Abstract
Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α–smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-β1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-β1–induced UPR and α-SMA and collagen type I induction. We also showed that TGF-β1–induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.
The endoplasmic reticulum (ER) is an intracellular organelle responsible for the folding and sorting of proteins (1). Various conditions that impair ER function lead to an accumulation of unfolded or misfolded proteins (ER stress) and then trigger the unfolded protein response (UPR). The UPR reduces ER stress by activating three adaptive pathways: (1) the transcriptional induction of ER chaperones to help protein folding; (2) the translational attenuation of protein synthesis, which blocks new protein synthesis; and (3) promotion of the degradation of misfolded or unfolded protein via the ubiquitin–proteasome system (2, 3). ER stress sensors perceive increases in unfolded proteins in ER and initiate UPR signaling cascades. Three ER stress sensors have been identified: IRE1 (inositol-requiring protein 1), PERK (PKR [double-stranded-RNA-dependent protein kinase]-like ER kinase), and ATF6 (activating transcription factor 6). eIF-2α and XBP-1 are down-stream targets of PERK and IRE1, respectively (2, 3). GRP78, also referred to as BiP, is an ER chaperone protein that promotes folding of proteins and prevents the aggregation of protein in the ER. When ER stress sensors such as IRE-1 or ATF6 are activated, GRP78 gene transcription is up-regulated (4). If the UPR is unable to rescue cells, the cells eventually enter apoptosis via increases in CHOP (CCAAT/enhancer-binding protein homologous protein) expression or the activation of ER-specific caspases (2, 3). Therefore, induction of GRP78 or CHOP is widely used as a marker for ER stress.
Chemical chaperones are a group of low-molecular-weight compounds known to stabilize protein conformation and improve ER folding capacity. They also serve as a type of quality control system, recognizing, retaining, and targeting misfolded proteins for their eventual degradation (5). One chemical chaperone, 4-phenyl butyric acid (4-PBA), is a nontoxic butyrate analog that was originally approved for clinical use as an ammonia scavenger in subjects with urea cycle disorders (6). Recent studies have suggested a potential role of chemical chaperones in the treatment of ER stress–related diseases such as Alzheimer's disease, Prion's disease, cystic fibrosis, and diabetes (7–10).
Reactive oxygen species (ROS) are oxygen-derived free radicals. In a variety of pathologic processes, ROS accumulation in cells and their involvement in the pathogenesis of various diseases has been reported. TGF-β1 stimulates production of ROS in various cell types, and ROS mediates many of TGF-β1’s fibrogenic effects (11). Previous studies have indicated that ROS may have the potential to induce the unfolded protein response (UPR) (12). On the contrary, prolonged UPR activation can also cause oxidative stress (13).
ER stress has been implicated in various diseases, such as neurodegenerative disease, cancer, diabetes, obesity, and inflammation (14–19). Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal form of interstitial lung disease. Despite extensive research efforts over the past few decades, the exact mechanism underlying the development of IPF remains unknown, and no effective therapies are available (20). Accumulating evidence suggests that type II alveolar epithelial cell (AEC) injury is important in the pathogenesis of IPF. Recent studies have indicated that ER stress is involved in the pathogenesis of pulmonary fibrosis by the induction of apoptosis of alveolar epithelial cells of patients with IPF (21). In addition to increased apoptosis of type II AECs, active synthesis and assembling of extracellular matrix proteins in fibroblasts are important in the development of fibrosis. Treatment of tunicamycin (TM) to human lung fibroblasts leads to the activation of UPR (22). Recently, the ER stress inducers thapsigargin (TG) and TM have been shown to increase α–smooth muscle actin (α-SMA) in AECs (23).
Based on the above reports, we hypothesized that ER stress is involved in the synthesis of extracellular matrix proteins and myofibroblastic differentiation of fibroblasts in pulmonary fibrosis. To test this hypothesis, we investigated the expression of ER stress sensor molecules in cultured human and mouse lung fibroblasts during TGF-β1–induced α-SMA and collagen type I production. We also investigated an association between the induction of ROS and UPR during TGF-β1–induced myofibroblastic differentiation.
Six-week-old female C57BL6 mice were purchased from Damul Science (Daejeon, Korea). Pulmonary fibrosis was induced by the endotracheal injection of 1.5 U/kg body weight of bleomycin (BLM) (Blenoxane; Mead Johnson, Jersey City, NJ) in sterile PBS as described elsewhere (24). The control group received the same volume of sterile PBS. At 7, 14, or 21 days after BLM injection, the animals were killed, and the lungs were removed. All experimental animals used in this study were maintained under the protocol approved by the Institutional Animal Care and Use Committee at Chonbuk National University.
Immunohistochemical staining of GRP78, vimentin, SMA, and TTF-1 was performed as described previously (25). Lung tissues from five patients with IPF and normal lung tissues were used for immunohistochemisty (see the online supplement for more details).
The mouse lung fibroblast cell line (MLg) was purchased from, and maintained as instructed by, the Korean Cell Line Bank (Seoul, Korea). Human lung fibroblasts were isolated from normal lung tissue (from lung resected because of lung cancer) and maintained in culture as described previously (26). Where indicated, recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) and/or a chemical chaperone, sodium 4-phenylbutyrate (4-PBA) (Calbiochem, EMD Chemicals Inc., Darmstadt, Germany), antioxidants, N-acetyl-l-cysteine (NAC) (Sigma-Aldrich, St. Louis, MO) and l-glutathione reduced (GSH) (Sigma-Aldrich), and TM or TG (Sigma-Aldrich) were added to the medium at the indicated concentrations and time points.
Lung homogenates or cell lysates were subjected to denaturating SDS-PAGE, followed by electroblotting and immunoblotting for anti-GRP78, anti-CHOP (Santa Cruz Biotechnology, Santa Cruz, CA), anti–α-SMA, anti-collagen type I (Sigma), anti-ATF6α (Abnova, Taipei City, Taiwan), anti–XBP-1 (Novus Biologicals, Littleton, CO), and anti-phospho eIF2α (Cell Signaling Technology, Danvers, MA). Blots were developed using corresponding HRP-conjugated secondary antibodies and detected using a chemiluminescent system (Amersham ECL Plus; GE Healthcare, Piscataway, NJ). Band intensities were quantified with the LAS-1000 plus system (Fuji Film, Japan).
The MLg cells were treated with TGF-β1 (2.5 ng/ml) with or without 5 mM NAC, 5 mM GSH, or 5 mM 4-PBA for the indicated time periods. The intracellular ROS level was measured as previously described (27). The each sample was analyzed by PAS Vantage flow cytometry (Partec, Münster, Germany) at 488 nm excitation and 525 nm emission wavelengths, respectively. The data were collected and analyzed using Partec software (Becton Dickinson, Franklin Lakes, NJ).
GRP78 siRNA (Santa Cruz Biotechnology) was transfected into human lung fibroblasts using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Then, the cells were treated with or without TGF-β1 or TM for 24 hours, and the subsequent experiments were performed. Transfected cells were screened on the basis of down-regulation of GRP78 expression by Western blotting.
The statistical significance of the data was analyzed using the SPSS software package, version 15.0 (SPSS, Seoul, Korea). Data were expressed as means ± SE. Statistical analyses were performed using one-way ANOVA for the comparison of data from different treatment groups. A P value < 0.05 was considered significant.
GRP78 is known as a master regulator of the UPR. To assess the potential biological role of the ER stress response in pulmonary fibrosis, we first examined the expression of GRP78 and other UPR markers in BLM-treated mouse lung tissue and in lung tissue from patients with IPF by Western blotting. In BLM-treated lung tissue, GRP78 and other UPR markers were up-regulated at Days 7, 14, and 21, compared with that of controls (Figures 1A and 1B). The UPR markers were also up-regulated in tissues with IPF but were very low in control tissues (Figure 1C). To support the Western blot results and to identify the cellular source of GRP78 in pulmonary fibrosis, IHC for GRP78 was done in lung tissues from normal control subjects or patients with IPF (Figure 2). GRP78 expression was increased in IPF compared with control lung tissue (for IHC results for control lung tissue, see Figure E1 in the online supplement). AECs lining airspaces in areas of fibroblastic foci showed strong staining for GRP78 in IPF lung tissue (Figure 2B). AECs also displayed immunoreactivity for TTF-1, which is a marker for the epithelial cells of lung (Figure 2C). GRP78 was also detected in fibroblasts or myofibroblasts in fibroblastic foci (Figure 2B). Although the staining intensity was weaker than that of AECs or macrophages, fibroblasts, or myofibroblasts in fibroblastic foci showed clear immunoreactivity for GRP78. GRP78-positive fibroblasts also showed immunoreactivity for α-SMA and vimentin (Figures 2D and 2E). In control lung tissue, there was no detectable signal for GRP78, except alveolar macrophages showed weak GRP78 expression (Figure E1B). Based on these results, we suggest that ER stress may play a role not only in AECs but also in fibroblasts. Then, we focused on evaluating the role of ER stress in fibroblasts.
Figure 1. Expression of unfolded protein response (UPR) markers in fibrotic lung tissue from mouse and human. C57BL6 mice were treated with an endotracheal injection of bleomycin (BLM) (1.5 U/kg) for the indicated number of days. The control group received the same volume of sterile phosphate-buffered saline. Whole-protein lysates from mice (A, B) and human (C, D) lungs (normal = normal lung tissue from resected lung for lung cancer) were subjected to Western blotting for GRP78, ATF-6α, XBP-1, CHOP (CCAAT/enhancer-binding protein homologous protein), and phospho-eIF2α expression. The expression of the UPR markers was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and is shown as mean ± SE (n = 5). *P < 0.001 compared with control. IPF = idiopathic pulmonary fibrosis.
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Figure 2. Expression of GRP78 in idiopathic pulmonary fibrosis (IPF) lung tissue. Representative hematoxylin-eosin (A) and immunohistochemical staining for GRP78 (B), thyroid transcription factor-1 (TTF-1) (C), smooth muscle actin (SMA) (D), and vimentin (E) in lung tissue from IPF are shown. Alveolar epithelial cells (arrows) adjacent to underlying fibroblastic focus showed strong staining for GRP78 in IPF lung tissue. GRP78 was also detected in fibroblasts or myofibroblasts (open arrows) in fibroblastic focus. SMA and vimentin were colocalized with GRP78 in fibroblasts or myofibroblasts.
[More] [Minimize]We analyzed UPR activation in fibroblasts by TGF-β1 treatment to support the hypothesis that ER stress is involved in the synthesis of extracellular matrix proteins and myofibroblastic differentiation of fibroblasts in pulmonary fibrosis. We first studied the expression of UPR markers, GRP78, CHOP, ATF6α (p60, the active spliced form of ATF6α), active spliced XBP-1, and phospho-eIF2α in mouse and human lung fibroblasts after TGF-β1 treatment for 2 to 24 hours by Western blotting. We also examined α-SMA and collagen type I expression levels in mouse and human lung fibroblasts after TGF-β1 treatment for 24 to 48 hours by Western blotting to show the relation between UPR activation and myofibroblastic differentiation or collagen type I expression. Expression of GRP78, ATF6α, and XBP-1 was increased time-dependently in mouse and human lung fibroblasts (Figures 3A, 3B, 3E, 3F). Up-regulation of the UPR targets was accompanied by α-SMA and collagen type I induction (Figures 3C, 3D, 3G, 3H). The two UPR targets, CHOP and phospho-eIF2α, were not significantly induced by TGF-β1 treatment in human and mouse lung fibroblasts (Figure 3A, 3B, 3E, 3F), suggesting that there is some selectivity in the UPR induced by ER stress during TGF-β1–induced myofibroblastic differentiation. To strengthen the present results that ER stress is involved in myofibroblastic differentiation, we examined the effect of TM or TG on myofibroblastic differentiation. Treatment of TM or TG to human lung fibroblasts induced up-regulation of GRP78, α-SMA, and collagen type I (Figure E2).
Figure 3. UPR activation during myofibroblastic differentiation of fibroblasts induced by TGF-β1. Human and mouse lung fibroblasts were treated with TGF-β1 (2.5 ng/ml) for the time intervals indicated. The expression of UPR targets (GRP78, CHOP, ATF-6α, XBP-1, and phospho-eIF2α) in human (A, B) and mouse lung fibroblasts (E, F) was analyzed by Western blotting. α-SMA and collagen type I were analyzed by Western blotting (C, D, G, H). The expression of GRP78, CHOP, ATF-6α, XBP-1, phospho-eIF2α, collagen type I, and α-SMA was normalized to that of GAPDH. The data shown represent the mean ± SE (n = 3). *Significant difference (P < 0.05) from the mean value for the control (None), as assessed by ANOVA.
[More] [Minimize]One chemical chaperone, 4-PBA, was shown to improve ER folding and to reduce ER stress (7, 8). We use this chemical as ER stress inhibitor to confirm that ER stress involved in myofibroblastic differentiation. We investigated the effect of 4-PBA on TGF-β1–induced UPR and TGF-β1–induced α-SMA and collagen type I production in fibroblasts. To determine the proper conditions for 4-PBA treatment, mouse lung fibroblasts were treated with TGF-β1 in the presence of various concentration of 4-PBA, and then the levels of GRP78 were analyzed by Western blotting. TGF-β1–induced GRP78 was efficiently reduced by pretreatment of the cells with 5 or 10 mM of 4-PBA for 4 hours before TGF-β1 treatment (Figure E3). Next, we investigated whether 4-PBA reduced the TGF-β1–induced ER stress in human and mouse lung fibroblasts. Pretreatment of human and mouse lung fibroblasts with 4-PBA (5 mM, 4 h) suppressed TGF-β1–induced GRP78, XBP-1, and ATF6α expression (Figures 4A, 4B, 4E, 4F). These results indicate that 4-PBA could reduce TGF-β1–induced ER stress. To confirm that ER stress is involved in induction of α-SMA and collagen type I by TGF-β1 stimulation, fibroblasts were treated with 4-PBA before TGF-β1 treatment. We then assessed α-SMA and collagen type I expression levels by Western blotting. Pretreatment with 4-PBA significantly reduced TGF-β1–induced α-SMA and collagen type I production compared with cells treated with TGF-β1 only in human and mouse lung fibroblasts (Figures 4C, 4D, 4G, 4H). To confirm the role of GRP78 in myofibroblastic differentiation, we used GRP78 siRNA to knockdown the expression of GRP78. TGF-β1 or TM-induced α-SMA and collagen type I expression was significantly blocked by knockdown of GRP78 (Figures 5A 5B). Together, the results suggest that ER stress is involved in the regulation of myofibroblastic differentiation. Also, with effective suppression of extracellular matrix production by 4-PBA, we suggest a role for 4-PBA as a novel therapeutic agent for pulmonary fibrosis.
Figure 4. Effects of chemical chaperones on TGF-β1–induced endoplasmic reticulum stress and myofibroblastic differentiation. Human (A–D) and mouse (E–H) lung fibroblasts were cultured in the absence or presence of a chemical chaperone, sodium 4-phenylbutyrate (4-PBA) (5 mM), for 4 hours and treated with TGF-β1 (2.5 ng/mL) for the times indicated. Whole-protein lysates from the cells were subjected to Western blotting using GRP78, ATF6α, and XBP-1–specific antibodies (A, E). α-SMA and collagen type I were analyzed by Western blotting (C, G). The expression of GRP78, ATF-6α, XBP-1, collagen type I, and α-SMA was normalized to that of GAPDH and is shown as mean ± SE (n = 3). *P < 0.001 and **P < 0.005 compared with control or TGF-β1, respectively.
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Figure 5. Knockdown of GRP78 leads to suppression of myofibroblastic differentiation. Human lung fibroblasts were transfected with or without GRP78 siRNA. The cells were treated with TGF-β1 or tumicamycin ™ for 24 hours and the cells were harvested for the identification of α-SMA and collagen type I by Western blotting. Data are expressed as mean ± SE of three independent experiments. *P < 0.05 and **P < 0.05 compared with the respective control.
[More] [Minimize]Previous studies have suggested that ROS and ER stress have the ability to influence each other (12, 13, 28, 29). We hypothesized that there is an association between the induction of ROS and UPR during TGF-β1–induced myofibroblastic differentiation. We first examined production of ROS in the MLg cells after treatment with TGF-β1. Cells were stimulated with TGF-β1, loaded with the ROS-responsive fluorescent probe 2′,7′-dichlorfluorescein-diacetate (DCF-DA), and subjected to flow cytometry. After exposure to TGF-β1, substantial induction of fluorescence was observed and showed a bimodal pattern (Figure E4). DCF-DA displayed a rapid increase in fluorescence intensity with the maximal intensity at 20 minutes after treatment with TGF-β1, and the intensity declined to the basal level by 2 hours. The delayed ROS production reached a maximum at 24 hours and normalized at 48 hours. Next, the effect of antioxidants on TGF-β1–induced α-SMA and collagen type I expression was analyzed. Cells were stimulated with TGF-β1 in the presence of NAC or GSH for 4 hours, and then ROS production and collagen type I and α-SMA expression were analyzed by flow cytometry and Western blotting, respectively. The level of intracellular ROS was markedly decreased by NAC or GSH (Figures 6A and 6B), and pretreatment with antioxidants also attenuated TGF-β1–induced α-SMA and collagen type I protein expression (Figures 6C and 6D). These results indicate that ROS is involved in TGF-β1–induced α-SMA and collagen type I production. Then, we investigated whether there is cross-talk between ROS and ER stress during TGF-β1–induced myofibroblastic differentiation. The MLg cells were treated with TGF-β1 in the presence of NAC or GSH for 4 hours, and the expression of GRP78 was analyzed by Western blotting. Pretreatment with antioxidants attenuated the expression of GRP78 (Figures 7A and 7B). This result suggests that ROS generation may be upstream UPR targets. However, in contrast to the above results, prolonged UPR activation leads to the accumulation of ROS (13). We next examined whether treatment with 4-PBA can affect ROS generation. The MLg cells were treated with TGF-β1 for 20 minutes or for 24 hours in the presence or absence of 5 mM 4-PBA, and ROS generation was analyzed by flow cytometry. We chose these two time points because ROS generation showed two peaks at these time points. ROS generation was suppressed by treatment with 4-PBA (Figures 7C and 7D). The effects of 4-PBA on ROS generation were different between these two time points after TGF-β1 stimulation. By treatment with 4-PBA, the effect of TGF-β1 on ROS generation at 20 minutes decreased to 80.22 ± 3.97% of that by treatment with TGF-β1 alone (P < 0.001); however, at 24 hours, ROS generation was markedly suppressed to 16.44 ± 2.28% of that by treatment with TGF-β1 alone (P < 0.001) (Figures 7C and 7D). These results suggest that the late induction of ROS may be affected by UPR. Consistent with the mouse lung fibroblasts findings, ROS and ER stress influenced each other in human lung fibroblasts (Figure E5). Taken together, these results suggest that ER stress and oxidative stress are mutually interactive during TGF-β1–induced myofibroblastic differentiation.
Figure 6. Effect of reactive oxygen species (ROS) on TGF-β1–induced collagen type I and α-SMA expression. (A) ROS assays on antioxidant-treated cells. MLg cells were treated with N-acetyl-l-cysteine (NAC) (5 mM) or l-glutathione reduced (GSH) (5 mM) for 30 minutes before treatment with TGF-β1 (2.5 ng/mL) for the indicated time periods and treated with 10 μM DCF-DA at 37°C for an additional 15-minute ROS accumulation was analyzed by flow cytometry. (B) Statistic diagram of ROS assays. Data are expressed as mean fluorescence intensity of three independent experiments. Values are represented as mean ± SE. *P < 0.001 and **P < 0.001 compared with control or TGF-β1, respectively. (C) Effect of antioxidants on collagen type I and α-SMA expression. The MLg cells were treated with NAC or GSH for 30 minutes before treatment with TGF-β1 for 24 hours. The expression of collagen type I and α-SMA was analyzed by Western blotting. (D) Statistic diagram of collagen type I and α-SMA expression. Data are normalized to GAPDH expression and expressed as relative intensity (mean ± SE) (n = 3). *P < 0.001 and **P < 0.001 compared with control or TGF-β1, respectively.
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Figure 7. Cross-talk between endoplasmic reticulum stress and oxidative stress. Effects of antioxidants on GRP78 in mouse (A, B) lung fibroblasts. Cells were treated with TGF-β1 for the indicated time points in the presence of NAC or GSH for 4 hours, and expression of GRP78 was analyzed by Western blotting. (B) Statistic diagram of GRP78 expression. Data are normalized to GAPDH expression and expressed as relative intensity (mean ± SE) (n = 3). *P < 0.001 and **P < 0.001 compared with control or TGF-β1, respectively. (C) Effects of chemical chaperone on ROS generation. Mouse lung fibroblasts were treated with 4-PBA (5 mM) for 4 hours before TGF-β1 treatment (2.5 ng/ml) for the indicated time points, and ROS accumulation was analyzed by flow cytometry. (D) Statistic diagram of ROS assays. Data are expressed as mean fluorescence intensity of three independent experiments. Values are represented as mean ± SE. *P < 0.001 and **P < 0.001 compared with control or TGF-β1, respectively.
[More] [Minimize]IPF is a fatal lung disease characterized by progressive interstitial fibrosis. Despite extensive research, its precise pathogenesis is unknown. Recently, ER stress has been implicated in AEC apoptosis in IPF and in the induction of α-SMA associated with epithelial–mesenchymal transition in AECs (21, 23, 30). We hypothesized that ER stress is also associated with α-SMA expression in fibroblasts (myofibroblastic differentiation of fibroblasts) and investigated the role of ER stress on myofibroblastic differentiation of fibroblasts. This study demonstrated that (1) GRP78 expression was up-regulated in fibroblasts of IPF lungs; (2) TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts; (3) 4-PBA suppressed ER stress and α-SMA and collagen type I expression in fibroblasts; and (4) ROS generation was involved in TGF-β1–induced UPR.
The previous studies related to the role of ER stress in IPF have been focused on AEC apoptosis (21, 30). Chronic epithelial injury has recently been suggested as a key event of IPF, and severe ER stress of AECs represents a major reason for apoptosis of these cells in IPF (21). In this study, we observed up-regulation of GRP78 not only in AECs but also in fibroblasts in lung tissue from patients with IPF, indicating that ER stress may also play a role in fibroblasts. Myofibroblastic differentiation of fibroblasts is one of the key processes mediating pulmonary fibrosis (31). Therefore, we first studied the expression of UPR targets during TGF-β1–induced myofibroblastic differentiation of fibroblasts. TGF-β1 up-regulated the expression of GRP78, ATF6α (p60), and XBP-1, which was accompanied by induction of α-SMA and collagen type I expression. ER stress inducers, TM, or TG increased expression of GRP78, α-SMA, and collagen type I in lung fibroblasts. 4-PBA supplementation and transfection of GRP78 siRNA decreased TGF-β1–induced UPR and myofibroblastic differentiation. These results clearly indicate that ER stress is involved in myofibroblastic differentiation of fibroblasts. Our study provides the first evidence implicating ER stress in myofibroblastic differentiation of fibroblasts during fibrosis. These results may be supported by the previous observation that ER stress is shown to be involved in cellular differentiation and in the induction of α-SMA (23, 32). XBP-1 has been shown to have a potential role in cellular differentiation, such as plasma cell differentiation, adipogenesis, T-cell differentiation, and the development of dendritic cells (33–35). ATF6 activation was also involved in cellular differentiation (36).
In this study, CHOP and phospho-eIF2α were not induced during TGF-β1–induced myofibroblastic differentiation. It is not clear why there is some selectivity in the UPR induced by ER stress during myofibroblastic differentiation. Under ER stress, CHOP and phospho-eIF2α are largely involved in apoptosis (19, 37–39) but are not induced during cellular differentiation (32). One of the functions of TGF-β1 is to create an antiapoptotic effect in myofibroblasts (40). We assume that the lack of induction of CHOP and phospho-eIF2α may be related to the antiapoptotic effect of TGF-β1.
An increasing number of studies have indicated that chemical chaperones can improve protein folding and reduce ER stress (7, 8). Recently, 4-PBA was reported to be capable of reducing ER stress and normalizing hyperglycemia in diabetic mice (8). In cultured retinal endothelial cells, inhibition of ER stress by 4-PBA ameliorated inflammation (41). We demonstrated that suppression of UPR by 4-PBA resulted in down-regulation of α-SMA and type I collagen expression. Although further evaluation of the therapeutic role of 4-PBA in pulmonary fibrosis is required, our results suggest that 4-PBA may have therapeutic potential for the treatment of IPF.
Previous studies have shown that ROS is a mediator of TGF-β1–induced fibrosis (11). Moreover, inhibitors of oxidative stress inhibited ROS formation and ER stress, and inhibitors of ER stress suppressed ROS production (12, 41). In the present study, we examined the possible connection between oxidative stress and UPR. Suppression of ROS accumulation by antioxidants resulted in a decrease in the expression of GRP78. Inhibition of ER stress with the chemical chaperone also resulted in reduced ROS generation. Thus, we suggest that oxidative stress and UPR were closely connected during TGF-β1–induced myofibroblastic differentiation. Regarding the sequence of events between ER stress and oxidant production, some studies have demonstrated that the ER stressor can trigger downstream oxidant generation (13). On the other hand, there are reports indicating ER stress downstream of oxidant production (42). In this study, the peak of ROS induction occurred as early as 20 minutes after treatment with TGF-β1, and pretreatment with antioxidants resulted in a marked decrease in GRP78 expression. Treatment with 4-PBA showed only a mild reduction in the ROS level at 20 minutes and a complete reduction of ROS at 24 hours after treatment with TGF-β1. Thus, we proposed that TGF-β1–induced UPR was mediated through the ROS generation and that late induction of ROS may be mediated by ER stress. Further studies need to be undertaken to confirm this proposed mechanism and to better understand the mechanistic link between ER stress and oxidative stress.
In conclusion, we demonstrated the involvement of the ER stress in myofibroblastic differentiation of fibroblast. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may help in improving our understanding of the pathogenesis of IPF and promoting the development of therapies for IPF.
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This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-E00042). The biospecimens for this study were provided by the Biobank of Chonbuk National University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare and Family Affairs.
Originally Published in Press as DOI: 10.1165/rcmb.2011-0121OC on August 18, 2011
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