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0Cellular Physiology Cel1 PhYsio1 B'ochem 2015;37:2085-2100 Open9access
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and Biochemistry Published online: November 25, 2015 Published by S. Karger AG, Basel 2085
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Original Paper
Peroxisome Proliferator-Activated Receptor Gamma Negatively Regulates the Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Toward Myofibroblasts in Liver Fibrogenesis
Shuangshuang Jia Xin Liu Weiyang Li Jieshi Xie Le Yang Liying Li
Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
Key Words
Peroxisome proliferator-activated receptor gamma • Bone marrow-derived mesenchymal stem cells • Differentiation • Fibrogenesis • Transforming growth factor pi
Abstract
Background/Aims: Bone marrow-derived mesenchymal stem cells (BMSCs) have been confirmed to have capacity to differentiate toward hepatic myofibroblasts, which contribute to fibrogenesis in chronic liver diseases. Peroxisome proliferator-activated receptor gamma (PPARy), a ligand-activated transcription factor, has gained a great deal of recent attention as it is involved in fibrosis and cell differentiation. However, whether it regulates the differentiation of BMSCs toward myofibroblasts remains to be defined. Methods: Carbon tetrachloride or bile duct ligation was used to induce mouse liver fibrosis. Expressions of PPARy, a-smooth muscle actin, collagen al (I) and collagen al (III) were detected by real-time RT-PCR and Western blot or immunofluorescence assay. Results: PPARy expression was decreased in mouse fibrotic liver. In addition, PPARy was declined during the differentiation of BMSCs toward myofibroblasts induced by transforming growth factor pi. Activation of PPARy stimulated by natural or synthetic ligands suppressed the differentiation of BMSCs. Additionally, knock down of PPARy by siRNA contributed to BMSC differentiation toward myofibroblasts. Furthermore, PPARy activation by natural ligand significantly inhibited the differentiation of BMSCs toward myofibroblasts in liver fibrogenesis and alleviated liver fibrosis. Conclusions: PPARy negatively regulates the differentiation of BMSCs toward myofibroblasts, which highlights a further mechanism implicated in the BMSC differentiation.
© 2015 The Author(s)
Published by S. Karger AG, Basel
Dr. Liying Li
KARG ER 125
Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, No.10 Xitoutiao, You An Men, Beijing 100069, (China); Tel./Fax 0086-10-83950101, E-Mail liliying@ccmu.edu.cn
DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
Introduction
Liver fibrosis is characterized by overproduction of collagen, especially collagen al (I] (Col al (I]] and collagen al (III] (Col al (III]], and other extracellular matrix (ECM] components [1, 2]. Myofibroblasts, characterized by the cytoskeletal protein a-smooth muscle actin (a-SMA], are known to be the predominant cells to generate Col al (I] and Col al (III], In addition to hepatic stellate cells (HSCs], portal fibroblasts, circulating fibrocytes and epithelial-mesenchymal transition (EMT], bone marrow (BM]-derived cells have recently been considered as another origin of hepatic myofibroblasts [2-4]. In particular, by using lethally irradiated female mice which receive male BM transplants, and tracking BM-derived cells through in situ hybridization for the Y chromosome, Russo et al. [5] found that 70% of hepatic myofibroblast populations are from BM-derived mesenchymal stem cells (BMSCs], In keeping with this finding, we also proved that a significant proportion of hepatic myofibroblasts is of BMSC-differentiation origin with a model of lethally irradiated mice receiving BM transplants from enhanced green fluorescent protein (EGFP] transgenic mice [6]. Considering the importance of BMSCs in liver fibrosis, identification of the molecular mechanisms underlying BMSC differentiation toward myofibroblast may represent an effective strategy for the treatment of fibrotic liver disease.
Transforming growth factor pi (TGF-pi] is a key profibrotic cytokine in the fibrogenesis [7-10], and can induce the differentiation of several sorts of fibroblasts toward myofibroblasts in vitro, such as fibroblasts from skin, lung, kidney and heart origin [11-14]. Our previous study has demonstrated that sphingosine kinase/sphingosine 1-phosphate (S1P]/S1P receptor axis is involved in the differentiation of BMSCs toward myofibroblasts induced by TGF-pi during liver fibrosis [9]. Nevertheless, it is still not clearly illustrated whether other molecules implicated in this process and the underlying mechanisms.
Peroxisome proliferator-activated receptors (PPARs] are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily [15], with three subtypes, PPARa, PPARp/S and PPARy, relate to multiple functions, including metabolism, immune responses and cellular proliferation [16]. PPARy is mainly known to regulate adipocyte differentiation and fatty-acid uptake and storage [17-19]. Some clinical studies reported that synthetic PPARy agonists, such as thiazolidinedione, have beneficial effects in patients with type 2 diabetes [20,21]. Besides its function in metabolism, a new action of PPARy in fibrosis has been revealed. It has been reported that activation of PPARy by specific agonists could ameliorate fibrosis in several organs, such as lung, kidney and heart [22]. In particular, several recent studies have illustrated that PPARy participates in the myofibroblast differentiation [11, 13, 23, 24]. However, whether PPARy is link to regulate the differentiation of BMSCs toward hepatic myofibroblasts and its molecular mechanisms remain obscure.
Based on the scientific background, the aim of this study is to elucidate the function of PPARy in BMSCs differentiation toward myofibroblasts in liver fibrogenesis. Here, we showed that PPARy expression was reduced in mouse liver fibrogenesis, as well as in the differentiation of primary mouse BMSCs toward myofibroblasts induced by TGF-pi in vitro. In addition, knock down of PPARy by siRNA contributed to the differentiation of BMSCs. Furthermore, activation of PPARy by ligands inhibited BMSCs differentiation toward myofibroblasts in vitro and in vivo, and ameliorated liver fibrosis. These observations strongly indicate that PPARy negatively governs the differentiation of BMSCs toward myofibroblasts, suggesting that PPARy-targeted therapy could be a potential strategy in the treatment of liver fibrosis.
Materials and Methods
Reagents
a-MEM was from Invitrogen (Grand Island, NY], Fetal bovine serum was from Hyclone/Thermo Scientific (Victoria, Australia], TGF-ßl came from PeproTech (London, UK], 15d-PGJ2 was from Cayman Chemi-
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DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
cal (Ann Arbor, MI], Troglitazone and ciglitazone were from Biomol (Tebu, France], Antibodies against PPARy, Col al (I], Col al (III] and tubulin were from Santa Cruz Biotechnology (CA, USA], Anti-glyceral-dehyde-3-phosphate dehydrogenase (GAPDH] monoclonal antibody was from Cell Signalling (Beverly, MA], PCR reagents were from Applied Biosystems (Foster City, CA], Anti-a-SMA antibody, bovine serum albumin (BSA] and other common reagents were from Sigma-Aldrich (St. Louis, MO],
BMSCs preparation
BM cells were isolated from bone marrow by flushing the tibias and femurs of ICR mice aged 3 weeks (Laboratory Animal Center, Capital Medical University] with culture medium using a 25-gauge needle. The cells were then passed through 70-mm nylon mesh and were washed three times with PBS containing 2% fetal bovine serum. BMSCs cultured as described previously [5]. I n brief, BM cells were cultured with BMSCs culture medium (a-MEM] containing 20% fetal bovine serum for 1 week at 37°C in 5% C02. To remove the nonadherent cells, the culture medium was replaced twice a week. a-MEM containing 15% fetal bovine serum was used to culture BMSCs after the first subculture. BMSCs are characterized by the phenotypes as positive for CD44, CD105 and CD166, but negative for CD14, CD34 and CD45 in flow cytometry analysis [6]. BMSCs of passage 3 to passage 5 were used in the experiments. All animal work was performed under the ethical guidelines ofthe Ethics Committee of Capital Medical University.
Mouse models of liver fibrosis
Mouse models of chronic liver fibrosis were performed by injection of carbon tetrachloride (CC1J or bile duct ligation (BDL], ICR mice aged 6 weeks received intraperitoneal injections of 1 |iL/g body weight of a CCl4/olive oil (00] mixture, 1:9 v/v, twice per week. 15d-PGJ2 (0.3mg/kg body weight] or saline was administrated the day before CC14 or 00 treatment, and then twice per week before CC14 or 00 treatment for indicated times. The mice were sacrificed at 1, 3 days or at 1, 2,4, or 8 weeks of CCl4treatment (n = 7 per group].
Another group of adult ICR mice received BDL operation as described previously [25]. In brief, mice were anesthetized to receive a midline laparotomy to expose the common bile duct which was ligated three times. One ligature was located in the distal portion ofthe bile duct and another two were placed in the proximal portion. Then, the bile duct was cut between the ligatures followed by closing the abdomen in layers. Sham-operated mice underwent a laparotomy with exposure, but no performance of ligation ofthe common bile duct, which used as controls. Mice were sacrificed at 3 days or at 1, 2 weeks of BDL (n = 7 per group],
BM transplantation
ICR mice received lethal irradiation (8 Grays], and then immediately received transplantation by a tail-vein injection of 1.5 x 107 whole BM cells obtained from 3-week-old EGFP transgenic mice. Four weeks later, mice received intraperitoneal injections of CC14 twice per week for 4 weeks. 15d-PGJ2 (0.3 mg/kgbody weight] or saline was applied the day before CC14 or 00 injection, and twice per week before CC14 or 00 treatment for 4 weeks (n = 7 per group].
Quantitative analysis of liver fibrosis
Liver tissues were fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Samples were cut into 5 |im slices and sections were stained for collagen visualization with Sirius Red.
Immunofluorescence and high content analysis
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% Triton X-100 for 15 minutes, blocked with 2% BSA for 1 hour, and incubated with antibodies to a-SMA (1:1000], Col al(I] (1:100], Col al(III] (1:200] and PPARy (1:100] overnight, followed by secondary antibody conjugated with Cy3 (1:500, Jackson Immunoresearch Laboratories, West Grove, PA] for 1 hour. Cells were washed twice with PBS after incubation with PBS containing 10|ig/mL DAPI for 5 minutes, and 200 |iL PBS were left in each well. For negative controls, cells were processed the same way, except the primary antibody was omitted. The plates were imaged on a Thermo Scientific Cell Insight personal cell imaging (PCI] platform (Cellomics, Inc., Thermo Fisher Scientific Inc., Waltham, MA], with a xio objective using the Thermo Scientific Cellomics iDEV Software.
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DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
High content analysis was performed as described previously [26]. In brief, at least 3,000 cells in thirty-six fields were automatically acquired by the software. The fluorescence intensity in each well was analyzed using Cellomics Cell Health Profiling BioApplication software.
Immunofluorescent detection for a-SMA in liver tissue was performed with a Vector M.O.M. (mouse-on-mouse] immunodetection kit (Vector Laboratories] [9]. Prepared liver sections were 5 |im and antibody to a-SMA was a 1:1000 dilution.
Western blot analysis
Western blot analysis of a-SMA, Col al(I], Col al(III] and PPARy in BMSCs or liver tissue were performed with 50 |ig of protein extract using primary antibodies of a-SMA (1:1000], Col al(I] (1:100], Col al(III] (1:200] and PPARy (1:500]. The bands were displayed using ODYSSEY and quantified by Odyssey v3.0 software. Results were normalized relative to the GAPDH (1:1000] or tubulin (1:1000] expression to correct for variations in protein loading and transfer.
RNA interference
The siRNA sequence specifically targeting mouse PPARy was synthesized by Dharmacon: L-040712-00-0005 (Thermo Scientific, Lafayette, CO], 40% to 50% confluent BMSCs were prepared. Transienttransfection of siRNA (40 nmol/L] was performed by using Invitrogen Lipofectamine RNAiMAX, as recommended by the manufacturer. Control cells were treated with 40 nmol/L RNAi Negative Control Duplexes (scramble siRNA], Cells were collected to perform real-time RT-PCR and Western blot analysis after 48 hours.
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 [6]. Primers (MWG Biotech, Ebersberg, Germany] used for real-time RT-PCR were as follows: 18s rRNA, sense, 5'-GTA ACC CGT TGA ACC CCA TT-3', and anti-sense, 5'-CCA TCC AAT CGG TAG TAG CG-3'; a-SMA: sense, 5'-ATG CTC CCA GGG CTG TTT T-3', and anti-sense, 5'-TTC CAA CCATTA CTC CCT GAT GT-3'; Col al(I]: sense, 5'-AGG GCG AGT GCT GTG CTT T-3', and anti-sense, 5'-CCC TCG ACT CCT ACA TCT TCT GA-3'; Col al(III]: sense, 5'-TGA AAC CCC AGC AAA ACA AAA-3', and anti-sense, 5'-TCA CTT GCA CTG GTT GAT AAG ATT AA-3'; TGF-pi: sense, 5'-TGC GCT TGC AGA GAT TAA AA-3', and anti-sense, 5'-TCA CTG GAG TTG TAC GGC AG-3'; PPARy: sense, 5'-GCC CAC CAA CTT CGG AAT C-3', and anti-sense, 5'-TGC GAG TGG TCT TCC ATC AC-3'.
Statistical analysis
All results were confirmed in three independent experiments. Data are expressed as mean ± SEM and were analyzed by Student's t-test or ANOVA for analysis of variance. Statistical significance was defined as P< 0.05.
Results
PPARy is down-regulated in mouse chronic liver fibrogenesis
There are recent reports that expression of PPARy is decreased in fibrogenesis [13, 27, 28]. We assessed PPARy content in liver tissue of mouse chronic liver fibrosis models induced by CC14 administration or BDL operation. The results showed that PPARy expression in both mRNA and protein levels were markedly reduced in CC14 administration group compared with OO-treated group (Fig. 1A and B], These findings were consistent with the results in BDL-induced liver fibrogenesis (Fig. 1C and D],
The expressions of main ECM, Col al (I] and Col al (III], were up-regulated in mouse liver fibrogenesis [29]. Here, we undertook correlation analysis between PPARy and the two molecules in liver tissue. Results presented in Fig. IE and F demonstrated that the mRNA level of PPARy had negative correlations with Col al (I] and Col al (III], respectively (correlation coefficient r = -0.503, P = 0.024; r = -0.525, P = 0.025, respectively]. In addition, the expression of a-SMA, a marker of myofibroblasts, which produces ECM predominantly,
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DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
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Fig. 1. PPARy is involved in the chronic liver fibrogenesis. Real time RT-PCR was performed to determine mRNA expression of PPARy in mouse fibrotic liver induced by CC14 treatment (A] or BDL operation (C] (n = 7 per group], PPARy protein expressions in CC14 treatment (4 weeks, B] or BDL operation (2 weeks, D] were detected by Western blot. The correlations between mRNA expression of PPARy and Col al (I] (E], Col al (III] (F], a-SMA (G] or TGF-pi (H] in liver tissue. *P < 0.05, compared with olive oil (00] or sham group.
was obviously increased as well after liver injury [29]. We also found a negative correlation between the mRNA levels of PPARy and a-SMA (correlation coefficient r = -0.505, P = 0.039] (Fig. 1G], Furthermore, there was a significant negative correlation between mRNA levels of PPARy and TGF-pi, a key profibrotic cytokine and contributing to BMSC differentiation toward myofibroblasts in fibrotic liver [9] (correlation coefficient r = -0.610, P = 0.009] (Fig. 1H], Collectively, these results suggested PPARy might play an essential role in liver fibrogenesis.
PPARy is down-regulated in the differentiation of BMSCs toward myofibroblasts In addition to representing the extent of liver fibrosis, presence of a-SMA and increased levels of Col al (I] and Col al (III] also denote the differentiation of BMSCs toward myofibroblasts [9]. Given that PPARy was negatively correlated with these molecules and TGF-pi in liver tissue, we then soughtto determine the effects of PPARy on the differentiation of BMSCs. In this regard, we explored the abundance of PPARy in the context of BMSC
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DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
Fig. 2. PPARy is implicated in the differentiation of BMSCs toward myofibroblasts. (A] Immunofluorescence analysis of PPARy (red] in BMSCs with (+] or without (-] TGF-pi (10 ng/mL], (B] PPARy mRNA in differentiated BMSCs induced by indicated doses of TGF-pi for 24 hours. (C] Protein level of PPARy in BMSCs treated with 10 ng/mL TGF-pi for 24 hours. Correlations between PPARy and a-SMA (D], Col al (I] (E] or Col al(III] (F] in BMSCs. All results were confirmed in three independent experiments. *P < 0.05, compared with control. Scale bars, 50 |im.
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differentiation toward myofibroblasts in vitro. Immunofluorescent staining showed that the degree of positive staining for PPARy was much weakened when BMSCs were induced differentiation toward myofibroblasts by TGF-pi (Fig. 2A], Furthermore, the mRNA level of PPARy was strikingly decreased during differentiation of BMSCs trigged by TGF-pi in a dose-dependent manner (Fig. 2B], In parallel, PPARy expression in protein level was attenuated, too (Fig. 2C], It was noteworthy that there were also inverse correlations between mRNA expressions of PPARy and a-SMA, Col al (I] or Col al (III] in BMSCs (Fig. 2D-F] (correlation coefficient r = -0.691, P = 0.000046; r = -0.792, P = 0.000001; r = -0.737, P = 0.000008, respectively]. These observations highlight the link between PPARy and the differentiation of BMSCs toward myofibroblasts.
PPARy negatively modulates the differentiation of BMSCs toward myofibroblasts To further characterize the function of PPARy in BMSC differentiation, we used an endogenous ligand (15d-PGJ2] and two synthetic ligands (troglitazone and ciglitazone] of PPARy. As illustrated in Fig. 3A, a-SMA mRNA in differentiated BMSCs triggered by TGF-pi
KAR.GER
DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
2015 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb
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Fig. 3. Activated PPARy negatively regulates a-SMA expression in BMSCs. BMSCs were exposed to 5 |imol/L endogenous PPARy ligand (15d-PGJ2] or 10 |imol/L PPARy synthetic agonists (troglitazone (Trog] or ciglita-zone (Cig]] for 1 hour, then challenged by 10 ng/mL TGF-pi for 24 hours. Real time RT-PCR was performed to detect a-SMA mRNA (A], a-SMA protein expression was evaluated by Western blot (B], The intensity of each band with a-SMA antibody was quantified and normalized to that with anti-p-tubulin antibody (C], (H] Immunofluorescence analysis of a-SMA (red], and total fluorescence intensity of a-SMA protein was counted with high content analysis (G], Cells were transfected with scrambled siRNA or PPARy siRNA for 48 hours. Protein level of PPARy was detected by Western blot (E] and mRNA expression of PPARy and a-SMA was measured by real-time RT-PCR (D and F], All results were confirmed in three independent experiments. *P < 0.05, compared with control. # P < 0.05, compared to TGF-pi group. Scale bars, 50 |im.
was obviously diminished when pretreatment with either natural or synthetic ligands of PPARy. In accord, a-SMA protein expression induced by TGF-pi was down-regulated by 15d-PGJ2, troglitazone or ciglitazone (Fig. 3B and C], Furthermore, siRNA technology was
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ö jjT
DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
2015 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb
□ TGF-pi(-)
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fold 1.00 2.75 1.43 1.30 1.32 Col a1 (I)
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Fig. 4. PPARy activation suppresses Col al (I] expression in BMSCs. BMSCs were incubated with 5 |imol/L 15d-PGJ2,10 |imol/Ltroglitazone (Trog] or 10 |imol/L ciglitazone (Cig] for 1 hour, followed by treatment of 10 ng/mL TGF-pi for 24 hours. mRNA expression of Col al(I] was examined by real-time RT-PCR (A], Col al(I] protein expression was detected by Western blot (B], immunofluorescence analysis (red] (E] and high content analysis (D], Arrows in (E] indicated that Col al (I] secreted into the medium. (C] Cells were trans-fected with scrambled siRNA or PPARy siRNA, and Col al (I] mRNA was evaluated 48 hours later. All results were confirmed in three independent experiments. *P < 0.05, compared with control; # P < 0.05, compared with TGF-pi group. Scale bars, 50 |im.
performed to knock-down of PPARy. Transfection of BMSCs with PPARy-siRNA decreased the mRNA expression of PPARy by 70% (Fig. 3D]. Meanwhile, Western blotanalysis indicated PPARy protein expression was also significantly reduced after knock-down of PPARy (Fig. 3E], The results showed that PPARy knock-down enhanced a-SMA mRNA expression, whereas scrambled siRNA (SCR siRNA] had no effects (Fig. 3F],
To further confirm the function of PPARy in BMSC differentiation, we performed immunofluorescent staining for a-SMA to reveal morphological changes in BMSCs. Isolated BMSCs showed very weak expression of a-SMA under normal condition. TGF-pi treatment resulted in the appearance of numerous bundles of actin microfilaments and dramatically increased the expression of a-SMA in BMSCs (Fig. 3H], However, when BMSCs were pretreated with ligands of PPARy, the ability of TGF-pi to enhance the expression of a-SMA was markedly blunted (Fig. 3H], Additionally, the total fluorescence intensity of a-SMA examined by high content analysis demonstrated that exposure to 15d-PGJ2, troglitazone or ciglitazone resulted in reducing a-SMA protein level in differentiated BMSCs initiated by TGF-pl (Fig. 3G],
KARGER.
DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
2015 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb
V) 5 o _ a, re 4
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Fig. 5. Activation of PPARy represses expression of Col al (III] in BMSCs. BMSCs were pretreated with 15d-PGJ2 (5 |imol/L], troglitazone (Trog, 10 ^mol/L] or ciglitazone (Cig, 10 |imol/L] for 1 hour, then cells were stimulated with TGF-ßl (10 ng/mL] for 24 hours. Col al (III] mRNA expression was assessed by real-time RT-PCR (A], Western blot (B], immunofluorescence analysis (red] (E] and high content analysis (D] were used to evaluated Col al (III] protein. Col al (III] secreted into the medium was shown as arrows in (E], (C] BMSCs were transfected with scrambled siRNA or PPARy siRNA for 48 hours, and Col al (I] mRNA was evaluated by real-time RT-PCR. All results were confirmed in three independent experiments. *P< 0.05, compared with control; # P < 0.05, compared with TGF-ßl group. Scale bars, 50 |im.
Myofibroblasts are the predominant collagen-producing cells in liver fibrosis. Up-regulation of Col al (I] and Col al (III] is an essential event during BMSC differentiate toward myofibroblasts. Therefore, we sought to characterize the effects of PPARy on the expression of Col al (I] and Col al (III] in BMSCs. As expected, all three PPARy agonists, 15d-PGJ2, troglitazone or ciglitazone blunted the increases of Col al(I] and Col al(III] mRNA expression in BMSCs treated with TGF-ßl (Fig. 4A and 5A], Western blot analysis exhibited similar results in protein level (Fig. 4B and 5B], In accord with a-SMA, mRNA expressions of Col al (I] and Col al (III] were also increased in PPARy-knock-down cells (Fig. 4C and 5C],
Furthermore, immunofluorescence was also performed to study the effects of PPARy on the protein expressions of Col al (I] and Col al (III] in BMSCs. The results showed that there were strong immunoreactivities for Col al (I] and Col al (III] in BMSCs after treatment with TGF-ßl (Fig. 4E and 5E], and collagen that was secreted into the medium was also detected by immunofluorescence (Fig. 4E and 5E, arrow]. Manipulation of PPARy ligands reduced the degree of positive staining for Col al (I] and Col al (III] induced by TGF-ßl (Fig. 4E and
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DOI: 10.1159/000438567
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Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
Fig. 6. Activated PPARy inhibits BMSCs differentiate toward myofibroblasts during liver injury. (A] Mouse liver fibrosis was induced by injection of CC14 twice per week for different time periods with or without 15d-PGJ2 administration (n = 7 per group], mRNA ofa-SMA in liver tissues was detected with real-time RT-PCR. (B-D] ICR mice were lethally irradiated and received whole BM transplants from EGFP transgenic mice, followed by CC14 injection twice per week for 4 weeks in the presence or absence of 15d-PGJ2 treatment (n = 7 per group]. The proportion of myofibroblasts from BMSCs was evaluated using Image-Pro Plus software (B], Representative immunofluorescence images ofa-SMA (red], EGFP (green] and merged fields to track myofibroblasts of BMSC origin (C and D], Insets in (C] were immunofluorescence images for olive oil (00]-treated livers, and in (D] were livers of 00 group with 15d-PGJ2. The nuclei were stained with DAPI (blue], * P < 0.05 compared with 00 group. # P < 0.05, compared with CC14 group without 15d-PGJ2. Scale bars, 50 |im.
5E], High content analysis indicated that the increased total fluorescence intensities of Col al (I] and Col al (III] were attenuated by 15d-PGJ2, roglitazone or ciglitazone (Fig. 4D and 5D], Altogether, these findings showed that PPARy negatively governed BMSCs differentiate toward myofibroblasts.
Activated PPARy inhibits the differentiation of BMSCs toward myofibroblasts in mouse
fibrotic liver
During liver injury, myofibroblasts are increased and its marker, a-SMA, is up-regulated to contribute to liver fibrogenesis [29]. In the present study, we found that treatment with the natural ligand of PPARy, 15d-PGJ2, reduced a-SMA mRNA in liver fibrosis induced by different
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DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
Fig. 7. PPARy activation suppresses collagen expression in liver fibrosis. Different time periods of CC14 was used to induce mouse liver fibrosis with or without 15d-PGJ2 administration (n = 7 per group], (A] Expression of Col al (I] and Col al(I-II] mRNA levels in liver tissues were measured by real-time RT-PCR. (B] Representative images of Sirius Red staining in the absence or presence of 15d-PGJ2 after CC14 administration. * P < 0.05 compared with olive oil (00] group. # P < 0.05, compared with CC14 group without 15d-PGJ2. Scale bars, 50 |im.
OO ••«•• 00+15d-PGJ2
CCL "•<*•• CCIj+ISd-PGJ
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41 ö .2 ë.
Col a1(l)
2 4 6 CCU(weeks)
30 25 20 15 10 5 0
Col a1 (III) *
0 2 4 6 8 CCU(weeks)
periods of CC14 injection (Fig. 6A], Given that myofibroblasts are mostly BMSC-derived, we further determinedthe effectofPPARy on BMSC differentiation toward myofibroblasts in vivo BM in the irradiated mice was reconstituted by transplanting BM cells from EGFP transgenic mice, and then the proportion of BMSC-derived myofibroblasts was measured. As shown in Fig. 6C, after CC14 treatment, there was a strong expression of a-SMA colocalizing with EGFP in the fibrotic areas, which indicated they were BMSC origin. However, 15d-PGJ2 significantly inhibited the differentiation of BMSCs toward myofibroblasts in liver fibrogenesis (Fig. 6D], and markedly decreased the proportion of BMSC-derived myofibroblasts compared with CC14 group without 15d-PGJ2 treatment (Fig. 6B], These results indicated that PPARy activation could suppress BMSCs differentiate toward myofibroblasts in vivo.
Activated PPARy attenuates hepatic fibrosis
Col al (I] and Col al (III] are the main ECM in fibrotic liver tissue [1, 2]. After injection of CC14, there were dynamic changes in mRNA expressions of Col al (I] and Col al (III] in liver tissue, which markedly up-regulated in a way correlated with the progression of liver fibrosis (Fig. 7A], As expected, PPARy activation by 15d-PGJ2 markedly decreased these mRNA levels (Fig. 7A], In addition, hepatic collagen deposition was further evaluated by morphometric analysis with Sirius red staining. As shown in Fig. 7B, collagen deposition was markedly attenuated after 15d-PGJ2 administration. These data suggest activation of PPARy had anti-fibrotic properties.
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DOI: 10.1159/000438567 and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
Discussion
Liver fibrosis is a common pathological course of chronic hepatic diseases. So far, there is no effective and safe strategy for the prevention or inhibition of fibrogenesis in clinical practice. Differentiation of BMSCs toward myofibroblasts is an essential event for liver fibrogenesis, thus, elucidating the regulatory mechanisms in this process may shed light on the treatment of liver fibrosis. In the current study, we delineated the potential role of PPARy in the differentiation of primary mouse BMSCs toward myofibroblasts. We found PPARy negatively regulated BMSC differentiation toward myofibroblasts in liver fibrogenesis. Expression of PPARy was reduced in fibrotic liver tissue. In addition, differentiation of BMSCs was accompanied by the decrease of PPARy expression. Activated PPARy by specific ligands could attenuate BMSC differentiation in vitro and in vivo, and alleviate liver fibrosis. These results critically revealed a mechanism underlying differentiation of BMSCs toward myofibroblasts.
Accumulating evidence suggests that levels of PPARy were dramatically reduced in the process of fibrosis [13,27,28,30,31], whereas overexpression of PPARy could ameliorate the degree of fibrosis [32]. In the present study, the abundance of PPARy was evidently reduced in fibrogenesis, similar to the reports in rats that PPARy expression was down-regulated in fibrotic liver tissue induced by 2 or 8 weeks of CC14 combined with ethyl alcohol treatment or caused by 10 days of BDL operation [28, 33, 34]. In addition, mRNA expressions of PPARy and those fibrotic related molecules, such as TGF-pi, a-SMA, Col al (I] and Col al (III], were negative correlations. Intriguingly, PPARy and plasminogen activator inhibitor-1 (PAI-1], associated with fibrosis and implicated in its pathogenesis, were inversely correlated in skin [13]. In heart, PPARy was also negatively correlated with tenascin-x, which was exclusively expressed in fibroblasts that can mediate fibrosis [35]. These results emphasize the importance of PPARy in the pathogenesis of fibrosis in different tissues.
Several studies reveal that PPARy was down-regulated in the differentiation of sorts of fibroblasts and myocytes toward myofibroblasts [11, 13, 36]. In our study, expression of PPARy was remarkably decreased during BMSC differentiation toward myofibroblasts induced by TGF-pi, similar to our previous report that expression of PPARy almost couldn't be detected in human hepatic myofibroblasts [37]. In addition, the down-regulation of PPARy by specific siRNA had favorable effect on the differentiation of BMSCs. In accordance, enhanced myofibroblast formation potential was observed in PPARy-deficient mouse fibroblasts [38]. BMSCs are multipotent cells with the potential to differentiate toward a variety of mesenchymal cells, such as adipocytes [39]. The decreased expression of PPARy, a transcription factor that related to fat-formation, might make BMSCs lost potential to differentiate toward adipocytes, but contribute to the differentiation toward myofibroblasts in the present study. This hypothesis is in agreement with the results in HSCs, another type fat-storing cell, that significant reduced expression of PPARy was paralleled by activation of HSCs [32, 40-42]. It has been reported that the necdin-wnt pathway and a combination of methyl-CpG binding protein 2 (MeCP2], enhancer of zeste homolog 2 (EZH2] and miR132 cause epigenetic PPARy repression and result in HSC activation [43, 44]. In addition, PPARy expression could be repressed by TGF-pi through increasing the binding of histone deacetylase 1 (HDAC1] and decreasing the levels of acetylated histone3 (AcH3] at the PPARy promoter or via smad binding elements in the PPARy gene promoter to negatively regulate the promoter activity of PPARy gene in cardiac fibroblasts or HSCs [11, 24]. However, the precise regulatory mechanism of PPARy expression in BMSCs is scarce and should be valued in further research.
PPARy needs to be activated by ligands to exert effects. Results presented here showed that either natural or synthetic ligands of PPARy could suppress BMSC differentiate toward myofibroblasts induced by TGF-pi in vitro, with alleviation of a-SMA, Col al (I] and Col al (III] expression. More importantly, we found activation of PPARy by natural ligand could inhibit the differentiation of BMSCs toward myofibroblasts in liver fibrogenesis and decreased the proportion of BMSC-derived myofibroblasts. These results provide strong evidence for
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DOI: 10.1159/000438567 and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
that activated PPARy can negatively regulate BMSC differentiate toward myofibroblasts. In particular, serum concentrations of 15d-PGJ2 in Caucasian patients with ongoing hepatic fibrogenesis and Chinese patients with hepatocellular carcinoma are elevated, which suggests that increased 15d-PGJ2 might trigger PPARy activity to attempt to execute its beneficial effects [45]. Additionally, PPARy activation by agonists attenuates skin and renal fibroblast differentiation [12, 46] and inhibits HSC activation [32, 42]. It is noteworthy that effects of activated PPARy by different agonists may have diverse and complex roles in regulating cell differentiation. For instance, Da et al. [47] reported that PPARy ligand, pioglitazone, did not suppress cultured HSC activation by assessment expression of a-SMA and Col al(I],
Our previous result has indicated that sphingosine kinase/SlP/SIP receptor axis participates in the differentiation of BMSCs toward myofibroblasts [9]. In the present study, we found PPARy negatively regulates BMSC differentiation toward myofibroblasts. There are several reports have linked PPARy with SIP signaling pathway. In renal mesangial cells, synthetic PPARy agonists thiazolidinediones up-regulate sphingosine kinase and intracellular SIP resulting in decrease expression of CTGF to causes an anti-fibrotic effect [48]. In addition, thiazolidinediones could increase the abundance of SIP receptor [49]. In turn, PPARy is an intracellular target for SIP and SIP could activate PPARy coactivator la (PGC-la] [50, 51]. These findings might partially explain the effect of PPARy on BMSC differentiation toward myofibroblasts and its inhibitory function in liver fibrosis. However, the relationship between PPARy and SIP pathway remains controversial. In 3T3-L1 preadipocytes, SIP down-regulates the expression of PPARy [52], which reflects the complexity of signaling cascades in a biological system.
An importantissue of PPARy is that PPARy carries outits function requiring translocation from cytoplasm to nucleus, such as in the study of PPARy governs the differentiation of skin fibroblasts [46]. Besides regulating target gene expression directly, PPARy could interact with other cell signaling pathways, including fibrotic-related events in cells. It has been reported that PPARy is capable of binding to Smad3 and p-Smad3, and prevents nuclear accumulation ofp-Smad3, resulting in disruption ofTGF-pi signaling [53]. Activated PPARy by rosiglitazone reduced the induction of the early-immediate transcription factor Egr-1, a mediator of non-Smad TGF-pi signaling, to exhibit its antifibrotic function [54]. Furthermore, albumin may be a downstream effector of PPARy in phenotypic switch of pancreatic stellate cells (PSCs] [55], and PPARy activation might ameliorate renal fibrotic lesions via hepatocyte growth factor (HGF] [56]. However, whether these molecular mechanisms mediate the effects of PPARy on BMSC differentiation toward myofibroblasts need to be further explored.
In summary, our results demonstrate that PPARy is a negative regulator in BMSC differentiation toward myofibroblasts in liver fibrogenesis. Decrease in PPARy expression is accompanied by the differentiation of BMSCs toward myofibroblasts, and activated PPARy by agonists inhibits BMSC differentiation in vitro and in vivo, and causes an attenuation of liver fibrosis. Consequently, these findings expand our knowledge on the regulatory mechanisms of BMSC differentiation, and make PPARy to be a promising target for treatment of liver fibrotic diseases.
Abbreviations
PPARy (peroxisome proliferator-activated receptor gamma]; BMSCs (bone marrow-derived mesenchymal stem cells]; a-SMA (a-smooth muscle actin]; 15d-PGJ2 (15-Deoxy-A1214-prostaglandin J2]; TGF-pi (transforming growth factor pi].
Acknowledgements
This work was supported by grants from the National Natural and Science Foundation of China (81430013, 81300335] and the Project of Construction of Innovative Teams and
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ö jjT
DOI: 10.1159/000438567
and Biochemistry Published online: November 25, 2015
Jia et al.: PPARy Negatively Regulates the Differentiation of BMSCs
Teacher Career Development for Universities and Colleges under Beijing Municipality (IDHT20150502).
Disclosure Statement
The authors declared that they have no conflicts of interest related to this work. References
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