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Smad signaling pathway
Biomedicine & Pharmacotherapy 89 (2017) 1387–1391
Available online at
ScienceDirect www.sciencedirect.com
Original research
CTRP3 attenuates hepatic stellate cell activation through transforming growth factor-b/Smad signaling pathway Chuantao Chenga,1, Shuo Yua,1, Ran Kongb , Qinggong Yuana , Yuefeng Mab , Wenbin Yanga , Gang Caoa,* , Liyi Xiec,* a b c
Department of General Surgery, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China Department of Thoracic Surgery, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China Department of Nephrology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China
A R T I C L E I N F O
Article history: Received 22 November 2016 Received in revised form 7 March 2017 Accepted 7 March 2017 Keywords: Liver fibrosis Hepatic stellate cells (HSCs) C1q/tumor necrosis factor-related protein 3 Extracellular matrix (ECM)
A B S T R A C T
Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of liver fibrosis. C1q/ tumor necrosis factor-related protein 3 (CTRP3), a member of CTRPs, was involved in fibrosis. However, little is known about the role of CTRP3 in liver fibrosis. This study aimed to determine its role in liver fibrosis and explore the possible mechanism. Our results demonstrated that CTRP3 was lowly expressed in liver fibrosis tissues and activated HSCs. Overexpression of CTRP3 inhibited the proliferation and migration of HSCs, as well as suppressed the expression of extracellular matrix (ECM) in transforming growth factor-b1 (TGF-b1)-stimulated HSC-T6 cells. Furthermore, CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGF-b1-stimulated HSC-T6 cells. In conclusion, the present study demonstrated that CTRP3 inhibited the proliferation and migration of TGFb1-induced HSC-T6 cells and attenuated liver fibrosis, at least in part, through inhibiting the Smad signaling pathway. These findings suggest that CTRP3 may be a promising therapeutic target for the treatment of liver fibrosis. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Liver fibrosis, a major cause of morbidity and mortality from hepatic diseases, is characterized by the excessive synthesis of extracellular matrix (ECM) proteins and eventually the development of hepatic cirrhosis [1]. Hepatic stellate cells (HSCs) are the major mesenchymal cells in liver, and the activation of HSCs plays a critical role in the fibrogenesis [2]. Previous studies demonstrated that HSC activation can be induced by a variety of factors, including transforming growth factor-b1 (TGF-b1) [3–5]. TGF-b1 generally induces HSC proliferation and ECM synthesis in vitro and liver fibrogenesis in vivo [6]. Thus, inhibition of HSC activation is of crucial importance for the treatment of liver fibrosis. C1q/tumor necrosis factor-related proteins (CTRPs) play important and distinct roles in regulating insulin sensitivity and energy balance [7]. CTRP3, a member of CTRPs, is identified as a novel adipokine [8]. Previous studies have shown that CTRP3 was
* Corresponding authors. E-mail addresses: [email protected] (G. Cao), [email protected] (L. Xie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.03.021 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
involved in multiple cellular processes, such as cell proliferation, apoptosis, immune-regulation, inflammation, glucose metabolism and lipid metabolism [9–12]. In addition, several studies suggest a role of CTRP3 in fibrosis disease [13,14]. It has been reported that the expression of CTRP3 was significantly decreased after myocardial infarction, and CTRP3 attenuated the proliferation, migration, and the expression of connective tissue growth factor, collagen I, and collagen III induced by TGF-b1 in cultured adult rat cardiac fibroblasts [14]. However, little is known about the role of CTRP3 in liver fibrosis. This study aimed to determine its role in liver fibrosis and explore the possible mechanism. Our data showed that CTRP3 attenuated liver fibrosis through inhibiting the TGF-b1/Smad signaling pathway. 2. Materials and methods 2.1. Specimen collection Liver biopsies were collected by transparietal puncture from 11 healthy individuals and 11 patients with liver fibrosis. All of the samples were stored at 80 C. For all of the patients who
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participated in this study, written informed consent was obtained. This study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University (China). 2.2. Cell culture The human HSC cell line (HSC-T6) was purchased from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) in the presence of penicillin/streptomycin (Sigma, St. Louis, MO, USA) at 37 C in a humidified 5% CO2 atmosphere.
2.7. Western blotting Total protein was extracted from the treated cells using RIPA lysis buffer (Beyotime, Nantong, China) according to the manufacturer’s instructions. Equal amounts of protein (30 mg) were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride membranes (Whatman Schleicher & Schuell, Middlesex, UK). After blocking with 5% non-fat milk in Tris-buffered saline
2.3. Construction of plasmids and cell transfection The full-length CTRP3 cDNA was cloned into the pcDNA3.1 vector (Genechem, Shanghai, China). HSC-T6 cells were transfected with CTRP3 or vector using LipofectamineTM2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. The transfection efficiency of CTRP3 was confirmed by quantitative real-time PCR and western blotting analysis. 2.4. Cell proliferation assay Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. In brief, the transfected HSC-T6 cells (1 105 cells/well) were seeded onto 96well plates for 24 h. Then, the MTT solution (5 mg/ml; Sigma) was added to each well and incubated for an additional 4 h. The supernatant was removed, and the crystals were dissolved in 150 ml dimethyl sulfoxide (DMSO; Sigma). The absorbance at 490 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). 2.5. Cell migration assay Cell migration was detected using Transwell migration chambers (8 mm pore size; BD Biosciences, Eugene, OR, USA). In brief, the transfected HSC-T6 cells (1 105 cells/well) were added to the upper chamber and the lower chambers were filled with DMEM containing 10% FBS. After incubating for 24 h at 37 C in the incubator supplemented with 5% CO2, non-migrating cells on the upper side of the filter were removed with a cotton swab, and cells that had migrated to the lower surface were fixed with 100% methanol and stained with 1% crystal violet in 20% methanol for 20 min. The number of cells that migrated to the bottom of the filter was counted in six random fields per well under a light microscope (100 magnification). 2.6. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from specimen tissues or HSCs using TRIzol reagents (Invitrogen), and 2 mg of total RNA were reverse transcribed by use of Superscript III (Invitrogen). Real-time PCR was performed according to the manufacturer’s instructions using SYBR1 Premix Ex TaqTM Kit (Takara, DRR041A, Japan) on the ABIPrism 7700. Primers used in real time PCR included the following: CTRP3, forward 50 -CGATTCACAGCCCCAGTCTC-30 and reverse 50 GTGCAGGCTGGCAGAAAAC-30 ; b-actin, forward 50 -GATCATTGCTCCTCCTGAGC-30 and reverse 50 -ACTCCTGCTTGCTGATCCAC-30 . The relative CTRP3 mRNA expression was calculated using the 2DDCt comparative method.
Fig. 1. CTRP3 is lowly expressed in liver fibrosis tissues and activated HSCs. A, The mRNA expression levels of CTRP3 in liver fibrotic tissues were detected using qRTPCR analysis. *P < 0.05 vs. control group. B, The mRNA expression levels of CTRP3 in quiescent and activated HSCs were detected using qRT-PCR analysis. C, The protein expression levels of CTRP3 in quiescent and activated HSCs were detected using western blotting, and GAPDH was used as a loading control. Data are representative of three independent experiments. *P < 0.05 vs. Quiescent group.
C. Cheng et al. / Biomedicine & Pharmacotherapy 89 (2017) 1387–1391
(TBS) with 0.1% Tween-20 (TBST) at room temperature for 1 h, the blots were incubated with primary antibodies against CTRP3, collagen I, a-SMA, p-Smad3, Smad3 and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (GE Healthcare, Buckinghamshire, UK). The signals were quantified by densitometry using Sion Image software (Scion Corporation, Frederick, MD, USA). 2.8. Statistics analysis Statistical analysis was done using SPSS 13.0 for windows (SPSS, Chicago, IL, USA). Data are expressed as mean standard deviation (SD). Statistical analysis involved using the Student’s t test for
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comparison of 2 groups or one-way analysis of variance (ANOVA) for multiple comparisons. P < 0.05 was considered to be statistically significant. 3. Results 3.1. CTRP3 is lowly expressed in liver fibrosis tissues and activated HSCs To investigate the role of CTRP3 in liver fibrosis, we measured the expression of CTRP3 in liver fibrosis tissues using qRT-PCR assay. The data showed that the mRNA expression levels of CTRP3 were significantly down-regulated in liver fibrosis tissues, as compared with the normal liver tissues (Fig. 1A). In addition, we examined the expression of CTRP3 in activated HSCs. As shown in Fig. 1B and C, the expression levels of CTRP3 at both mRNA and protein were obviously down-regulated in activated HSCs.
Fig. 2. Overexpression of CTRP3 inhibits the proliferation and migration of HSCs. HSC-T6 cells were transfected with CTRP3 or vector for 24 h, respectively. A, The mRNA expression level of CTRP3 was detected using qRT-PCR analysis. B, The protein expression level of CTRP3 was detected using western blotting analysis, and GAPDH was used as a loading control. HSC-T6 cells were transfected with CTRP3 or vector, and then stimulated with TGF-b1 (10 ng/ml) for 24 h. C, Cell proliferation was determined by the MTT assay. D, Cell migration was evaluated using the Transwell migration assay. Data are representative of three independent experiments. *P < 0.05 vs. control group, #P < 0.05 vs. TGF-b1 + vector group.
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3.2. Overexpression of CTRP3 inhibits the proliferation and migration of HSCs
3.4. Overexpression of CTRP3 inhibits the activation of TGF-b1/Smad pathway in HSCs
In order to investigate the effects of CTRP3 on HSC proliferation and migration, we used HSC-T6 to establish a stable cell line that constitutively overexpressed the CTRP3 protein. The results demonstrated that overexpression of CTRP3 resulted in marked increase in CTRP3 at both the mRNA (Fig. 2A) and protein (Fig. 2B) levels compared with the vector group. Then, we examined the effect of CTRP3 on HSCs proliferation. As shown in Fig. 2C, the proliferation assay demonstrated that TGF-b1 significantly promoted HSC proliferation. However, CTRP3 overexpression dramatically suppressed the proliferation of HSCs, compared with the TGF-b1 group. In addition, the results of Transwell migration assay indicated that CTRP3 overexpression markedly suppressed the number of migrated cells in TGF-b1-stimulated HSC-T6 cells (Fig. 2D).
TGF-b1/Smad signaling pathway plays a critical role in liver fibrosis. Thus, to further understand the molecular mechanisms responsible for the inhibition of HSCs activation, we detected the effect of CTRP3 on the level of p-Smad3 in HSCs using western blotting. As shown in Fig. 4, TGF-b1 treatment significantly increased the expression of phosphorylated Smad3, compared with the control group. However, CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGFb1-stimulated HSC-T6 cells.
3.3. Overexpression of CTRP3 inhibits the expression of ECM in HSCs Next, we examined the effect of CTRP3 on ECM expression in HSCs. As shown in Fig. 3, the protein expression levels of collagen I and a-SMA were significantly increased by TGF-b1, compared with the control group. However, CTRP3 overexpression dramatically suppressed the protein expression levels of collagen I and a-SMA in HSC-T6 cells, as compared with the TGF-b1 group.
Fig. 3. Overexpression of CTRP3 inhibits the expression of ECM in HSCs. HSC-T6 cells were transfected with CTRP3 or vector, and then stimulated with TGF-b1 (10 ng/ml) for 24 h. A, The protein expression levels of collagen I and a-SMA were detected using western blotting, and GAPDH was used as a loading control. B, The graph shows the quantification of the band intensity. Data are representative of three independent experiments.*P < 0.05 vs. control group, #P < 0.05 vs. TGFb1 + vector group.
4. Discussion In this study, we provided the first evidence that CTRP3 was lowly expressed in liver fibrosis tissues and activated HSCs. Overexpression of CTRP3 inhibited the proliferation and migration of HSCs, as well as suppressed the expression of ECM in TGF-b1stimulated HSC-T6 cells. Furthermore, CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGF-b1-stimulated HSC-T6 cells. CTRP3 plays an important role in regulation of fibrosis. Wu et al. reported that the expression of CTRP3 was significantly decreased after myocardial infarction [15]. Similarly, in this study, we observed that CTRP3 was lowly expressed in hepatic fibrosis tissues and activated HSCs. These data suggest that CTRP3 may play an important role in the process of liver fibrosis.
Fig. 4. Overexpression of CTRP3 inhibits the activation of TGF-b1/Smad pathway in HSCs. HSC-T6 cells were transfected with CTRP3 or vector, and then stimulated with TGF-b1 (10 ng/ml) for 1 h. A, The expression levels of p-Smad3 and Smad3 were detected using western blotting. B, The graph shows the quantification of the band intensity. Data are representative of three independent experiments. *P < 0.05 vs. control group, #P < 0.05 vs. TGF-b1 + vector group.
C. Cheng et al. / Biomedicine & Pharmacotherapy 89 (2017) 1387–1391
A number of studies have demonstrated that the proliferation and migration of HSCs are the key events in the progression of liver fibrosis [16–18]. TGF-b1 is considered to be the main stimuli factor responsible for the proliferation and migration of HSCs. In addition, it was reported that the expression level of TGF-b1 was greatly upregulated in liver fibrosis tissues, and its expression was closely related with the degree of liver fibrosis [19]. In this study, we found that TGF-b1 treatment significantly promoted HSC proliferation and migration. However, CTRP3 overexpression dramatically suppressed TGF-b1-induced HSC proliferation and migration. These data suggest that CTRP3 attenuated liver fibrosis through inhibition of TGF-b1-induced HSC proliferation and migration. Liver fibrosis is characterized by the excessive synthesis of ECM proteins. The expression of a-SMA in the liver tissues is an indicator of HSCs activation, which is recognized as being critical in liver fibrosis [20]. In addition, TGF-b1 can induce the activation of HSCs to produce a large amount of ECM components, including collagen I and a-SMA [21,22]. In line with the results of previous studies, herein, we observed that the protein expression levels of collagen I and a-SMA were significantly increased by TGF-b1. However, CTRP3 overexpression dramatically suppressed the protein expression levels of collagen I and a-SMA in TGF-b1stimualted HSCs. These data suggest that CTRP3 can protect against liver fibrosis through inhibition of ECM production in HSC-T6 cells. TGF-b1/Smad signaling pathway plays a critical role in the development of liver fibrosis [23–25]. During liver fibrosis, TGF-b1 exerts its biological functions through interaction with TGF-b1 receptors. Then, Smad 2/3 is phosphorylated and binds with Smad4 to form multimers, and the Smad complex translocates into the nucleus and regulates the expression of target genes, including type I collagen and a-SMA [26]. Smad3 is pathogenic because mice null for Smad3 are protected against dimethylnitrosamineinduced liver fibrosis [27]. In addition, it has been reported that deletion of Smad3 significantly suppressed type I collagen expression and blocked epithelial-myofibroblast transition [28]. The results of this study showed that CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGFb1-stimulated HSC-T6 cells. These results suggest that CTRP3 attenuated liver fibrosis, at least in part, through inhibiting the TGF-b1/Smad signaling pathway. In summary, the present study demonstrated that CTRP3 inhibited the proliferation and migration of TGF-b1-induced HSCT6 cells and attenuated liver fibrosis, at least in part, through inhibiting the Smad signaling pathway. These findings suggest that CTRP3 may be a promising therapeutic target for the treatment of liver fibrosis. References [1] R. Bataller, D.A. Brenner, Liver fibrosis, J. Clin. Invest. 115 (2005) 209–218. [2] S. Aleksic’Kova9 cevic’, V. Kukolj, B. Kureljušic’, et al., Role of hepatic stellate cells (HSCs) in the development of hepatic fibrosis in cats with polycystic kidney disease (PKD), Acta Vet. 60 (2010) 391–400. [3] X. Lin, L.N. Kong, C. Huang, et al., Hesperetin derivative-7 inhibits PDGF-BBinduced hepatic stellate cell activation and proliferation by targeting Wnt/ b-catenin pathway, Int. Immunopharmacol. 25 (2015) 311–320. [4] H. Yong, H. Cheng, S. Xu, et al., MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4, Cell Signal. 24 (2012) 1923–1930.
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[5] T. Knittel, L. Müller, B. Saile, G. Ramadori, Effect of tumour necrosis factoralpha on proliferation, activation and protein synthesis of rat hepatic stellate cells, J. Hepatol. 27 (1997) 1067–1080. [6] L.D. Presser, S. Mcrae, G. Waris, Activation of TGF-b1 promoter by hepatitis C virus-Induced AP-1 and sp1: role of TGF-b1 in hepatic stellate cell activation and invasion, PLoS One 8 (2013) e56367. [7] G.W. Wong, S.A. Krawczyk, C. Kitidismitrokostas, et al., Molecular, biochemical and functional characterizations of C1q/TNF family members: adipose-tissueselective expression patterns, regulation by PPAR-g agonist, cysteinemediated oligomerizations, combinatorial associations and metabolic functions, Biochem. J. 416 (2008) 161–177. [8] T. Maeda, M. Abe, K. Kurisu, A. Jikko, S. Furukawa, Molecular cloning and characterization of a novel gene, CORS26, encoding a putative secretory protein and its possible involvement in skeletal development, J. Biol. Chem. 276 (2001) 3628–3634. [9] Q. Hou, J. Lin, W. Huang, et al., CTRP3 stimulates proliferation and antiapoptosis of prostate cells through PKC signaling pathways, PLoS One 10 (2015) e0134006. [10] P.S. Petersen, R.M. Wolf, L. Xia, J.M. Peterson, G.W. Wong, Immunomodulatory roles of CTRP3 in endotoxemia and metabolic stress, Physiol. Rep. 4 (2016) 2016. [11] M.A. Murayama, S. Kakuta, T. Maruhashi, et al., CTRP3 plays an important role in the development of collagen-induced arthritis in mice, Biochem. Biophys. Res. Commun. 443 (2014) 42–48. [12] R.M. Wolf, X. Lei, Z.C. Yang, et al., CTRP3 deficiency reduces liver size and alters IL-6 and TGF-b levels in obese mice, Am. J. Physiol. Endocrinol. Metab. 310 (2016) E322–E345. [13] X. Lei, Q. Li, S. Rodriguez, et al., Thromboxane synthase deficiency improves insulin action and attenuates adipose tissue fibrosis, Am. J. Physiol. Endocrinol. Metab. 308 (2015) E792–804. [14] D. Wu, H. Lei, J.Y. Wang, et al., CTRP3 attenuates post-infarct cardiac fibrosis by targeting Smad3 activation and inhibiting myofibroblast differentiation, J. Mol. Med. 93 (2015) 1311–1325. [15] D. Wu, H. Lei, J.Y. Wang, et al., CTRP3 attenuates post-infarct cardiac fibrosis by targeting Smad3 activation and inhibiting myofibroblast differentiation, J. Mol. Med. 93 (2015) 1311–1325. [16] F. Qi, J.F. Hu, B.H. Liu, et al., Mi R-9a-5p regulates proliferation and migration of hepatic stellate cells under pressure through inhibition of Sirt1, World J. Gastroenterol. 21 (2015) 9900–9915. [17] R.F. Schwabe, R. Bataller, D.A. Brenner, Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (2003) G949–958. [18] F.P. Wang, L. Li, J. Li, et al., High mobility group box-1 promotes the proliferation and migration of hepatic stellate cells via TLR4-dependent signal pathways of PI3K/Akt and JNK, PLoS One 8 (2013) e64373. [19] F.B. Li, H. Zhao, K.R. Peng, et al., Expression of transforming growth factor-b1 and connective tissue growth factor in congenital biliary atresia and neonatal hepatitis liver tissue, Genet. Mol. Res. 15 (2016) 15017217. [20] Y. Liu, J. Brymora, H. Zhang, et al., Leptin and acetaldehyde synergistically promotes aSMA expression in hepatic stellate cells by an interleukin 6dependent mechanism, Alcohol. Clin. Exp. Res. 35 (2011) 921–928. [21] C. Balta, H. Herman, O.M. Boldura, et al., Chrysin attenuates liver fibrosis and hepatic stellate cell activation through TGF-b/Smad signaling pathway, Chem. Biol. Interact. 240 (2015) 94–101. [22] E. Wiercinska, L. Wickert, B. Denecke, et al., Id1 is a critical mediator in TGFb–induced transdifferentiation of rat hepatic stellate cells, Hepatology 43 (2006) 1032–1041. [23] Y. Qu, L. Zong, M. Xu, Y. Dong, L. Lu, Effects of 18a-glycyrrhizin on TGF-b1/ Smad signaling pathway in rats with carbon tetrachloride-induced liver fibrosis, Int. J. Clin. Exp. Pathol. 8 (2014) 1292–1301. [24] W.L. Xie, R. Jiang, X.L. Shen, Z.Y. Chen, X.M. Deng, Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-b/Smad signaling pathway, Int. J. Clin. Exp. Med. 8 (2015) 20323–20329. [25] L. Fang, C. Huang, X. Meng, et al., TGF-b1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-b1/Smad pathway, Toxicol. Appl. Pharmacol. 280 (2014) 335–344. [26] K. Yoshida, K. Matsuzaki, Differential regulation of TGF-b/Smad signaling in hepatic stellate cells between acute and chronic liver injuries, Front. Physiol. 3 (2012) 53–59. [27] G. Latella, A. Vetuschi, R. Sferra, et al., Targeted disruption of Smad3 confers resistance to the development of dimethylnitrosamine-induced hepatic fibrosis in mice, Liver Int. 29 (2009) 997–1009. [28] F. Xu, C. Liu, D. Zhou, L. Zhang, TGF-b/SMAD pathway and its regulation in hepatic fibrosis, J. Histochem. Cytochem. 64 (2016) 157–167.
Available online at
ScienceDirect www.sciencedirect.com
Original research
CTRP3 attenuates hepatic stellate cell activation through transforming growth factor-b/Smad signaling pathway Chuantao Chenga,1, Shuo Yua,1, Ran Kongb , Qinggong Yuana , Yuefeng Mab , Wenbin Yanga , Gang Caoa,* , Liyi Xiec,* a b c
Department of General Surgery, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China Department of Thoracic Surgery, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China Department of Nephrology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China
A R T I C L E I N F O
Article history: Received 22 November 2016 Received in revised form 7 March 2017 Accepted 7 March 2017 Keywords: Liver fibrosis Hepatic stellate cells (HSCs) C1q/tumor necrosis factor-related protein 3 Extracellular matrix (ECM)
A B S T R A C T
Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of liver fibrosis. C1q/ tumor necrosis factor-related protein 3 (CTRP3), a member of CTRPs, was involved in fibrosis. However, little is known about the role of CTRP3 in liver fibrosis. This study aimed to determine its role in liver fibrosis and explore the possible mechanism. Our results demonstrated that CTRP3 was lowly expressed in liver fibrosis tissues and activated HSCs. Overexpression of CTRP3 inhibited the proliferation and migration of HSCs, as well as suppressed the expression of extracellular matrix (ECM) in transforming growth factor-b1 (TGF-b1)-stimulated HSC-T6 cells. Furthermore, CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGF-b1-stimulated HSC-T6 cells. In conclusion, the present study demonstrated that CTRP3 inhibited the proliferation and migration of TGFb1-induced HSC-T6 cells and attenuated liver fibrosis, at least in part, through inhibiting the Smad signaling pathway. These findings suggest that CTRP3 may be a promising therapeutic target for the treatment of liver fibrosis. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Liver fibrosis, a major cause of morbidity and mortality from hepatic diseases, is characterized by the excessive synthesis of extracellular matrix (ECM) proteins and eventually the development of hepatic cirrhosis [1]. Hepatic stellate cells (HSCs) are the major mesenchymal cells in liver, and the activation of HSCs plays a critical role in the fibrogenesis [2]. Previous studies demonstrated that HSC activation can be induced by a variety of factors, including transforming growth factor-b1 (TGF-b1) [3–5]. TGF-b1 generally induces HSC proliferation and ECM synthesis in vitro and liver fibrogenesis in vivo [6]. Thus, inhibition of HSC activation is of crucial importance for the treatment of liver fibrosis. C1q/tumor necrosis factor-related proteins (CTRPs) play important and distinct roles in regulating insulin sensitivity and energy balance [7]. CTRP3, a member of CTRPs, is identified as a novel adipokine [8]. Previous studies have shown that CTRP3 was
* Corresponding authors. E-mail addresses: [email protected] (G. Cao), [email protected] (L. Xie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.03.021 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
involved in multiple cellular processes, such as cell proliferation, apoptosis, immune-regulation, inflammation, glucose metabolism and lipid metabolism [9–12]. In addition, several studies suggest a role of CTRP3 in fibrosis disease [13,14]. It has been reported that the expression of CTRP3 was significantly decreased after myocardial infarction, and CTRP3 attenuated the proliferation, migration, and the expression of connective tissue growth factor, collagen I, and collagen III induced by TGF-b1 in cultured adult rat cardiac fibroblasts [14]. However, little is known about the role of CTRP3 in liver fibrosis. This study aimed to determine its role in liver fibrosis and explore the possible mechanism. Our data showed that CTRP3 attenuated liver fibrosis through inhibiting the TGF-b1/Smad signaling pathway. 2. Materials and methods 2.1. Specimen collection Liver biopsies were collected by transparietal puncture from 11 healthy individuals and 11 patients with liver fibrosis. All of the samples were stored at 80 C. For all of the patients who
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participated in this study, written informed consent was obtained. This study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University (China). 2.2. Cell culture The human HSC cell line (HSC-T6) was purchased from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) in the presence of penicillin/streptomycin (Sigma, St. Louis, MO, USA) at 37 C in a humidified 5% CO2 atmosphere.
2.7. Western blotting Total protein was extracted from the treated cells using RIPA lysis buffer (Beyotime, Nantong, China) according to the manufacturer’s instructions. Equal amounts of protein (30 mg) were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride membranes (Whatman Schleicher & Schuell, Middlesex, UK). After blocking with 5% non-fat milk in Tris-buffered saline
2.3. Construction of plasmids and cell transfection The full-length CTRP3 cDNA was cloned into the pcDNA3.1 vector (Genechem, Shanghai, China). HSC-T6 cells were transfected with CTRP3 or vector using LipofectamineTM2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. The transfection efficiency of CTRP3 was confirmed by quantitative real-time PCR and western blotting analysis. 2.4. Cell proliferation assay Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. In brief, the transfected HSC-T6 cells (1 105 cells/well) were seeded onto 96well plates for 24 h. Then, the MTT solution (5 mg/ml; Sigma) was added to each well and incubated for an additional 4 h. The supernatant was removed, and the crystals were dissolved in 150 ml dimethyl sulfoxide (DMSO; Sigma). The absorbance at 490 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). 2.5. Cell migration assay Cell migration was detected using Transwell migration chambers (8 mm pore size; BD Biosciences, Eugene, OR, USA). In brief, the transfected HSC-T6 cells (1 105 cells/well) were added to the upper chamber and the lower chambers were filled with DMEM containing 10% FBS. After incubating for 24 h at 37 C in the incubator supplemented with 5% CO2, non-migrating cells on the upper side of the filter were removed with a cotton swab, and cells that had migrated to the lower surface were fixed with 100% methanol and stained with 1% crystal violet in 20% methanol for 20 min. The number of cells that migrated to the bottom of the filter was counted in six random fields per well under a light microscope (100 magnification). 2.6. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from specimen tissues or HSCs using TRIzol reagents (Invitrogen), and 2 mg of total RNA were reverse transcribed by use of Superscript III (Invitrogen). Real-time PCR was performed according to the manufacturer’s instructions using SYBR1 Premix Ex TaqTM Kit (Takara, DRR041A, Japan) on the ABIPrism 7700. Primers used in real time PCR included the following: CTRP3, forward 50 -CGATTCACAGCCCCAGTCTC-30 and reverse 50 GTGCAGGCTGGCAGAAAAC-30 ; b-actin, forward 50 -GATCATTGCTCCTCCTGAGC-30 and reverse 50 -ACTCCTGCTTGCTGATCCAC-30 . The relative CTRP3 mRNA expression was calculated using the 2DDCt comparative method.
Fig. 1. CTRP3 is lowly expressed in liver fibrosis tissues and activated HSCs. A, The mRNA expression levels of CTRP3 in liver fibrotic tissues were detected using qRTPCR analysis. *P < 0.05 vs. control group. B, The mRNA expression levels of CTRP3 in quiescent and activated HSCs were detected using qRT-PCR analysis. C, The protein expression levels of CTRP3 in quiescent and activated HSCs were detected using western blotting, and GAPDH was used as a loading control. Data are representative of three independent experiments. *P < 0.05 vs. Quiescent group.
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(TBS) with 0.1% Tween-20 (TBST) at room temperature for 1 h, the blots were incubated with primary antibodies against CTRP3, collagen I, a-SMA, p-Smad3, Smad3 and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (GE Healthcare, Buckinghamshire, UK). The signals were quantified by densitometry using Sion Image software (Scion Corporation, Frederick, MD, USA). 2.8. Statistics analysis Statistical analysis was done using SPSS 13.0 for windows (SPSS, Chicago, IL, USA). Data are expressed as mean standard deviation (SD). Statistical analysis involved using the Student’s t test for
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comparison of 2 groups or one-way analysis of variance (ANOVA) for multiple comparisons. P < 0.05 was considered to be statistically significant. 3. Results 3.1. CTRP3 is lowly expressed in liver fibrosis tissues and activated HSCs To investigate the role of CTRP3 in liver fibrosis, we measured the expression of CTRP3 in liver fibrosis tissues using qRT-PCR assay. The data showed that the mRNA expression levels of CTRP3 were significantly down-regulated in liver fibrosis tissues, as compared with the normal liver tissues (Fig. 1A). In addition, we examined the expression of CTRP3 in activated HSCs. As shown in Fig. 1B and C, the expression levels of CTRP3 at both mRNA and protein were obviously down-regulated in activated HSCs.
Fig. 2. Overexpression of CTRP3 inhibits the proliferation and migration of HSCs. HSC-T6 cells were transfected with CTRP3 or vector for 24 h, respectively. A, The mRNA expression level of CTRP3 was detected using qRT-PCR analysis. B, The protein expression level of CTRP3 was detected using western blotting analysis, and GAPDH was used as a loading control. HSC-T6 cells were transfected with CTRP3 or vector, and then stimulated with TGF-b1 (10 ng/ml) for 24 h. C, Cell proliferation was determined by the MTT assay. D, Cell migration was evaluated using the Transwell migration assay. Data are representative of three independent experiments. *P < 0.05 vs. control group, #P < 0.05 vs. TGF-b1 + vector group.
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3.2. Overexpression of CTRP3 inhibits the proliferation and migration of HSCs
3.4. Overexpression of CTRP3 inhibits the activation of TGF-b1/Smad pathway in HSCs
In order to investigate the effects of CTRP3 on HSC proliferation and migration, we used HSC-T6 to establish a stable cell line that constitutively overexpressed the CTRP3 protein. The results demonstrated that overexpression of CTRP3 resulted in marked increase in CTRP3 at both the mRNA (Fig. 2A) and protein (Fig. 2B) levels compared with the vector group. Then, we examined the effect of CTRP3 on HSCs proliferation. As shown in Fig. 2C, the proliferation assay demonstrated that TGF-b1 significantly promoted HSC proliferation. However, CTRP3 overexpression dramatically suppressed the proliferation of HSCs, compared with the TGF-b1 group. In addition, the results of Transwell migration assay indicated that CTRP3 overexpression markedly suppressed the number of migrated cells in TGF-b1-stimulated HSC-T6 cells (Fig. 2D).
TGF-b1/Smad signaling pathway plays a critical role in liver fibrosis. Thus, to further understand the molecular mechanisms responsible for the inhibition of HSCs activation, we detected the effect of CTRP3 on the level of p-Smad3 in HSCs using western blotting. As shown in Fig. 4, TGF-b1 treatment significantly increased the expression of phosphorylated Smad3, compared with the control group. However, CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGFb1-stimulated HSC-T6 cells.
3.3. Overexpression of CTRP3 inhibits the expression of ECM in HSCs Next, we examined the effect of CTRP3 on ECM expression in HSCs. As shown in Fig. 3, the protein expression levels of collagen I and a-SMA were significantly increased by TGF-b1, compared with the control group. However, CTRP3 overexpression dramatically suppressed the protein expression levels of collagen I and a-SMA in HSC-T6 cells, as compared with the TGF-b1 group.
Fig. 3. Overexpression of CTRP3 inhibits the expression of ECM in HSCs. HSC-T6 cells were transfected with CTRP3 or vector, and then stimulated with TGF-b1 (10 ng/ml) for 24 h. A, The protein expression levels of collagen I and a-SMA were detected using western blotting, and GAPDH was used as a loading control. B, The graph shows the quantification of the band intensity. Data are representative of three independent experiments.*P < 0.05 vs. control group, #P < 0.05 vs. TGFb1 + vector group.
4. Discussion In this study, we provided the first evidence that CTRP3 was lowly expressed in liver fibrosis tissues and activated HSCs. Overexpression of CTRP3 inhibited the proliferation and migration of HSCs, as well as suppressed the expression of ECM in TGF-b1stimulated HSC-T6 cells. Furthermore, CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGF-b1-stimulated HSC-T6 cells. CTRP3 plays an important role in regulation of fibrosis. Wu et al. reported that the expression of CTRP3 was significantly decreased after myocardial infarction [15]. Similarly, in this study, we observed that CTRP3 was lowly expressed in hepatic fibrosis tissues and activated HSCs. These data suggest that CTRP3 may play an important role in the process of liver fibrosis.
Fig. 4. Overexpression of CTRP3 inhibits the activation of TGF-b1/Smad pathway in HSCs. HSC-T6 cells were transfected with CTRP3 or vector, and then stimulated with TGF-b1 (10 ng/ml) for 1 h. A, The expression levels of p-Smad3 and Smad3 were detected using western blotting. B, The graph shows the quantification of the band intensity. Data are representative of three independent experiments. *P < 0.05 vs. control group, #P < 0.05 vs. TGF-b1 + vector group.
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A number of studies have demonstrated that the proliferation and migration of HSCs are the key events in the progression of liver fibrosis [16–18]. TGF-b1 is considered to be the main stimuli factor responsible for the proliferation and migration of HSCs. In addition, it was reported that the expression level of TGF-b1 was greatly upregulated in liver fibrosis tissues, and its expression was closely related with the degree of liver fibrosis [19]. In this study, we found that TGF-b1 treatment significantly promoted HSC proliferation and migration. However, CTRP3 overexpression dramatically suppressed TGF-b1-induced HSC proliferation and migration. These data suggest that CTRP3 attenuated liver fibrosis through inhibition of TGF-b1-induced HSC proliferation and migration. Liver fibrosis is characterized by the excessive synthesis of ECM proteins. The expression of a-SMA in the liver tissues is an indicator of HSCs activation, which is recognized as being critical in liver fibrosis [20]. In addition, TGF-b1 can induce the activation of HSCs to produce a large amount of ECM components, including collagen I and a-SMA [21,22]. In line with the results of previous studies, herein, we observed that the protein expression levels of collagen I and a-SMA were significantly increased by TGF-b1. However, CTRP3 overexpression dramatically suppressed the protein expression levels of collagen I and a-SMA in TGF-b1stimualted HSCs. These data suggest that CTRP3 can protect against liver fibrosis through inhibition of ECM production in HSC-T6 cells. TGF-b1/Smad signaling pathway plays a critical role in the development of liver fibrosis [23–25]. During liver fibrosis, TGF-b1 exerts its biological functions through interaction with TGF-b1 receptors. Then, Smad 2/3 is phosphorylated and binds with Smad4 to form multimers, and the Smad complex translocates into the nucleus and regulates the expression of target genes, including type I collagen and a-SMA [26]. Smad3 is pathogenic because mice null for Smad3 are protected against dimethylnitrosamineinduced liver fibrosis [27]. In addition, it has been reported that deletion of Smad3 significantly suppressed type I collagen expression and blocked epithelial-myofibroblast transition [28]. The results of this study showed that CTRP3 overexpression greatly inhibited the expression level of phosphorylation of Smad3 in TGFb1-stimulated HSC-T6 cells. These results suggest that CTRP3 attenuated liver fibrosis, at least in part, through inhibiting the TGF-b1/Smad signaling pathway. In summary, the present study demonstrated that CTRP3 inhibited the proliferation and migration of TGF-b1-induced HSCT6 cells and attenuated liver fibrosis, at least in part, through inhibiting the Smad signaling pathway. These findings suggest that CTRP3 may be a promising therapeutic target for the treatment of liver fibrosis. References [1] R. Bataller, D.A. Brenner, Liver fibrosis, J. Clin. Invest. 115 (2005) 209–218. [2] S. Aleksic’Kova9 cevic’, V. Kukolj, B. Kureljušic’, et al., Role of hepatic stellate cells (HSCs) in the development of hepatic fibrosis in cats with polycystic kidney disease (PKD), Acta Vet. 60 (2010) 391–400. [3] X. Lin, L.N. Kong, C. Huang, et al., Hesperetin derivative-7 inhibits PDGF-BBinduced hepatic stellate cell activation and proliferation by targeting Wnt/ b-catenin pathway, Int. Immunopharmacol. 25 (2015) 311–320. [4] H. Yong, H. Cheng, S. Xu, et al., MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4, Cell Signal. 24 (2012) 1923–1930.
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