A. M. Mackay, S. C. Beck et al., “Multilineage potential of adult human mesenchymal stem cells,” Science, vol. 284, no. 5411, pp. 143–147, 1999.
View at: Publisher Site | Google Scholar
K. Pelttari, E. Steck, and W. Richter, “The use of mesenchymal stem cells for chondrogenesis,” Injury, vol. 39, supplement 1, pp. S58–S65, 2008.
View at: Publisher Site | Google Scholar
M. Horie, H. Choi, R. H. Lee et al., “Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen,” Osteoarthritis and Cartilage, vol. 20, no. 10, pp. 1197–1207, 2012.
View at: Publisher Site | Google Scholar
L. Yang, N. Chang, X. Liu et al., “Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis,” The American Journal of Pathology, vol. 181, no. 1, pp. 85–97, 2012.
View at: Publisher Site | Google Scholar
F. P. Russo, M. R. Alison, B. W. Bigger et al., “The bone marrow functionally contributes to liver fibrosis,” Gastroenterology, vol. 130, no. 6, pp. 1807–1821, 2006.
View at: Publisher Site | Google Scholar
L. Yuan, N. Sakamoto, G. Song, and M. Sato, “Low-level shear stress induces human mesenchymal stem cell migration through the SDF-1/CXCR4 axis via MAPK signaling pathways,” Stem Cells and Development, vol. 22, no. 17, pp. 2384–2393, 2013.
View at: Publisher Site | Google Scholar
S. G. Ball, C. A. Shuttleworth, and C. M. Kielty, “Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors,” Journal of Cellular and Molecular Medicine, vol. 11, no. 5, pp. 1012–1030, 2007.
View at: Publisher Site | Google Scholar
C. Schichor, T. Birnbaum, N. Etminan et al., “Vascular endothelial growth factor A contributes to glioma-induced migration of human marrow stromal cells (hMSC),” Experimental Neurology, vol. 199, no. 2, pp. 301–310, 2006.
View at: Publisher Site | Google Scholar
A. Schmidt, D. Ladage, T. Schinköthe et al., “Basic fibroblast growth factor controls migration in human mesenchymal stem cells,” Stem Cells, vol. 24, no. 7, pp. 1750–1758, 2006.
View at: Publisher Site | Google Scholar
L. Xu, S. Huang, Y. Hou et al., “Sox11-modified mesenchymal stem cells (MSCs) accelerate bone fracture healing: Sox11 regulates differentiation and migration of MSCs,” The FASEB Journal, vol. 29, no. 4, pp. 1143–1152, 2015.
View at: Publisher Site | Google Scholar
K.-M. Chung, S.-C. Hsu, Y.-R. Chu et al., “Fibroblast activation protein (FAP) is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation,” PLoS ONE, vol. 9, no. 2, Article ID e88772, 2014.
View at: Publisher Site | Google Scholar
R. Sangani, C. D. Pandya, M. H. Bhattacharyya et al., “Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells,” Stem Cell Research, vol. 12, no. 2, pp. 354–363, 2014.
View at: Publisher Site | Google Scholar
M. Zhang, L. Sun, X. Wang et al., “Activin B promotes BMSC-mediated cutaneous wound healing by regulating cell migration via the JNK-ERK signaling pathway,” Cell Transplantation, vol. 23, no. 9, pp. 1061–1073, 2014.
View at: Publisher Site | Google Scholar
C. Li, Y. Kong, H. Wang et al., “Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis,” Journal of Hepatology, vol. 50, no. 6, pp. 1174–1183, 2009.
View at: Publisher Site | Google Scholar
D. S. Straus and C. K. Glass, “Cyclopentenone prostaglandins: new insights on biological activities and cellular targets,” Medicinal Research Reviews, vol. 21, no. 3, pp. 185–210, 2001.
View at: Publisher Site | Google Scholar
T. S. Farnesi-de-Assunção, C. F. Alves, V. Carregaro et al., “PPAR-γ agonists, mainly 15d-PGJ₂, reduce eosinophil recruitment following allergen challenge,” Cellular Immunology, vol. 273, no. 1, pp. 23–29, 2012.
View at: Publisher Site | Google Scholar
M. H. Napimoga, S. M. Vieira, D. Dal-Secco et al., “Peroxisome proliferator-activated receptor-γ ligand, 15-deoxy-Δ^12,14-prostaglandin J₂, reduces neutrophil migration via a nitric oxide pathway,” Journal of Immunology, vol. 180, no. 1, pp. 609–617, 2008.
View at: Publisher Site | Google Scholar
J. Stephen, C. Delvecchio, N. Spitale et al., “PPAR ligands decrease human airway smooth muscle cell migration and extracellular matrix synthesis,” European Respiratory Journal, vol. 41, no. 2, pp. 425–432, 2013.
View at: Publisher Site | Google Scholar
A. R. Diers, B. P. Dranka, K. C. Ricart et al., “Modulation of mammary cancer cell migration by 15-deoxy-Δ^12,14-prostaglandin J₂: implications for anti-metastatic therapy,” Biochemical Journal, vol. 430, no. 1, pp. 69–78, 2010.
View at: Publisher Site | Google Scholar
Z. Han, T. Zhu, X. Liu et al., “15-Deoxy-delta12,14-prostaglandin J2 reduces recruitment of bone marrow-derived monocyte/macrophages in chronic liver injury in mice,” Hepatology, vol. 56, no. 1, pp. 350–360, 2012.
View at: Publisher Site | Google Scholar
B. Wickstead and K. Gull, “The evolution of the cytoskeleton,” Journal of Cell Biology, vol. 194, no. 4, pp. 513–525, 2011.
View at: Publisher Site | Google Scholar
M. T. Abreu-Blanco, J. J. Watts, J. M. Verboon, and S. M. Parkhurst, “Cytoskeleton responses in wound repair,” Cellular and Molecular Life Sciences, vol. 69, no. 15, pp. 2469–2483, 2012.
View at: Publisher Site | Google Scholar
T. D. Pollard and G. G. Borisy, “Cellular motility driven by assembly and disassembly of actin filaments,” Cell, vol. 112, no. 4, pp. 453–465, 2003.
View at: Publisher Site | Google Scholar
Y. Zhang, J. Zheng, Z. Zhou et al., “Fractalkine promotes chemotaxis of bone marrow-derived mesenchymal stem cells towards ischemic brain lesions through Jak2 signaling and cytoskeletal reorganization,” FEBS Journal, vol. 282, no. 5, pp. 891–903, 2015.
View at: Publisher Site | Google Scholar
X. Liu, S. Yue, C. Li, L. Yang, H. You, and L. Li, “Essential roles of sphingosine 1-phosphate receptor types 1 and 3 in human hepatic stellate cells motility and activation,” Journal of Cellular Physiology, vol. 226, no. 9, pp. 2370–2377, 2011.
View at: Publisher Site | Google Scholar
M. Cadamuro, G. Nardo, S. Indraccolo et al., “Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma,” Hepatology, vol. 58, no. 3, pp. 1042–1053, 2013.
View at: Publisher Site | Google Scholar
G. J. Kastanis, Z. Hernandez-Nazara, N. Nieto et al., “The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 301, no. 3, pp. G464–G474, 2011.
View at: Publisher Site | Google Scholar
Y. Lu, N. Lin, Z. Chen, and R. Xu, “Hypoxia-induced secretion of platelet-derived growth factor-BB by hepatocellular carcinoma cells increases activated hepatic stellate cell proliferation, migration and expression of vascular endothelial growth factor-A,” Molecular Medicine Reports, vol. 11, no. 1, pp. 691–697, 2014.
View at: Publisher Site | Google Scholar
A. K. Ghosh, S. Bhattacharyya, G. Lakos, S.-J. Chen, Y. Mori, and J. Varga, “Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma,” Arthritis and Rheumatism, vol. 50, no. 4, pp. 1305–1318, 2004.
View at: Publisher Site | Google Scholar
A. Chawla, Y. Barak, L. Nagy, D. Liao, P. Tontonoz, and R. M. Evans, “PPAR-γ dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation,” Nature Medicine, vol. 7, no. 1, pp. 48–52, 2001.
View at: Publisher Site | Google Scholar
W. Wang, F. Liu, and N. Chen, “Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists attenuate the profibrotic response induced by TGF-β1 in renal interstitial fibroblasts,” Mediators of Inflammation, vol. 2007, Article ID 62641, 10 pages, 2007.
View at: Publisher Site | Google Scholar
L. Li, J. Tao, J. Davaille et al., “15-Deoxy-Δ12,14-prostaglandin J2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors,” The Journal of Biological Chemistry, vol. 276, no. 41, pp. 38152–38158, 2001.
View at: Google Scholar
X. Liu, H. Yu, L. Yang, C. Li, and L. Li, “15-Deoxy-Δ^12,14-prostaglandin J₂ attenuates the biological activities of monocyte/macrophage cell lines,” European Journal of Cell Biology, vol. 91, no. 8, pp. 654–661, 2012.
View at: Publisher Site | Google Scholar
P. J. M. van Haastert and P. N. Devreotes, “Chemotaxis: signalling the way forward,” Nature Reviews Molecular Cell Biology, vol. 5, no. 8, pp. 626–634, 2004.
View at: Publisher Site | Google Scholar
B. K. A. Seong, J. Lau, T. Adderley et al., “SATB2 enhances migration and invasion in osteosarcoma by regulating genes involved in cytoskeletal organization,” Oncogene, vol. 34, no. 27, pp. 3582–3592, 2015.
View at: Publisher Site | Google Scholar
V. Dormoy-Raclet, I. Ménard, E. Clair et al., “The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the β-actin mRNA in a U-rich-element-dependent manner,” Molecular and Cellular Biology, vol. 27, no. 15, pp. 5365–5380, 2007.
View at: Publisher Site | Google Scholar
R. M. Evans, “The steroid and thyroid hormone receptor superfamily,” Science, vol. 240, no. 4854, pp. 889–895, 1988.
View at: Publisher Site | Google Scholar
E.-H. Kim and Y.-J. Surh, “15-Deoxy-Δ^12,14-prostaglandin J₂ as a potential endogenous regulator of redox-sensitive transcription factors,” Biochemical Pharmacology, vol. 72, no. 11, pp. 1516–1528, 2006.
View at: Publisher Site | Google Scholar
J. W. Fuseler and M. T. Valarmathi, “Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide,” Biomaterials, vol. 33, no. 4, pp. 1032–1043, 2012.
View at: Publisher Site | Google Scholar
Y. Jiang and S.-K. He, “The effect of oxidative stress on retinal pigment epithelial cell migration,” Zhonghua Yan Ke Za Zhi, vol. 41, no. 2, pp. 100–105, 2005.
View at: Google Scholar
W.-S. Wu, J.-R. Wu, and C.-T. Hu, “Signal cross talks for sustained MAPK activation and cell migration: the potential role of reactive oxygen species,” Cancer and Metastasis Reviews, vol. 27, no. 2, pp. 303–314, 2008.
View at: Publisher Site | Google Scholar
A. Stanley, K. Thompson, A. Hynes, C. Brakebusch, and F. Quondamatteo, “NADPH oxidase complex-derived reactive oxygen species, the actin cytoskeleton, and Rho GTPases in cell migration,” Antioxidants and Redox Signaling, vol. 20, no. 13, pp. 2026–2042, 2014.
View at: Publisher Site | Google Scholar
T. R. Hurd, M. DeGennaro, and R. Lehmann, “Redox regulation of cell migration and adhesion,” Trends in Cell Biology, vol. 22, no. 2, pp. 107–115, 2012.
View at: Publisher Site | Google Scholar
L. Tochhawng, S. Deng, S. Pervaiz, and C. T. Yap, “Redox regulation of cancer cell migration and invasion,” Mitochondrion, vol. 13, no. 3, pp. 246–253, 2013.
View at: Publisher Site | Google Scholar
J. M. Zock, “Applications of high content screening in life science research,” Combinatorial Chemistry and High Throughput Screening, vol. 12, no. 9, pp. 870–876, 2009.
View at: Publisher Site | Google Scholar
M. Pignatelli, J. Sánchez-Rodríguez, A. Santos, and A. Perez-Castillo, “15-Deoxy-Δ-^12,14-prostaglandin J₂ induces programmed cell death of breast cancer cells by a pleiotropic mechanism,” Carcinogenesis, vol. 26, no. 1, pp. 81–92, 2005.
View at: Publisher Site | Google Scholar
H. Gao, W. Priebe, J. Glod, and D. Banerjee, “Activation of signal transducers and activators of transcription 3 and focal adhesion kinase by stromal cell-derived factor 1 is required for migration of human mesenchymal stem cells in response to tumor cell-conditioned medium,” Stem Cells, vol. 27, no. 4, pp. 857–865, 2009.
View at: Publisher Site | Google Scholar
Y. Li, X. Li, K.-R. Liu, J.-N. Zhang, Y. Liu, and Y. Zhu, “Visfatin derived from ascites promotes ovarian cancer cell migration through Rho/ROCK signaling-mediated actin polymerization,” European Journal of Cancer Prevention, vol. 24, no. 3, pp. 231–239, 2015.
View at: Publisher Site | Google Scholar

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Copyright © 2015 Xin Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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PPAR Research

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury via Redox Pathway

Abstract

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. BMSCs Preparation

2.3. Mouse Models

2.4. Immunofluorescence and High Content Analysis

2.5. Fluorescent Measurement of Intracellular Reactive Oxygen Species

2.6. Flow Cytometric Analysis

2.7. Cell Migration Assay

2.8. Real-Time RT-PCR

2.9. Statistical Analysis

3. Results

3.1. 15d-PGJ2 Inhibits Homing of BMSCs to the Injured Liver

3.2. 15d-PGJ2 Inhibits Migration of BMSCs In Vitro

3.3. 15d-PGJ2 Inhibits BMSCs Migration via ROS-Dependent Pathway, Independently of PPARγ

3.4. 15d-PGJ2 Inhibiting Migration of BMSCs Requires F-Actin Remodeling

4. Discussion

5. Conclusions

Conflict of Interests

Authors’ Contribution

Acknowledgments

References

Copyright

15-Deoxy-Δ^12,14-Prostaglandin J₂ Inhibits Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury via Redox Pathway

3.1. 15d-PGJ₂ Inhibits Homing of BMSCs to the Injured Liver

3.2. 15d-PGJ₂ Inhibits Migration of BMSCs In Vitro

3.3. 15d-PGJ₂ Inhibits BMSCs Migration via ROS-Dependent Pathway, Independently of PPARγ

3.4. 15d-PGJ₂ Inhibiting Migration of BMSCs Requires F-Actin Remodeling