Original Paper Received: November 7, 2013 Accepted after revision: February 14, 2014 Published online: May 20, 2014
Pharmacology 2014;93:185–192 DOI: 10.1159/000360638
Transforming Growth Factor-Beta Inhibits Heme Oxygenase-1 Expression in Lung Fibroblasts through Nuclear Factor-Kappa-B-Dependent Pathway Ying Xie a Ye Wang b Chuanyue Zong a Jiawen Cheng a, c
a
Department of Anesthesiology, Huai’an Hospital Affiliated with Xuzhou Medical College and Huai’an Second People’s Hospital, Huaian, b Department of Anesthesiology, Wuxi Ninth People’s Hospital, Wuxi, and c Department of anesthesiology, The First People’s Hospital of Suqian, Suqian, China
Abstract Background: Heme oxygenase-1 (HO-1) contributes to the pathogenesis of pulmonary fibrosis. However, the expression of HO-1 in fibroblasts under fibrotic conditions has not been studied. Methods: This study was conducted to investigate the expression of HO-1 in lung fibroblasts from mice and humans under fibrotic conditions by Western blot. Results: We found that the expression of HO-1 was significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice in comparison with control mice. Transforming growth factor-β (TGF-β) inhibited HO-1 expression and induced differentiation in human lung fibroblasts. Pretreatment with nuclear factor-κB (NF-κB) activation inhibitor or knockdown of the NF-κB p65 subunit attenuated TGF-βinduced inhibition of HO-1 expression and differentiation in human lung fibroblasts. Similarly, lysophosphatidic acid (LPA) induced TGF-β expression and decreased HO-1 expression in human lung fibroblasts. Interestingly, pretreatment with neutralized anti-TGF-β antibody attenuated LPA effects in human lung fibroblasts. Conclusion: These data suggested that TGF-β inhibited HO-1 expression in human lung fibroblasts through activation of NF-κB. © 2014 S. Karger AG, Basel
Introduction
Pulmonary fibrosis is a chronic and progressive interstitial lung disease [1, 2]. Alveolar epithelial cell injury, accumulation and differentiation of fibroblasts [3, 4], and deposition of extracellular matrix proteins contribute to the progress of pulmonary fibrosis [5, 6]. Currently, lung transplantation is the only effective cure for pulmonary fibrosis [7], thus investigations of the pathological mechanisms and efficacious therapeutic approaches for pulmonary fibrosis are extremely urgent. Since fibroblast accumulation leads to an excessive scarring of lung tissue and to progressive and irreversible destruction of lung architecture [4], investigations of fibroblast functions are critical for understanding the molecular mechanisms of pulmonary fibrosis. Investigations demonstrated that transforming growth factor-β (TGF-β) and lysophosphatidic acid (LPA) are extensively involved in the fibrogenesis of different organs, such as heart, liver and lung [4, 5, 8, 9]. TGF-β induces lung fibroblast differentiation through both Smad-dependent and independent pathways, and followed by increased expression of α-smooth muscle actin (α-SMA) and extracellular matrix proteins [2]. LPA also induces fibroblast recruitment, migration and differentiation via activation of LPA receptors [5, 8]. Interestingly, a recent report demonstrated that LPA also induces TGF-β expression in lung fibroYing Xie and Ye Wang contributed equally to this work.
© 2014 S. Karger AG, Basel 0031–7012/14/0934–0185$39.50/0 E-Mail [email protected] www.karger.com/pha
Jiawen Cheng, MD Department of Anesthesiology The First People’s Hospital Of Suqian 120 Suzhi Road, Suqian, Jiangshu 223800 (China) E-Mail chengjiawen2013 @ 163.com
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Key Words Heme oxygenase-1 · Lysophosphatidic acid · Transforming growth factor-β · Nuclear factor-κB · Pulmonary fibrosis
Materials and Methods Reagents and Kits Bleomycin sulfate was from Hospira Inc. (Lakeforest, Ill., USA), and neutralizing chicken anti-TGF-β1 antibody and control chicken IgG were obtained from R&D Systems (Minneapolis, Minn., USA). Oleoyl lysophosphatidic acid (18:1 LPA) was obtained from Avanti Polar Lipids (Alabaster, Ala., USA), and cell lysis buffer was purchased from Cell Signaling Technology Inc. (Danvers, Mass., USA). Recombinant human TGF-β1 was obtained from Prepro Tech Inc. (Rocky Hill, N.J., USA). Horseradish peroxidase-linked anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories Inc. (Hercules, Calif., USA). Rabbit anti-fibronection (FN), anti-HO-1 and anti-p65 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif., USA). Mouse anti-α-SMA and anti-β-actin antibodies and Bay 11-7082 (NF-κB inhibitor) were from Sigma-Aldrich (St. Louis, Mo., USA).
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Experimental Pulmonary Fibrosis Model The animal experiment of pulmonary fibrosis was designed as described before [5, 6]. Briefly, C57/BL6 mice (male, age 8 weeks) purchased from Jackson Laboratory (Bar Harbor, Me., USA) were used for bleomycin-induced fibrosis. C57/BL6 mice were anesthetized (with a 3 ml/kg mixture of 25 mg/kg of ketamine in 2.5 ml of xylazine), followed by treatment with saline or bleomycin sulfate (1.5 U/kg of body weight, ∼0.03 U/animal) in saline by intratracheal injection in a total volume of 50 μl. Twenty-one days after bleomycin administration, animals were killed, and lungs were removed for isolation of lung fibroblasts. All animal protocols conformed to the standards of Xuzhou Medical College and were in accordance with Chinese animal operation regulations. Isolation of Mouse Primary Fibroblasts and Cell Culture Mouse lung fibroblasts were isolated from mice with or without bleomycin challenge as described previously [5, 22]. Human lung fibroblast cell line (WI-38) was ordered from ATCC. Mouse primary lung fibroblasts and human lung fibroblasts were grown and maintained in 6-well dishes with DMEM medium containing 10% fetal bovine serum. siRNA Transfection Scrambled RNA and targeting human NF-κB p65 subunit smart pool (si-p65) were from Dharmacom Inc. (Lafayette, Colo., USA). Briefly, WI-38 cells cultured on 6-well plates (50–60% confluence) were transiently transfected with scrambled RNA and sip65 (200 nmol/l) according to manufacturer’s instructions (Qiagen, Md., USA). After dilution with 900 μl of basal DMEM medium, transfection complex was directly added to the cells. Six hours after treatment, transfection medium was replaced by complete DMEM medium (with 10% fetal bovine serum), and cells were analyzed (48 h after treatment) by Western blot. Treatment of Neutralizing Antibodies or NF-κB Inhibitor Serum-starved (for 24 h) human lung fibroblasts (WI-38, ∼90% confluence) were pretreated with neutralized anti-TGF-β antibody or control IgG antibody (5 μg/ml, 1 h). In case of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor (Bay 11-7082), the compound was pretreated with a final concentration of 10 μmol/l for 1 h [23]. Next, cells were challenged with 18:1 LPA (10 μmol/l) or TGF-β (5 ng/ml) for 48 h, and cell lysates (20 μg protein) were subjected to SDS-PAGE and Western blot. SDS-PAGE and Western Blot SDS-PAGE and Western blot were performed as described previously [5]. Blots were developed using the ECL chemiluminescence kit, and integrated density of pixels in each membrane was quantified and normalized to actin by using Image Quant 5.2 software (Molecular Dynamics, Sunnyvale, Calif., USA). Immunofluorescence Staining Immunofluorescence microscopy to determine protein expression was performed as described [5]. Briefly, primary mouse lung fibroblasts were grown in slide chambers for 24 h. Cells were fixed, incubated with primary antibodies (1: 200 dilutions in blocking buffer) for 1 h and with Alexa Fluor secondary antibodies (1:200 dilutions in blocking buffer) for another 1 h, followed
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blasts through activation of LPA receptor type 2 [5]. However, the crosstalk between TGF-β and LPA pathways in fibroblast differentiation is still elusive. Heme oxygenase-1 (HO-1) is an inducible stress protein, belongs to the heat shock protein family [10], and induces the degradation of heme to biliverdin and the generation of carbon monoxide [11]. Its expression has been proved to accompany the pathogenesis of various lung injury-induced diseases, including asthma [12], acute lung injury [13, 14], cystic fibrosis [11, 15] and lung transplant rejection [16]. Previous investigations indicated that endogenous HO-1 is involved in the resolving of inflammatory reactions and migration of neutrophil cells in experimental models of acute lung injury [13, 17]. Recent investigations also suggested that HO-1 plays a protecting role during pulmonary fibrogenesis [11, 18–20]. Jin et al. [21] indicated that bleomycin treatment increased the HO-1 expression in mouse lung tissue. Immunohistochemical staining of lung samples from patients with pulmonary fibrosis and mice treated with bleomycin indicated that HO-1 was strongly expressed in macrophages, respiratory epithelial cells and endothelial cells [11, 20, 21], while the expression of HO-1 in lung fibroblasts during fibrogenesis has not been investigated. Clinical investigations suggested that HO-1 deficiency correlates with increased fibrosis [10, 15], and up-regulation of HO-1 expression protects against renal injury and fibrosis through antiapoptotic pathway modulation [11]. However, the expression of HO-1 in pulmonary fibroblasts during fibrogenesis is still elusive, and the potential mechanism is not clear. Here, we found that HO-1 expression was dramatically decreased in lung fibroblasts from humans and mice under fibrotic conditions, and the expression of HO-1 was regulated by activation of nuclear factor-κB (NF-κB).
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by mounting; then they were examined under a Nikon Eclipse TE 2000-S fluorescence microscope with a ×60 oil immersion objective lens. Statistical Analysis All data are expressed as means ± SEMs from at least three independent sets of experiments, and results were subjected to statistical analysis using one-way ANOVA or a two-tailed Student t test. Values of p < 0.05 were considered significant.
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cells from control mice. e Immunofluorescence staining of HO-1 and FN in lung fibroblasts from mice with or without BLM challenge. Images were examined by immunofluorescence microscopy and recorded by using a ×60 oil objective. The FN (red) and HO-1 (green) images showed matched cell fields for each condition.
Results
Expression of HO-1 in Lung Fibroblasts from Mice with or without Bleomycin Challenge To study the expression of HO-1 in fibroblasts during fibrogenesis, lung fibroblasts were isolated from C57/BL6 mice with or without bleomycin challenge. As shown in figure 1a–c, the expression of α-SMA and fibronectin (FN), Pharmacology 2014;93:185–192 DOI: 10.1159/000360638
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Fig. 1. Expression of α-SMA, FN and HO-1 in lung fibroblasts from mice with or without bleomycin (BLM) challenge. Representative Western blot (a) and quantification of FN (b), α-SMA (c) and HO-1 (d) expression in mouse lung fibroblasts. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs.
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biomarkers of fibroblast differentiation, was dramatically increased in lung fibroblasts isolated from bleomycin-challenged mice compared with that from control mice; however, the expression of HO-1 was significantly decreased (fig. 1a, d) and immunofluorescence staining also indicated that the expression of HO-1 was significantly decreased in lung fibroblasts from bleomycin-challenged mice when compared with that from control mice (fig. 1e). HO-1 Is Down-Regulated by TGF-β in Human Lung Fibroblasts To check whether the expression of HO-1 is related to lung fibroblast activation and differentiation, we checked the expression of HO-1 in TGF-β-induced human lung fibroblast differentiation. Western blot analysis revealed that TGF-β challenge (5 ng/ml, 48 h) dramatically increased fibroblast differentiation, characterized as the expression of α-SMA and FN (fig. 2a–c), while it significantly inhibited the expression of HO-1 in lung fibroblasts (fig. 2a, d). TGF-β Inhibits HO-1 Expression through NF-κB To further examine the molecular mechanisms of TGF-β-induced inhibition of HO-1 expression in lung fibroblasts, we pretreated human lung fibroblast cells with NF-κB inhibitor (10 μmol/l) for 1 h before TGF-β challenge. As shown in figure 3, TGF-β up-regulated α-SMA and FN expression, and pretreatment with NFκB inhibitor dramatically blocked TGF-β effects. Inter188
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3 2 1 0
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estingly, the inhibition effect of TGF-β on HO-1 expression in lung fibroblasts was also significantly suppressed (fig. 3a, d). Additionally, knockdown of the expression of the NF-κB p65 subunit by si-p65 dramatically attenuated the expression of p65 in human lung fibroblasts (fig. 4a, b), and it also inhibited TGF-β-induced differentiation of lung fibroblasts (fig. 4a, c, d). Similarly, knockdown of the expression of the p65 subunit also increased HO-1 expression under TGF-β challenge (fig. 4a, e). Together, these data indicated that TGF-β inhibited HO-1 expression through activation of NF-κB signaling pathways. LPA-Induced Differentiation of Fibroblasts and Suppression of HO-1 Expression Is Attenuated by Anti-TGF-β Antibody In our studies, we proved that LPA induces TGF-β expression in WI-38 cells in a dose-dependent manner (fig. 5a, b). Next, we turned to check LPA effects on HO-1 expression in lung fibroblasts. LPA challenge also induced expression of α-SMA and FN, and inhibited HO-1 expression in human lung fibroblasts (fig. 5c–f). Additionally, to block the effect of secreted TGF-β under LPA challenge, cells were pretreated with an anti-TGF-β antibody. As shown in figure 5g–j, pretreatment with antiTGF-β1 antibody dramatically inhibited LPA-induced differentiation of human lung fibroblasts, and it significantly increased HO-1 expression under LPA treatment (fig. 5g–j). These results suggest that LPA also suppressed Xie/Wang/Zong/Cheng
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FN, α-SMA and HO-1 in human lung fibroblasts. Human lung fibroblasts challenged with TGF-β (5 ng/ml) were applied for Western blot as described in ‘Materials and Methods’. Representative Western blot (a) and quantification of the expression of FN (b), α-SMA (c) and HO-1 (d) in human lung fibroblasts with or without TGF-β challenge. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs. cells without TGF-β challenge.
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NF-κB p65 subunit attenuates TGF-βinduced inhibition of HO-1 expression in human lung fibroblasts. WI-38 cells with transient transfection of scrambled RNA and p65 siRNA (200 nmol/l) according to ‘Materials and Methods’ followed by TGF-β (5 ng/ml, 48 h) challenge. Representative Western blot (a) and quantification of the expression of p65 (b), FN (c), α-SMA (d) and HO-1 (e) in human lung fibroblasts. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs. cells without si-p65 and TGF-β challenge; # p < 0.05 vs. cells without si-p65 treatment but with TGF-β challenge.
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induced inhibition of HO-1 expression in human lung fibroblasts. Human lung fibroblasts with pretreatment of NF-κB inhibitor (Bay 11-7082, 10 μmol/l, 1 h) were challenged with TGF-β (5 ng/ml, 48 h), and protein expression was analyzed by Western blot as described in ‘Materials and Methods’. Representative Western blot (a) and quantification of the expression of FN (b), α-SMA (c) and HO-1 (d) in human lung fibroblasts with or without TGF-β challenge. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs. cells without TGF-β challenge; # p < 0.05 vs. cells without NF-κB inhibitor treatment and with TGF-β challenge.
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Fig. 3. NF-κB inhibitor attenuates TGF-β-
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hibits HO-1 expression in human lung fibroblasts. Starved WI-38 cells with pretreatment of anti-TGF-β antibody or control IgG antibody (5 μg/ml, 1 h), and further challenged with 18:1 LPA (0, 10, 20, 30 μmol/l) for 48 h. Representative Western blot (a) and quantification of LPA (0–30 μmol/l)-induced TGF-β expression in WI38 cells (b). * p < 0.05 vs. cells without LPA treatment. Representative Western blot (c), quantification of the expression of FN (d), α-SMA (e) and HO-1 (f) induced by LPA (10 μmol/l, 48 h) in hu-
the expression of HO-1 in human lung fibroblasts, and this effect was, at least partly, due to LPA-induced expression of TGF-β.
Discussion
Since pulmonary fibrosis is a fatal lung disease without effective pharmacological treatment, identification of new therapeutic strategies for pulmonary fibrosis is urgent. Accumulated reports reveal that inflammatory cells, epithelial cells and fibroblasts are involved in pulmonary fibrogenesis [4]. During pulmonary fibrogenesis, the contents of various lipid ligands, cytokines and chemokines are changed and followed by regulation of potential 190
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man lung fibroblasts. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs. cells without LPA treatment. Effect of anti-TGF-β antibody on LPA-induced inhibition of HO-1 expression in WI-38 cells: representative Western blot (g) and quantification of the expression of FN (h), α-SMA (i) and HO-1 (j) in human lung fibroblasts. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs. cells with control antibody but without LPA treatment; # p < 0.05 vs. LPAchallenged cells with pretreatment of control antibody.
pathways [6, 24]. Among them, TGF-β is a key factor for fibrogenesis; it regulates the activation and differentiation of fibroblasts [2]. Thus, investigation of TGF-β effects on fibrogenesis is essential for understanding pulmonary fibrosis. HO-1 is an inducible heat shock protein, and clinical study in pulmonary fibrosis patients indicated that HO-1 expression contributes to pulmonary fibrosis [15]. Based on the staining of lung tissue from pulmonary fibrosis, a recent report demonstrated that HO-1 expression in macrophages was increased compared with normal control subjects [11]. Quantitative reverse transcriptionpolymerase chain reaction data further indicated that the expression of HO-1 was increased in lung tissue from pulmonary fibrosis patients compared with control subjects Xie/Wang/Zong/Cheng
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[11]. In an animal model of pulmonary fibrosis, HO-1 expression was also increased by intratracheal instillation of rats with crystalline silica, and most of the HO-1-positive cells were alveolar macrophages [25]. Additionally, in vitro treatment with curcumin, a chemopreventive agent, increased the expression of HO-1, blocked the radiation-induced generation of reactive oxygen species and boosted antioxidant defenses both in lung endothelial and fibroblast cells [19]. These studies suggested that expression of HO-1 in macrophages positively correlates with the progress of pulmonary fibrosis. Although previous reports strongly suggested the protecting role of HO-1 during pulmonary fibrosis, the effects of HO-1 in lung fibroblasts is still elusive. A recent investigation indicated that treatment with quercetin, a flavonoid with a wide variety of cytoprotective functions, increased the expression of HO-1 in fibroblasts and further attenuated TGF-β-induced expression of collagen via stimulating carbon monoxide production [20]. Since activation of NF-κB is linked to aberrant expression of numerous genes during the progression of inflammatory and fibrotic diseases, it is essential to fibroblast differentiation [26]. Both inhibition of NF-κB activation or knockdown of the expression of the NF-κB p65 subunit dramatically increased the HO-1 expression under TGF-β challenge and further inhibited TGF-β-induced fibro-
blast differentiation. Interestingly, inhibition of the NFκB signaling pathway dramatically ameliorated profibrotic events in gammaherpesvirus-induced pulmonary fibrosis in the mouse [27], and specific inhibition of the NF-κB signaling pathway in T cells also attenuated the progression of lung fibrosis [28]. Various lipid ligands were also involved in inflammation reactions [29–35] and pulmonary fibrosis [5, 6]. Especially, LPA induced the recruitment and differentiation of fibroblasts [5, 8] and regulated endothelial permeability and epithelial barrier functions via activation of its receptors during fibrogenesis. LPA also induced the expression of TGF-β in human lung fibroblasts in a dose-dependent manner, and this effect is due to LPA-induced activation of LPA receptor type 2 in lung fibroblasts [5]. Taken together, this study indicated that LPA induced inhibition of HO-1 expression in human lung fibroblasts through TGF-β. In conclusion, we revealed that TGF-β inhibits HO-1 expression through the NF-κB signaling pathway, which suggests that targeting HO-1 inhibit pulmonary fibrogenesis and may be a novel therapeutic strategy for this disease.
Disclosure Statement None.
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29 Huang LS, Hung ND, Sok DE, Kim MR: Lysophosphatidylcholine containing docosahexaenoic acid at the sn-1 position is anti-inflammatory. Lipids 2010;45:225–236. 30 Huang LS, Kim MR, Sok DE: Regulation of lipoxygenase activity by polyunsaturated lysophosphatidylcholines or their oxygenation derivatives. J Agric Food Chem 2008; 56: 7808–7814. 31 Huang LS, Kang JS, Kim MR, Sok DE: Oxygenation of arachidonoyl lysophospholipids by lipoxygenases from soybean, porcine leukocyte, or rabbit reticulocyte. J Agric Food Chem 2008;56:1224–1232. 32 Huang LS, Kim MR, Jeong TS, Sok DE: Linoleoyl lysophosphatidic acid and linoleoyl lysophosphatidylcholine are efficient substrates for mammalian lipoxygenases. Biochim Biophys Acta 2007;1770:1062–1070. 33 Huang LS, Kim MR, Sok DE: Linoleoyl lysophosphatidylcholine is an efficient substrate for soybean lipoxygenase-1. Arch Biochem Biophys 2006;455:119–126. 34 Huang LS, Kim MR, Sok DE: Oxygenation of 1-docosahexaenoyl lysophosphatidylcholine by lipoxygenases; conjugated hydroperoxydiene and dihydroxytriene derivatives. Lipids 2007;42:981–990. 35 Huang LS, Kim MR, Sok DE: Enzymatic reduction of polyunsaturated lysophosphatidylcholine hydroperoxides by glutathione peroxidase-1. Eur J Lipid Sci Tech 2009;111:584–592.
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17 Wu SY, Li MH, Ko FC, Wu GC, Huang KL, Chu SJ: Protective effect of hypercapnic acidosis in ischemia-reperfusion lung injury is attributable to upregulation of heme oxygenase-1. PLoS One 2013;8:e74742. 18 Chen Y, Nie YC, Luo YL, Lin F, Zheng YF, Cheng GH, Wu H, Zhang KJ, Su WW, Shen JG, Li PB: Protective effects of naringin against paraquat-induced acute lung injury and pulmonary fibrosis in mice. Food Chem Toxicol 2013;58:133–140. 19 Lee JC, Kinniry PA, Arguiri E, Serota M, Kanterakis S, Chatterjee S, Solomides CC, Javvadi P, Koumenis C, Cengel KA, Christofidou-Solomidou M: Dietary curcumin increases antioxidant defenses in lung, ameliorates radiation-induced pulmonary fibrosis, and improves survival in mice. Radiat Res 2010;173:590–601. 20 Nakamura T, Matsushima M, Hayashi Y, Shibasaki M, Imaizumi K, Hashimoto N, Shimokata K, Hasegawa Y, Kawabe T: Attenuation of transforming growth factor-beta-stimulated collagen production in fibroblasts by quercetin-induced heme oxygenase-1. Am J Respir Cell Mol Biol 2011;44:614–620. 21 Jin C, Zhou D, Lv F: Beneficial effects of early (but not late) intervention of heme oxygenase-1 on bleomycin-induced pulmonary fibrosis in mice. Respir Physiol Neurobiol 2011;175:239–246.
Pharmacology 2014;93:185–192 DOI: 10.1159/000360638
Transforming Growth Factor-Beta Inhibits Heme Oxygenase-1 Expression in Lung Fibroblasts through Nuclear Factor-Kappa-B-Dependent Pathway Ying Xie a Ye Wang b Chuanyue Zong a Jiawen Cheng a, c
a
Department of Anesthesiology, Huai’an Hospital Affiliated with Xuzhou Medical College and Huai’an Second People’s Hospital, Huaian, b Department of Anesthesiology, Wuxi Ninth People’s Hospital, Wuxi, and c Department of anesthesiology, The First People’s Hospital of Suqian, Suqian, China
Abstract Background: Heme oxygenase-1 (HO-1) contributes to the pathogenesis of pulmonary fibrosis. However, the expression of HO-1 in fibroblasts under fibrotic conditions has not been studied. Methods: This study was conducted to investigate the expression of HO-1 in lung fibroblasts from mice and humans under fibrotic conditions by Western blot. Results: We found that the expression of HO-1 was significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice in comparison with control mice. Transforming growth factor-β (TGF-β) inhibited HO-1 expression and induced differentiation in human lung fibroblasts. Pretreatment with nuclear factor-κB (NF-κB) activation inhibitor or knockdown of the NF-κB p65 subunit attenuated TGF-βinduced inhibition of HO-1 expression and differentiation in human lung fibroblasts. Similarly, lysophosphatidic acid (LPA) induced TGF-β expression and decreased HO-1 expression in human lung fibroblasts. Interestingly, pretreatment with neutralized anti-TGF-β antibody attenuated LPA effects in human lung fibroblasts. Conclusion: These data suggested that TGF-β inhibited HO-1 expression in human lung fibroblasts through activation of NF-κB. © 2014 S. Karger AG, Basel
Introduction
Pulmonary fibrosis is a chronic and progressive interstitial lung disease [1, 2]. Alveolar epithelial cell injury, accumulation and differentiation of fibroblasts [3, 4], and deposition of extracellular matrix proteins contribute to the progress of pulmonary fibrosis [5, 6]. Currently, lung transplantation is the only effective cure for pulmonary fibrosis [7], thus investigations of the pathological mechanisms and efficacious therapeutic approaches for pulmonary fibrosis are extremely urgent. Since fibroblast accumulation leads to an excessive scarring of lung tissue and to progressive and irreversible destruction of lung architecture [4], investigations of fibroblast functions are critical for understanding the molecular mechanisms of pulmonary fibrosis. Investigations demonstrated that transforming growth factor-β (TGF-β) and lysophosphatidic acid (LPA) are extensively involved in the fibrogenesis of different organs, such as heart, liver and lung [4, 5, 8, 9]. TGF-β induces lung fibroblast differentiation through both Smad-dependent and independent pathways, and followed by increased expression of α-smooth muscle actin (α-SMA) and extracellular matrix proteins [2]. LPA also induces fibroblast recruitment, migration and differentiation via activation of LPA receptors [5, 8]. Interestingly, a recent report demonstrated that LPA also induces TGF-β expression in lung fibroYing Xie and Ye Wang contributed equally to this work.
© 2014 S. Karger AG, Basel 0031–7012/14/0934–0185$39.50/0 E-Mail [email protected] www.karger.com/pha
Jiawen Cheng, MD Department of Anesthesiology The First People’s Hospital Of Suqian 120 Suzhi Road, Suqian, Jiangshu 223800 (China) E-Mail chengjiawen2013 @ 163.com
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Key Words Heme oxygenase-1 · Lysophosphatidic acid · Transforming growth factor-β · Nuclear factor-κB · Pulmonary fibrosis
Materials and Methods Reagents and Kits Bleomycin sulfate was from Hospira Inc. (Lakeforest, Ill., USA), and neutralizing chicken anti-TGF-β1 antibody and control chicken IgG were obtained from R&D Systems (Minneapolis, Minn., USA). Oleoyl lysophosphatidic acid (18:1 LPA) was obtained from Avanti Polar Lipids (Alabaster, Ala., USA), and cell lysis buffer was purchased from Cell Signaling Technology Inc. (Danvers, Mass., USA). Recombinant human TGF-β1 was obtained from Prepro Tech Inc. (Rocky Hill, N.J., USA). Horseradish peroxidase-linked anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories Inc. (Hercules, Calif., USA). Rabbit anti-fibronection (FN), anti-HO-1 and anti-p65 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif., USA). Mouse anti-α-SMA and anti-β-actin antibodies and Bay 11-7082 (NF-κB inhibitor) were from Sigma-Aldrich (St. Louis, Mo., USA).
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Experimental Pulmonary Fibrosis Model The animal experiment of pulmonary fibrosis was designed as described before [5, 6]. Briefly, C57/BL6 mice (male, age 8 weeks) purchased from Jackson Laboratory (Bar Harbor, Me., USA) were used for bleomycin-induced fibrosis. C57/BL6 mice were anesthetized (with a 3 ml/kg mixture of 25 mg/kg of ketamine in 2.5 ml of xylazine), followed by treatment with saline or bleomycin sulfate (1.5 U/kg of body weight, ∼0.03 U/animal) in saline by intratracheal injection in a total volume of 50 μl. Twenty-one days after bleomycin administration, animals were killed, and lungs were removed for isolation of lung fibroblasts. All animal protocols conformed to the standards of Xuzhou Medical College and were in accordance with Chinese animal operation regulations. Isolation of Mouse Primary Fibroblasts and Cell Culture Mouse lung fibroblasts were isolated from mice with or without bleomycin challenge as described previously [5, 22]. Human lung fibroblast cell line (WI-38) was ordered from ATCC. Mouse primary lung fibroblasts and human lung fibroblasts were grown and maintained in 6-well dishes with DMEM medium containing 10% fetal bovine serum. siRNA Transfection Scrambled RNA and targeting human NF-κB p65 subunit smart pool (si-p65) were from Dharmacom Inc. (Lafayette, Colo., USA). Briefly, WI-38 cells cultured on 6-well plates (50–60% confluence) were transiently transfected with scrambled RNA and sip65 (200 nmol/l) according to manufacturer’s instructions (Qiagen, Md., USA). After dilution with 900 μl of basal DMEM medium, transfection complex was directly added to the cells. Six hours after treatment, transfection medium was replaced by complete DMEM medium (with 10% fetal bovine serum), and cells were analyzed (48 h after treatment) by Western blot. Treatment of Neutralizing Antibodies or NF-κB Inhibitor Serum-starved (for 24 h) human lung fibroblasts (WI-38, ∼90% confluence) were pretreated with neutralized anti-TGF-β antibody or control IgG antibody (5 μg/ml, 1 h). In case of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor (Bay 11-7082), the compound was pretreated with a final concentration of 10 μmol/l for 1 h [23]. Next, cells were challenged with 18:1 LPA (10 μmol/l) or TGF-β (5 ng/ml) for 48 h, and cell lysates (20 μg protein) were subjected to SDS-PAGE and Western blot. SDS-PAGE and Western Blot SDS-PAGE and Western blot were performed as described previously [5]. Blots were developed using the ECL chemiluminescence kit, and integrated density of pixels in each membrane was quantified and normalized to actin by using Image Quant 5.2 software (Molecular Dynamics, Sunnyvale, Calif., USA). Immunofluorescence Staining Immunofluorescence microscopy to determine protein expression was performed as described [5]. Briefly, primary mouse lung fibroblasts were grown in slide chambers for 24 h. Cells were fixed, incubated with primary antibodies (1: 200 dilutions in blocking buffer) for 1 h and with Alexa Fluor secondary antibodies (1:200 dilutions in blocking buffer) for another 1 h, followed
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blasts through activation of LPA receptor type 2 [5]. However, the crosstalk between TGF-β and LPA pathways in fibroblast differentiation is still elusive. Heme oxygenase-1 (HO-1) is an inducible stress protein, belongs to the heat shock protein family [10], and induces the degradation of heme to biliverdin and the generation of carbon monoxide [11]. Its expression has been proved to accompany the pathogenesis of various lung injury-induced diseases, including asthma [12], acute lung injury [13, 14], cystic fibrosis [11, 15] and lung transplant rejection [16]. Previous investigations indicated that endogenous HO-1 is involved in the resolving of inflammatory reactions and migration of neutrophil cells in experimental models of acute lung injury [13, 17]. Recent investigations also suggested that HO-1 plays a protecting role during pulmonary fibrogenesis [11, 18–20]. Jin et al. [21] indicated that bleomycin treatment increased the HO-1 expression in mouse lung tissue. Immunohistochemical staining of lung samples from patients with pulmonary fibrosis and mice treated with bleomycin indicated that HO-1 was strongly expressed in macrophages, respiratory epithelial cells and endothelial cells [11, 20, 21], while the expression of HO-1 in lung fibroblasts during fibrogenesis has not been investigated. Clinical investigations suggested that HO-1 deficiency correlates with increased fibrosis [10, 15], and up-regulation of HO-1 expression protects against renal injury and fibrosis through antiapoptotic pathway modulation [11]. However, the expression of HO-1 in pulmonary fibroblasts during fibrogenesis is still elusive, and the potential mechanism is not clear. Here, we found that HO-1 expression was dramatically decreased in lung fibroblasts from humans and mice under fibrotic conditions, and the expression of HO-1 was regulated by activation of nuclear factor-κB (NF-κB).
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cells from control mice. e Immunofluorescence staining of HO-1 and FN in lung fibroblasts from mice with or without BLM challenge. Images were examined by immunofluorescence microscopy and recorded by using a ×60 oil objective. The FN (red) and HO-1 (green) images showed matched cell fields for each condition.
Results
Expression of HO-1 in Lung Fibroblasts from Mice with or without Bleomycin Challenge To study the expression of HO-1 in fibroblasts during fibrogenesis, lung fibroblasts were isolated from C57/BL6 mice with or without bleomycin challenge. As shown in figure 1a–c, the expression of α-SMA and fibronectin (FN), Pharmacology 2014;93:185–192 DOI: 10.1159/000360638
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Fig. 1. Expression of α-SMA, FN and HO-1 in lung fibroblasts from mice with or without bleomycin (BLM) challenge. Representative Western blot (a) and quantification of FN (b), α-SMA (c) and HO-1 (d) expression in mouse lung fibroblasts. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs.
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biomarkers of fibroblast differentiation, was dramatically increased in lung fibroblasts isolated from bleomycin-challenged mice compared with that from control mice; however, the expression of HO-1 was significantly decreased (fig. 1a, d) and immunofluorescence staining also indicated that the expression of HO-1 was significantly decreased in lung fibroblasts from bleomycin-challenged mice when compared with that from control mice (fig. 1e). HO-1 Is Down-Regulated by TGF-β in Human Lung Fibroblasts To check whether the expression of HO-1 is related to lung fibroblast activation and differentiation, we checked the expression of HO-1 in TGF-β-induced human lung fibroblast differentiation. Western blot analysis revealed that TGF-β challenge (5 ng/ml, 48 h) dramatically increased fibroblast differentiation, characterized as the expression of α-SMA and FN (fig. 2a–c), while it significantly inhibited the expression of HO-1 in lung fibroblasts (fig. 2a, d). TGF-β Inhibits HO-1 Expression through NF-κB To further examine the molecular mechanisms of TGF-β-induced inhibition of HO-1 expression in lung fibroblasts, we pretreated human lung fibroblast cells with NF-κB inhibitor (10 μmol/l) for 1 h before TGF-β challenge. As shown in figure 3, TGF-β up-regulated α-SMA and FN expression, and pretreatment with NFκB inhibitor dramatically blocked TGF-β effects. Inter188
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3 2 1 0
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estingly, the inhibition effect of TGF-β on HO-1 expression in lung fibroblasts was also significantly suppressed (fig. 3a, d). Additionally, knockdown of the expression of the NF-κB p65 subunit by si-p65 dramatically attenuated the expression of p65 in human lung fibroblasts (fig. 4a, b), and it also inhibited TGF-β-induced differentiation of lung fibroblasts (fig. 4a, c, d). Similarly, knockdown of the expression of the p65 subunit also increased HO-1 expression under TGF-β challenge (fig. 4a, e). Together, these data indicated that TGF-β inhibited HO-1 expression through activation of NF-κB signaling pathways. LPA-Induced Differentiation of Fibroblasts and Suppression of HO-1 Expression Is Attenuated by Anti-TGF-β Antibody In our studies, we proved that LPA induces TGF-β expression in WI-38 cells in a dose-dependent manner (fig. 5a, b). Next, we turned to check LPA effects on HO-1 expression in lung fibroblasts. LPA challenge also induced expression of α-SMA and FN, and inhibited HO-1 expression in human lung fibroblasts (fig. 5c–f). Additionally, to block the effect of secreted TGF-β under LPA challenge, cells were pretreated with an anti-TGF-β antibody. As shown in figure 5g–j, pretreatment with antiTGF-β1 antibody dramatically inhibited LPA-induced differentiation of human lung fibroblasts, and it significantly increased HO-1 expression under LPA treatment (fig. 5g–j). These results suggest that LPA also suppressed Xie/Wang/Zong/Cheng
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NF-κB p65 subunit attenuates TGF-βinduced inhibition of HO-1 expression in human lung fibroblasts. WI-38 cells with transient transfection of scrambled RNA and p65 siRNA (200 nmol/l) according to ‘Materials and Methods’ followed by TGF-β (5 ng/ml, 48 h) challenge. Representative Western blot (a) and quantification of the expression of p65 (b), FN (c), α-SMA (d) and HO-1 (e) in human lung fibroblasts. Data are expressed as means ± SEMs of three independent experiments. * p < 0.05 vs. cells without si-p65 and TGF-β challenge; # p < 0.05 vs. cells without si-p65 treatment but with TGF-β challenge.
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the expression of HO-1 in human lung fibroblasts, and this effect was, at least partly, due to LPA-induced expression of TGF-β.
Discussion
Since pulmonary fibrosis is a fatal lung disease without effective pharmacological treatment, identification of new therapeutic strategies for pulmonary fibrosis is urgent. Accumulated reports reveal that inflammatory cells, epithelial cells and fibroblasts are involved in pulmonary fibrogenesis [4]. During pulmonary fibrogenesis, the contents of various lipid ligands, cytokines and chemokines are changed and followed by regulation of potential 190
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pathways [6, 24]. Among them, TGF-β is a key factor for fibrogenesis; it regulates the activation and differentiation of fibroblasts [2]. Thus, investigation of TGF-β effects on fibrogenesis is essential for understanding pulmonary fibrosis. HO-1 is an inducible heat shock protein, and clinical study in pulmonary fibrosis patients indicated that HO-1 expression contributes to pulmonary fibrosis [15]. Based on the staining of lung tissue from pulmonary fibrosis, a recent report demonstrated that HO-1 expression in macrophages was increased compared with normal control subjects [11]. Quantitative reverse transcriptionpolymerase chain reaction data further indicated that the expression of HO-1 was increased in lung tissue from pulmonary fibrosis patients compared with control subjects Xie/Wang/Zong/Cheng
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[11]. In an animal model of pulmonary fibrosis, HO-1 expression was also increased by intratracheal instillation of rats with crystalline silica, and most of the HO-1-positive cells were alveolar macrophages [25]. Additionally, in vitro treatment with curcumin, a chemopreventive agent, increased the expression of HO-1, blocked the radiation-induced generation of reactive oxygen species and boosted antioxidant defenses both in lung endothelial and fibroblast cells [19]. These studies suggested that expression of HO-1 in macrophages positively correlates with the progress of pulmonary fibrosis. Although previous reports strongly suggested the protecting role of HO-1 during pulmonary fibrosis, the effects of HO-1 in lung fibroblasts is still elusive. A recent investigation indicated that treatment with quercetin, a flavonoid with a wide variety of cytoprotective functions, increased the expression of HO-1 in fibroblasts and further attenuated TGF-β-induced expression of collagen via stimulating carbon monoxide production [20]. Since activation of NF-κB is linked to aberrant expression of numerous genes during the progression of inflammatory and fibrotic diseases, it is essential to fibroblast differentiation [26]. Both inhibition of NF-κB activation or knockdown of the expression of the NF-κB p65 subunit dramatically increased the HO-1 expression under TGF-β challenge and further inhibited TGF-β-induced fibro-
blast differentiation. Interestingly, inhibition of the NFκB signaling pathway dramatically ameliorated profibrotic events in gammaherpesvirus-induced pulmonary fibrosis in the mouse [27], and specific inhibition of the NF-κB signaling pathway in T cells also attenuated the progression of lung fibrosis [28]. Various lipid ligands were also involved in inflammation reactions [29–35] and pulmonary fibrosis [5, 6]. Especially, LPA induced the recruitment and differentiation of fibroblasts [5, 8] and regulated endothelial permeability and epithelial barrier functions via activation of its receptors during fibrogenesis. LPA also induced the expression of TGF-β in human lung fibroblasts in a dose-dependent manner, and this effect is due to LPA-induced activation of LPA receptor type 2 in lung fibroblasts [5]. Taken together, this study indicated that LPA induced inhibition of HO-1 expression in human lung fibroblasts through TGF-β. In conclusion, we revealed that TGF-β inhibits HO-1 expression through the NF-κB signaling pathway, which suggests that targeting HO-1 inhibit pulmonary fibrogenesis and may be a novel therapeutic strategy for this disease.
Disclosure Statement None.
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