Mol Cell Biochem (2015) 408:181–189 DOI 10.1007/s11010-015-2494-z
Autophagy regulates hyperoxia-induced intracellular accumulation of surfactant protein C in alveolar type II cells Liang Zhang1 • Shuang Zhao2 • Li-Jie Yuan3 • Hong-Min Wu1 • Hong Jiang4 Shi-Meng Zhao1 • Gang Luo4 • Xin-Dong Xue5
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Received: 24 March 2015 / Accepted: 18 June 2015 / Published online: 30 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Surfactant protein C (SP-C) deficiency is a risk factor for hyperoxia-induced bronchopulmonary dysplasia in newborn infants. However, the role of SP-C deficiency in the process is unclear. Here, using neonatal rat BPD model and MLE-12, mouse alveolar epithelial type II cell, we examined the changes of SP-C levels during hyperoxia. Immunohistochemistry, immunofluorescence, and ELISA analysis showed SP-C accumulation in alveolar epithelial type II cells. Electron microscopy further demonstrated the accumulation of lamellar bodies and the co-localization of lamellar bodies with autophagosomes in the cytoplasm of alveolar epithelial type II cells. The inhibition of autophagy with 3-Methyladenine and knockdown of Atg7 abolished hyperoxia-induced SP-C accumulation in the cytoplasm. Furthermore, inhibition of JNK signaling with SP600125 suppressed hyperoxia-induced Atg7 expression and SP-C accumulation. These findings suggest that hyperoxia triggers autophagy via JNK signaling-mediated Atg7 & Hong-Min Wu [email protected] 1
Department of Neonatology, The First Affiliated Hospital of China Medical University, 155 Nan Jing Northern Street, Shenyang 110001, Liaoning, China
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Department of Pediatrics, The Fourth People Hospital of Shenyang, 20 Huang He Street, Shenyang 110003, Liaoning, China
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Department of Biochemistry and Molecular Biology, Harbin Medical University, Daqing Campus, Daqing 163319, China
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Department of Pediatrics, The First Affiliated Hospital of China Medical University, 155 Nan Jing Northern Street, Shenyang 110001, Liaoning, China
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Department of Pediatrics, Shengjing Hospital of China Medical University, 36 San Hao Street, Shenyang 110004, Liaoning, China
expression, which promotes the accumulation of SP-C within alveolar epithelial type II cells. Our data provide a potential approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway. Keywords Hyperoxia Autophagy Surfactant protein C Alveolar type II cells
Introduction Supplemental oxygen therapy is an important clinical application, but oxygen toxicity in the lung resulting from hyperoxia is a major impediment in the use of oxygen therapy [1]. Hyperoxia is administered to premature newborn infants, which is believed to be a factor in the development of bronchopulmonary dysplasia (BPD) [2]. High oxygen results in inflammation, alveolar epithelial cell apoptosis, reactive proliferation of type II pneumocytes, altered expression of surfactant proteins, broadening of alveolar septa, and parenchymal consolidation [3, 4]. Alveolar type II cells are primary target of hyperoxia-induced lung injury [5]. Surfactant deficiency and immaturity are pivotal risk factors for developing BPD in preterm infants [6, 7]. The pulmonary surfactant proteins (SP) are secreted by alveolar type II cells, and lower surface tension and prevent atelectasis at end-expiration. There are four surfactantspecific proteins: SP-A and SP-D participate in host defense in the lung, whereas SP-B and SP-C contribute to the surface tension-lowering activity [8]. Pulmonary surfactant has been implicated in the protection of the lung against hyperoxic injury, and elevated expression of certain surfactant proteins occurs in the lungs of adult rats during adaptation to sub-lethal oxygen [9, 10]. Recent study
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indicated that all surfactant proteins show high level of expression at birth and decline during neonatal development in normoxia, whereas the expression of SP-A, -B, and -D but not SP-C is increased in hyperoxia [11]. The hydrophobic SP-C has been characterized for its effect on lamellar body formation and secretion, which is critical for the formation of surfactant monolayer to lower surface tension at the air/water interface [12]. However, SP-C expression and distribution in alveolar type II cells during hyperoxia are not well understood. In this study, we aimed to investigate the expression of SP-C in the lung exposed to hyperoxia. Our results demonstrated that hyperoxia induced the accumulation of SP-C in neonatal rat lung tissues and MLE-12 mouse lung epithelial type II cells. Furthermore, we found that hyperoxia induced the upregulation of autophagy gene Atg7 through the activation of c-jun N-terminal kinase (JNK) signaling, which contribute to the accumulation of SP-C in MLE-12 cells.
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inhibitors U0126 (100 nM), SB203580 (100 nM) for 1 h.
SP600125 (100 nM),
or
Animal experiments Wistar rats were bred and maintained in the animal facility of China Medical University Experimental Animal Center. The protocol was described previously [13]. Within 12 h of birth, pups were divided into two groups, group A hyperoxia-exposed group and group B normoxia-exposed group. Group A was pooled in Plexiglas chambers (Biosperix, NY, USA) into which oxygen was continuously delivered to keep a constant level of 90 % oxygen and CO2 concentration \0.5 %. The oxygen concentration was continuously monitored using Proox110-O2 and CO2 level was monitored with Proox-CO2 (Biospherix, NY, USA). The chamber contained soda lime in a container for the removal of excess CO2. Temperature and relative humidity were maintained at 22–27 and 50–70 %. Pups were sacrificed and the lungs were harvested at the end of 1, 3, 5, 7, and 14 days of exposure.
Materials and methods Cell lines and reagents MLE-12 mouse lung epithelial type II cells were purchased from ATCC (Manassas, VA, USA). SP-C rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JNK and phosphoJNK polyclonal antibodies were from Cell Signaling Technology (Danvers, MA, USA). ECL Plus Western Blotting Detection Reagents were from GE Healthcare (Diegem, Belgium). BCA Protein Assay Reagent Kit was obtained from Pierce (Rockford, IL, USA). ELISA assay kit for SP-C was purchased from Uscn Life Science Inc (Wuhan, China). Complete protease inhibitor cocktail was purchased from Roche (Indianapolis, IN, USA). U0126, SP600125, and SB203580 were purchased from Calbiochem (San Diego, CA, USA). 3-Methyladenine was purchased from Sigma Aldrich (St. Louis, MO, USA). Cells culture and cell treatment MLE-12 Cells were cultured in HITES (Hydrocortisone, Insulin, Transferrin, Estrogen) media containing RPMI1640, 2 % FBS, insulin (5 mg/mL), transferring (10 mg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), b-estradiol (10 nM), and HEPES (10 nM). Cells were seeded at 100,000 cells/ well in 30 mm plate and incubated in 5 % CO2 at 37 °C. After reaching the confluence, cells were exposed to hyperoxia (80 % oxygen ?5 % CO2) for the indicated time. For inhibition experiments, MLE-12 cells were pretreated with the
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Bronchoalveolar lavage and measurement of SP-C in by ELISA After general anesthesia with intraperitoneal injection of pentobarbital in the rats, a tracheostomy was placed with a 22-gage catheter. 1 ml normal saline was infused and removed gently with a tuberculin syringe. Lavage fluid was centrifuged (300 g, 10 min, 4 °C) to sediment cells and cell debris, and the supernatant was recovered. The concentration of SP-C in cell-free supernatant was determined using a mouse SP-C ELISA quantification kit according to the manufacturer’s instruction. Western blot analysis MLE-12 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktails. The cell lysates were centrifuged at 100009g at 4 °C. Supernatants were collected and the protein content in each sample was estimated using BCA Protein Assay Reagent Kit. Equal amounts of protein sample were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes, which were incubated with the indicated antibodies overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibody and developed with ECL Plus Western Blotting Detection Reagents. Immunohistochemistry All the left upper lobes were cut for immunohistochemical staining. The tissues were fixed in 10 % neutral formalin,
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embedded in paraffin, and cut into serial sections of 5 lm. Tissue sections were deparaffinized and rehydrated. Then, sections were heated to retrieve antigenicity. After blocking nonspecific binding with 10 % normal goat serum, sections were incubated overnight at 4 °C with rabbit antiSP-C, and then stained with EnVision-HRP kit (Dako, Glostrup, Denmark). The sections were lightly counterstained with hematoxylin, mounted with Permount (Thermo Fisher Scientific, Waltham, MA), and examined by light microscopy. Immunofluorescence MLE-12 cells were seeded on glass coverslips and fixed with 4 % paraformaldehyde. Next the cells were permeabilized with 0.1 % Triton X-100 and incubated with rabbit SP-C antibody for 1 h, followed by incubation with FITCconjugated secondary antibody. Cell nuclei were stained with DAPI (blue). The glass slides were analyzed using immunofluorescence microscopy (Olympus, Japan). Electron microscopy Rats were transcardially perfused with a fixation buffer containing 2.5 % glutaraldehyde and 4 % paraformaldehyde, followed by a perfusion with cold phosphate-buffered saline (PBS) at room temperature. The lungs were then kept in the fixation buffer for 48 h at room temperature. The lungs were cut as sections of 0.5 lm and stained with uranyl acetate and lead citrate. Cells exposed to normal oxygen or hyperoxia were washed with cold PBS at pH 7.2, fixed in 2.5 % glutaraldehyde at room temperature and fixed in 1.5 % osmium overnight. Cells were then stained with 2 % tannic acid and dehydrated in graded ethanol, and infiltrated with resin. All ultrathin sections were viewed under a transmission electron microscope (‘‘Leo 906’’; Zeiss). RNA interference MLE-12 cells were transfected with 25 nM Atg7 and JNK siRNAs, respectively. Non-targeting siRNAs were used as negative control. The siRNA sequences for Atg7 and JNK were as previously described [14, 15]. Transfections were carried out according to the guidelines for the DharmaFECTÒ siRNA Transfection Reagents (Dharmacon, Lafayette, CO). At 72 h after transfection, the levels of Atg 7 and JNK in MLE-12 cells were examined by Western blot analysis. Statistical analysis The values are expressed as mean ± SD. Statistical significance of the differences was analyzed by Student’s t test
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for comparisons between two groups and by a one-way ANOVA for comparisons among more than three groups. P \ 0.05 was considered statistically significant.
Results Hyperoxia induces the accumulation of SP-C in neonatal rat lung tissues To examine the expression of SP-C during hyperoxia, a preterm rat mode was established and the temporal expression of SP-C was detected by Western blot analysis. We observed significant increase in SP-C expression in a time-dependent manner after exposure to hyperoxia compared to control groups (Fig. 1a). However, ELISA assay of bronchoalveolar lavage fluid (BALF) indicated that hyperoxia led to a significant reduction of SP-C level in BALF (Fig. 1b). Next, the expression of SP-C in the lung tissue was detected by immunohistochemical staining. There was obvious increase in SP-A protein level after exposure to hyperoxia on day 5 and 7 (Fig. 1c). Because SP-C is produced by Type II alveolar epithelial cells (AECIIs), and kept in the lamellar bodies, a well marker of AECIIs, the ultrastructural structure was examined with electron micrography. The results showed multilamellar bodies and the autophagosomes in the cytoplasm of AECIIs after exposure to hyperoxia for 14 days. In addition, electron microscopy revealed some lamellar bodies within mature autophagosomes (Fig. 1d). These data suggest that hyperoxia induces the accumulation of SP-C in neonatal rat lung tissues. Hyperoxia induces the accumulation of SP-C in mouse alveolar epithelial type II cells To further confirm the above results, murine epithelial cell line MLE-12 was used as a model in vitro. In MLE-12 cells exposed to hyperoxia, we found that hyperoxia led to increased SP-C protein level in a time-dependent manner (Fig. 2a). Furthermore, ELISA assay of cell supernatants indicated that hyperoxia inhibited the release of SP-C from the cells (Fig. 2b). Immunofluorescence analysis showed significant increase of SP-C protein level in MLE-12 cells after exposure to hyperoxia for 24 h (Fig. 2c). More importantly, electron microscopy indicated not only the accumulation of lamellar bodies but also the co-localization of lamellar bodies and autophagosomes in MLE-12 cells (Fig. 2d). These results suggest that hyperoxia induces the accumulation of SP-C in mouse alveolar epithelial type II cell and autophagy might be involved in this process.
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Fig. 1 Hyperoxia induces the accumulation of SP-C in neonatal rat lung tissues. a The levels of SP-C proteins in hyperoxia-exposed and normal oxygen-exposed neonatal rat lungs on days 1, 3, 5, 7, and 14 were determined by Western blot analysis (n = 3). b The levels of SP-C proteins in bronchoalveolar lavage fluid (BALF) from hyperoxia-exposed and normal oxygen-exposed on days neonatal rat lungs on days 1, 3, 5, 7, and 14 were determined by ELISA. c The expression of SP-C was detected by immunohistochemical staining on formaldehyde fixed paraffin sections of neonatal rat lungs during normal postnatal development at birth day 7 and after exposure to
hyperoxia on day 5, 7, and 14. Magnification 9200. d Transmission electron micrographs of alvolar epithelial type II cells. a After exposure to normal oxygen for 7 days, alveolar epithelial type II cells were characterized by lamellar body (arrows) in the cytoplasm. b After exposure to hyperoxia for 7 days, many lamellar bodies (arrows) appeared in the cytoplasm of alveolar epithelial type II cells. c After exposure to hyperoxia for 7 days, some lamellar bodies within mature autophagosomes (arrows) were observed in the cytoplasm of alveolar epithelial type II cells. Scale bar 2 lm
Autophagy is involved in the accumulation of SP-C induced by hyperoxia
Atg7 regulates the accumulation of SP-C in MLE-12 cells
To confirm that autophagy is involved in the accumulation of SP-C induced by hyperoxia, 3-Methyladenine (3-MA), an inhibitor for autophagy, was used to inhibit the activation of autophagy signaling. We found that 3-MA inhibited hyperoxia-induced increase of SP-C protein level in the cytoplasm of MLE-12 cells (Fig. 3a, b). Further results from cell morphological examinations revealed that the inhibition of autophagy by 3-MA abolished the effects of hyperoxia on the distribution of SP-C and the appearance of lamellar bodies in MLE-12 cells (Fig. 3c, d). These data suggest that autophagy is involved in the accumulation of SP-C induced by hyperoxia.
To further demonstrate that autophagy was involved in the accumulation of SP-C induced by hyperoxia, we examined the expression of autophagy-related gene Atg7 in MLE-12 cells during hyperoxia. The results showed that the expression of Atg7 was increased in a time-dependent manner in MLE-12 cells exposed to hyperoxia (Fig. 4a). Next, we used Atg7 siRNAs to knockdown the expression of Atg7 in MLE-12 cells (Fig. 4b). Knockdown of Atg7 inhibited hyperoxia-induced increase of SP-C protein level in the cytoplasm (Fig. 4c), cell-free culture supernatant (Fig. 4d), and the distribution of SP-C (Fig. 4e) in MLE-12 cells. These results suggest that hyperoxia modulates the
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Fig. 2 Hyperoxia induces the accumulation of SP-C in mice alveolar epithelial type II cells. MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for the indicated time. The expression of SP-C in MLE-12 cells was examined by Western blot analysis (a, n = 3) and the levels of SP-C proteins in cell culture supernatant were determined by ELISA (b, n = 3). c MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The distribution of SP-C was detected by immunofluorescence (Green). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm. d Transmission electron
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microscopy of MLE-12 cells. a After exposure to normal oxygen for 24 h, MLE-12 cells were characterized by lamellar body (arrows) in the cytoplasm. b After exposure to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h, lamellar bodies (arrows) accumulated in the cytoplasm of MLE-12 cells. c After exposure to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h, some lamellar bodies within mature autophagosomes (arrows) were observed in the cytoplasm of MLE-12 cells. Scale bar 20 lm. (Color figure online)
Fig. 3 Autophagy is involved in the accumulation of SP-C induced by hyperoxia. MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h in the presence or absence of 3-Methyladenine (10 mM) in the culture medium. a The levels of SP-C in MLE-12 cells were determined by Western blot analysis (n = 3). b The levels of SP-C cell culture supernatant were determined by ELISA (n = 3). c The distribution of SP-C was detected by immunofluorescence (Green). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm. d Transmission electron microscopy of MLE-12 cells exposed to hyperoxia. Scale bar 20 lm. **P \ 0.01. (Color figure online)
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Fig. 4 Atg7 regulates the accumulation of SP-C in MLE-12 cells. a MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for the indicated time and the levels of Atg 7 were detected by Western blot analysis (n = 3). b MLE-12 cells were transfected with non-silencing siRNA or Atg 7 siRNAs and the levels of Atg7 in transfected cells were examined by Western blot analysis (n = 3). After transfection with siRNAs, MLE-12 cells were exposed to
hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The levels of SP-C in MLE-12 cells were detected by Western blot analysis (c, n = 3); the levels of SP-C in cell culture supernatant were detected by ELISA (d, n = 3); and the distribution of SP-C was detected by immunofluorescence (e). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm. *P \ 0.05, **P \ 0.01. (Color figure online)
expression of autophagy-related gene Atg7 to induce the accumulation of SP-C in alveolar epithelial type II cells.
increase of SP-C protein level in the cytoplasm (Fig. 5a), cell-free culture supernatant (Fig. 5b), and the distribution of SP-C (Fig. 5c) in MLE-12 cells. However, these effects were not abolished by p38 MAPK inhibitor SB203580 or ERK1/2 inhibitors U0126. These data suggest that the activation of JNK, rather than p38 MAPK and ERK1/2, is involved in the signaling cascade triggered by hyperoxia to induce the accumulation of SP-C in MLE-12 cells. To further demonstrate that MAPK-mediated activation of autophagy contribute to hyperoxia-induced SP-C accumulation, we examined the changes of Atg7 expression in MLE-12 cells when JNK signaling was inhibited by SP600125 or JNK siRNA. The results showed that both SP600125 and JNK siRNA inhibited hyperoxia-induced Atg7 expression in MLE-12 cells (Fig. 5d). Taken together, these data provide evidence to demonstrate that
Hyperoxia induces the upregulation of Atg7 through the activation of JNK signaling in MLE-12 cells To elucidate the signaling mechanisms underlying hyperoxia-induced SP-C accumulation in mouse alveolar epithelial type II cells, we focused on MAPK signaling pathways because previous studies have demonstrated that hydrogen peroxide induced ‘‘non-canonical autophagy’’ dependent on MAPK-mediated activation of Atg7 [16, 17]. Therefore, we pretreated MLE-12 cells with specific inhibitors for MAPK signaling U0126, SP600125, and SB203580 prior to hyperoxia stimulation. We found that JNK inhibitor SP600125 blocked hyperoxia-induced
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Fig. 5 Hyperoxia induces the upregulation of Atg7 through the activation of JNK in MLE12 cells. MLE-12 cells were pretreated with specific inhibitors U0126 (5 lM), SP600125 (5 lM), and SB203580 (5 lM) for 1 h, followed by exposure to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The levels of SPC in MLE-12 cells were examined by Western blot analysis (a, n = 3); and the levels of SP-C in cell culture supernatant were examined by ELISA (b, n = 3). MLE-12 cells were transfected with JNK siRNA or treated with SP600125 (5 lM), and then exposed to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The distribution of SP-C was detected by immunofluorescence (c). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm; and the levels of Atg7 were determined by Western blot analysis (d, n = 3). *P \ 0.05,**P \ 0.01. (Color figure online)
hyperoxia induces the upregulation of Atg7 through the activation of JNK, leading to hyperoxia-induced SP-C accumulation in mouse alveolar epithelial type II cells.
Discussion In this study, we reported that hyperoxia induced the accumulation of SP-C in neonatal rat lung tissues and mouse lung epithelial type II cells. Inhibition of autophagy signaling by specific inhibitor or the deletion of Atg7 resulted in impaired accumulation of SP-C in lung epithelial type II cells. In addition, we demonstrated that hyperoxia-induced autophagy in lung epithelial type II cells via JNK-mediated upregulation of Atg7. Surfactant is a developmentally and hormonally regulated, phospholipid-rich lipoprotein, and it is synthesized mainly by alveolar epithelial type II cells. A number of hormones, growth factors, and cytokines could regulate surfactant synthesis and modulate fetal lung development and maturation [18–21]. Previous studies demonstrated that chronic
exposure to hyperoxia caused structural changes in type II pneumocyte morphology, resulting in increased phospholipid synthesis, storage, and secreted phospholipid pools [22, 23]. In addition, endotoxin induced a rather uniform response of type II pneumocytes, with increased lamellar body sizes after short-term injury followed by increased surfactant storage [24, 25]. Similar to previous observations, by histological, immunohistochemical, and cytological analysis and the detection of SP-C levels in BALF and supernatants of MEL12 cells, we confirmed SP-C accumulation in alveolar epithelial type II cells. Therefore, our in vitro and in vivo data demonstrate that hyperoxia induces the accumulation of SPC in alveolar epithelial type II cells. Autophagy, a cellular homeostatic process responsible for lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues [26, 27]. In this study, we found lamellar bodies and autophagosomes within alveolar epithelial type II cells. Moreover, the contents of autophagosomes revealed the presence of lamellar bodies. These findings indicate the possibility of the involvement of autophagy in hyperoxia-
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induced SP-C accumulation. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states. During autophagy, cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded and the breakdown products are released for reutilization [27]. In addition to its known role in intracellular recycling, autophagy participates in protein secretion in certain specialized cell types. For example, conditional deletion of Atg7 in pancreatic beta cells led to the impairment of insulin secretion [28, 29]. Similarly, impaired granular exocytosis was observed after the disruption of autophagy in mast cells and osteoclasts [30, 31]. Although we found lamellar bodies within autophagosomes, the mechanisms by which autophagy potentially plays a role during high oxygen stress remain elusive. To better understand the mechanisms by which hyperoxia affects SP-C production in mouse lung epithelial cells, autophagy-related gene Atg7 was inhibited by siRNA. The results indicate that Atg7 knockdown led to the accumulation of SP-C within alveolar epithelial type II cells. Furthermore, the upregulation of Atg7 occurred in alveolar epithelial type II cells during hyperoxia. These data suggest that Atg7 mediates hyperoxia-induced SP-C accumulation. Mitogen-activated protein kinases (MAPKs) have been extensively connected with reactive oxygen species (ROS)mediated macroautophagy [16, 17]. In this study, we found that hyperoxia strongly induced the phosphorylation of JNK in alveolar epithelial type II cells, and specific inhibitor of JNK, SP600125, obviously abolished hyperoxiainduced upregulation of Atg7. Simultaneously, hyperoxiainduced accumulation of SP-C was abrogated by SP600125. These data indicate that hyperoxia induces the upregulation of autophagy-related gene Atg7 via the activation of JNK signaling, which promotes the accumulation of SP-C within alveolar epithelial type II cells. In summary, our findings suggest that hyperoxia triggers autophagy via JNK signaling mediated upregulation of Atg7, leading to the accumulation of SP-C in alveolar epithelial type II cells. Our data provide a new approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81170605).
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Autophagy regulates hyperoxia-induced intracellular accumulation of surfactant protein C in alveolar type II cells Liang Zhang1 • Shuang Zhao2 • Li-Jie Yuan3 • Hong-Min Wu1 • Hong Jiang4 Shi-Meng Zhao1 • Gang Luo4 • Xin-Dong Xue5
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Received: 24 March 2015 / Accepted: 18 June 2015 / Published online: 30 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Surfactant protein C (SP-C) deficiency is a risk factor for hyperoxia-induced bronchopulmonary dysplasia in newborn infants. However, the role of SP-C deficiency in the process is unclear. Here, using neonatal rat BPD model and MLE-12, mouse alveolar epithelial type II cell, we examined the changes of SP-C levels during hyperoxia. Immunohistochemistry, immunofluorescence, and ELISA analysis showed SP-C accumulation in alveolar epithelial type II cells. Electron microscopy further demonstrated the accumulation of lamellar bodies and the co-localization of lamellar bodies with autophagosomes in the cytoplasm of alveolar epithelial type II cells. The inhibition of autophagy with 3-Methyladenine and knockdown of Atg7 abolished hyperoxia-induced SP-C accumulation in the cytoplasm. Furthermore, inhibition of JNK signaling with SP600125 suppressed hyperoxia-induced Atg7 expression and SP-C accumulation. These findings suggest that hyperoxia triggers autophagy via JNK signaling-mediated Atg7 & Hong-Min Wu [email protected] 1
Department of Neonatology, The First Affiliated Hospital of China Medical University, 155 Nan Jing Northern Street, Shenyang 110001, Liaoning, China
2
Department of Pediatrics, The Fourth People Hospital of Shenyang, 20 Huang He Street, Shenyang 110003, Liaoning, China
3
Department of Biochemistry and Molecular Biology, Harbin Medical University, Daqing Campus, Daqing 163319, China
4
Department of Pediatrics, The First Affiliated Hospital of China Medical University, 155 Nan Jing Northern Street, Shenyang 110001, Liaoning, China
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Department of Pediatrics, Shengjing Hospital of China Medical University, 36 San Hao Street, Shenyang 110004, Liaoning, China
expression, which promotes the accumulation of SP-C within alveolar epithelial type II cells. Our data provide a potential approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway. Keywords Hyperoxia Autophagy Surfactant protein C Alveolar type II cells
Introduction Supplemental oxygen therapy is an important clinical application, but oxygen toxicity in the lung resulting from hyperoxia is a major impediment in the use of oxygen therapy [1]. Hyperoxia is administered to premature newborn infants, which is believed to be a factor in the development of bronchopulmonary dysplasia (BPD) [2]. High oxygen results in inflammation, alveolar epithelial cell apoptosis, reactive proliferation of type II pneumocytes, altered expression of surfactant proteins, broadening of alveolar septa, and parenchymal consolidation [3, 4]. Alveolar type II cells are primary target of hyperoxia-induced lung injury [5]. Surfactant deficiency and immaturity are pivotal risk factors for developing BPD in preterm infants [6, 7]. The pulmonary surfactant proteins (SP) are secreted by alveolar type II cells, and lower surface tension and prevent atelectasis at end-expiration. There are four surfactantspecific proteins: SP-A and SP-D participate in host defense in the lung, whereas SP-B and SP-C contribute to the surface tension-lowering activity [8]. Pulmonary surfactant has been implicated in the protection of the lung against hyperoxic injury, and elevated expression of certain surfactant proteins occurs in the lungs of adult rats during adaptation to sub-lethal oxygen [9, 10]. Recent study
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indicated that all surfactant proteins show high level of expression at birth and decline during neonatal development in normoxia, whereas the expression of SP-A, -B, and -D but not SP-C is increased in hyperoxia [11]. The hydrophobic SP-C has been characterized for its effect on lamellar body formation and secretion, which is critical for the formation of surfactant monolayer to lower surface tension at the air/water interface [12]. However, SP-C expression and distribution in alveolar type II cells during hyperoxia are not well understood. In this study, we aimed to investigate the expression of SP-C in the lung exposed to hyperoxia. Our results demonstrated that hyperoxia induced the accumulation of SP-C in neonatal rat lung tissues and MLE-12 mouse lung epithelial type II cells. Furthermore, we found that hyperoxia induced the upregulation of autophagy gene Atg7 through the activation of c-jun N-terminal kinase (JNK) signaling, which contribute to the accumulation of SP-C in MLE-12 cells.
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inhibitors U0126 (100 nM), SB203580 (100 nM) for 1 h.
SP600125 (100 nM),
or
Animal experiments Wistar rats were bred and maintained in the animal facility of China Medical University Experimental Animal Center. The protocol was described previously [13]. Within 12 h of birth, pups were divided into two groups, group A hyperoxia-exposed group and group B normoxia-exposed group. Group A was pooled in Plexiglas chambers (Biosperix, NY, USA) into which oxygen was continuously delivered to keep a constant level of 90 % oxygen and CO2 concentration \0.5 %. The oxygen concentration was continuously monitored using Proox110-O2 and CO2 level was monitored with Proox-CO2 (Biospherix, NY, USA). The chamber contained soda lime in a container for the removal of excess CO2. Temperature and relative humidity were maintained at 22–27 and 50–70 %. Pups were sacrificed and the lungs were harvested at the end of 1, 3, 5, 7, and 14 days of exposure.
Materials and methods Cell lines and reagents MLE-12 mouse lung epithelial type II cells were purchased from ATCC (Manassas, VA, USA). SP-C rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JNK and phosphoJNK polyclonal antibodies were from Cell Signaling Technology (Danvers, MA, USA). ECL Plus Western Blotting Detection Reagents were from GE Healthcare (Diegem, Belgium). BCA Protein Assay Reagent Kit was obtained from Pierce (Rockford, IL, USA). ELISA assay kit for SP-C was purchased from Uscn Life Science Inc (Wuhan, China). Complete protease inhibitor cocktail was purchased from Roche (Indianapolis, IN, USA). U0126, SP600125, and SB203580 were purchased from Calbiochem (San Diego, CA, USA). 3-Methyladenine was purchased from Sigma Aldrich (St. Louis, MO, USA). Cells culture and cell treatment MLE-12 Cells were cultured in HITES (Hydrocortisone, Insulin, Transferrin, Estrogen) media containing RPMI1640, 2 % FBS, insulin (5 mg/mL), transferring (10 mg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), b-estradiol (10 nM), and HEPES (10 nM). Cells were seeded at 100,000 cells/ well in 30 mm plate and incubated in 5 % CO2 at 37 °C. After reaching the confluence, cells were exposed to hyperoxia (80 % oxygen ?5 % CO2) for the indicated time. For inhibition experiments, MLE-12 cells were pretreated with the
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Bronchoalveolar lavage and measurement of SP-C in by ELISA After general anesthesia with intraperitoneal injection of pentobarbital in the rats, a tracheostomy was placed with a 22-gage catheter. 1 ml normal saline was infused and removed gently with a tuberculin syringe. Lavage fluid was centrifuged (300 g, 10 min, 4 °C) to sediment cells and cell debris, and the supernatant was recovered. The concentration of SP-C in cell-free supernatant was determined using a mouse SP-C ELISA quantification kit according to the manufacturer’s instruction. Western blot analysis MLE-12 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktails. The cell lysates were centrifuged at 100009g at 4 °C. Supernatants were collected and the protein content in each sample was estimated using BCA Protein Assay Reagent Kit. Equal amounts of protein sample were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes, which were incubated with the indicated antibodies overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibody and developed with ECL Plus Western Blotting Detection Reagents. Immunohistochemistry All the left upper lobes were cut for immunohistochemical staining. The tissues were fixed in 10 % neutral formalin,
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embedded in paraffin, and cut into serial sections of 5 lm. Tissue sections were deparaffinized and rehydrated. Then, sections were heated to retrieve antigenicity. After blocking nonspecific binding with 10 % normal goat serum, sections were incubated overnight at 4 °C with rabbit antiSP-C, and then stained with EnVision-HRP kit (Dako, Glostrup, Denmark). The sections were lightly counterstained with hematoxylin, mounted with Permount (Thermo Fisher Scientific, Waltham, MA), and examined by light microscopy. Immunofluorescence MLE-12 cells were seeded on glass coverslips and fixed with 4 % paraformaldehyde. Next the cells were permeabilized with 0.1 % Triton X-100 and incubated with rabbit SP-C antibody for 1 h, followed by incubation with FITCconjugated secondary antibody. Cell nuclei were stained with DAPI (blue). The glass slides were analyzed using immunofluorescence microscopy (Olympus, Japan). Electron microscopy Rats were transcardially perfused with a fixation buffer containing 2.5 % glutaraldehyde and 4 % paraformaldehyde, followed by a perfusion with cold phosphate-buffered saline (PBS) at room temperature. The lungs were then kept in the fixation buffer for 48 h at room temperature. The lungs were cut as sections of 0.5 lm and stained with uranyl acetate and lead citrate. Cells exposed to normal oxygen or hyperoxia were washed with cold PBS at pH 7.2, fixed in 2.5 % glutaraldehyde at room temperature and fixed in 1.5 % osmium overnight. Cells were then stained with 2 % tannic acid and dehydrated in graded ethanol, and infiltrated with resin. All ultrathin sections were viewed under a transmission electron microscope (‘‘Leo 906’’; Zeiss). RNA interference MLE-12 cells were transfected with 25 nM Atg7 and JNK siRNAs, respectively. Non-targeting siRNAs were used as negative control. The siRNA sequences for Atg7 and JNK were as previously described [14, 15]. Transfections were carried out according to the guidelines for the DharmaFECTÒ siRNA Transfection Reagents (Dharmacon, Lafayette, CO). At 72 h after transfection, the levels of Atg 7 and JNK in MLE-12 cells were examined by Western blot analysis. Statistical analysis The values are expressed as mean ± SD. Statistical significance of the differences was analyzed by Student’s t test
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for comparisons between two groups and by a one-way ANOVA for comparisons among more than three groups. P \ 0.05 was considered statistically significant.
Results Hyperoxia induces the accumulation of SP-C in neonatal rat lung tissues To examine the expression of SP-C during hyperoxia, a preterm rat mode was established and the temporal expression of SP-C was detected by Western blot analysis. We observed significant increase in SP-C expression in a time-dependent manner after exposure to hyperoxia compared to control groups (Fig. 1a). However, ELISA assay of bronchoalveolar lavage fluid (BALF) indicated that hyperoxia led to a significant reduction of SP-C level in BALF (Fig. 1b). Next, the expression of SP-C in the lung tissue was detected by immunohistochemical staining. There was obvious increase in SP-A protein level after exposure to hyperoxia on day 5 and 7 (Fig. 1c). Because SP-C is produced by Type II alveolar epithelial cells (AECIIs), and kept in the lamellar bodies, a well marker of AECIIs, the ultrastructural structure was examined with electron micrography. The results showed multilamellar bodies and the autophagosomes in the cytoplasm of AECIIs after exposure to hyperoxia for 14 days. In addition, electron microscopy revealed some lamellar bodies within mature autophagosomes (Fig. 1d). These data suggest that hyperoxia induces the accumulation of SP-C in neonatal rat lung tissues. Hyperoxia induces the accumulation of SP-C in mouse alveolar epithelial type II cells To further confirm the above results, murine epithelial cell line MLE-12 was used as a model in vitro. In MLE-12 cells exposed to hyperoxia, we found that hyperoxia led to increased SP-C protein level in a time-dependent manner (Fig. 2a). Furthermore, ELISA assay of cell supernatants indicated that hyperoxia inhibited the release of SP-C from the cells (Fig. 2b). Immunofluorescence analysis showed significant increase of SP-C protein level in MLE-12 cells after exposure to hyperoxia for 24 h (Fig. 2c). More importantly, electron microscopy indicated not only the accumulation of lamellar bodies but also the co-localization of lamellar bodies and autophagosomes in MLE-12 cells (Fig. 2d). These results suggest that hyperoxia induces the accumulation of SP-C in mouse alveolar epithelial type II cell and autophagy might be involved in this process.
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Fig. 1 Hyperoxia induces the accumulation of SP-C in neonatal rat lung tissues. a The levels of SP-C proteins in hyperoxia-exposed and normal oxygen-exposed neonatal rat lungs on days 1, 3, 5, 7, and 14 were determined by Western blot analysis (n = 3). b The levels of SP-C proteins in bronchoalveolar lavage fluid (BALF) from hyperoxia-exposed and normal oxygen-exposed on days neonatal rat lungs on days 1, 3, 5, 7, and 14 were determined by ELISA. c The expression of SP-C was detected by immunohistochemical staining on formaldehyde fixed paraffin sections of neonatal rat lungs during normal postnatal development at birth day 7 and after exposure to
hyperoxia on day 5, 7, and 14. Magnification 9200. d Transmission electron micrographs of alvolar epithelial type II cells. a After exposure to normal oxygen for 7 days, alveolar epithelial type II cells were characterized by lamellar body (arrows) in the cytoplasm. b After exposure to hyperoxia for 7 days, many lamellar bodies (arrows) appeared in the cytoplasm of alveolar epithelial type II cells. c After exposure to hyperoxia for 7 days, some lamellar bodies within mature autophagosomes (arrows) were observed in the cytoplasm of alveolar epithelial type II cells. Scale bar 2 lm
Autophagy is involved in the accumulation of SP-C induced by hyperoxia
Atg7 regulates the accumulation of SP-C in MLE-12 cells
To confirm that autophagy is involved in the accumulation of SP-C induced by hyperoxia, 3-Methyladenine (3-MA), an inhibitor for autophagy, was used to inhibit the activation of autophagy signaling. We found that 3-MA inhibited hyperoxia-induced increase of SP-C protein level in the cytoplasm of MLE-12 cells (Fig. 3a, b). Further results from cell morphological examinations revealed that the inhibition of autophagy by 3-MA abolished the effects of hyperoxia on the distribution of SP-C and the appearance of lamellar bodies in MLE-12 cells (Fig. 3c, d). These data suggest that autophagy is involved in the accumulation of SP-C induced by hyperoxia.
To further demonstrate that autophagy was involved in the accumulation of SP-C induced by hyperoxia, we examined the expression of autophagy-related gene Atg7 in MLE-12 cells during hyperoxia. The results showed that the expression of Atg7 was increased in a time-dependent manner in MLE-12 cells exposed to hyperoxia (Fig. 4a). Next, we used Atg7 siRNAs to knockdown the expression of Atg7 in MLE-12 cells (Fig. 4b). Knockdown of Atg7 inhibited hyperoxia-induced increase of SP-C protein level in the cytoplasm (Fig. 4c), cell-free culture supernatant (Fig. 4d), and the distribution of SP-C (Fig. 4e) in MLE-12 cells. These results suggest that hyperoxia modulates the
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Fig. 2 Hyperoxia induces the accumulation of SP-C in mice alveolar epithelial type II cells. MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for the indicated time. The expression of SP-C in MLE-12 cells was examined by Western blot analysis (a, n = 3) and the levels of SP-C proteins in cell culture supernatant were determined by ELISA (b, n = 3). c MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The distribution of SP-C was detected by immunofluorescence (Green). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm. d Transmission electron
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microscopy of MLE-12 cells. a After exposure to normal oxygen for 24 h, MLE-12 cells were characterized by lamellar body (arrows) in the cytoplasm. b After exposure to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h, lamellar bodies (arrows) accumulated in the cytoplasm of MLE-12 cells. c After exposure to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h, some lamellar bodies within mature autophagosomes (arrows) were observed in the cytoplasm of MLE-12 cells. Scale bar 20 lm. (Color figure online)
Fig. 3 Autophagy is involved in the accumulation of SP-C induced by hyperoxia. MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h in the presence or absence of 3-Methyladenine (10 mM) in the culture medium. a The levels of SP-C in MLE-12 cells were determined by Western blot analysis (n = 3). b The levels of SP-C cell culture supernatant were determined by ELISA (n = 3). c The distribution of SP-C was detected by immunofluorescence (Green). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm. d Transmission electron microscopy of MLE-12 cells exposed to hyperoxia. Scale bar 20 lm. **P \ 0.01. (Color figure online)
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Fig. 4 Atg7 regulates the accumulation of SP-C in MLE-12 cells. a MLE-12 cells were exposed to hyperoxia (80 % oxygen ? 5 % CO2) for the indicated time and the levels of Atg 7 were detected by Western blot analysis (n = 3). b MLE-12 cells were transfected with non-silencing siRNA or Atg 7 siRNAs and the levels of Atg7 in transfected cells were examined by Western blot analysis (n = 3). After transfection with siRNAs, MLE-12 cells were exposed to
hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The levels of SP-C in MLE-12 cells were detected by Western blot analysis (c, n = 3); the levels of SP-C in cell culture supernatant were detected by ELISA (d, n = 3); and the distribution of SP-C was detected by immunofluorescence (e). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm. *P \ 0.05, **P \ 0.01. (Color figure online)
expression of autophagy-related gene Atg7 to induce the accumulation of SP-C in alveolar epithelial type II cells.
increase of SP-C protein level in the cytoplasm (Fig. 5a), cell-free culture supernatant (Fig. 5b), and the distribution of SP-C (Fig. 5c) in MLE-12 cells. However, these effects were not abolished by p38 MAPK inhibitor SB203580 or ERK1/2 inhibitors U0126. These data suggest that the activation of JNK, rather than p38 MAPK and ERK1/2, is involved in the signaling cascade triggered by hyperoxia to induce the accumulation of SP-C in MLE-12 cells. To further demonstrate that MAPK-mediated activation of autophagy contribute to hyperoxia-induced SP-C accumulation, we examined the changes of Atg7 expression in MLE-12 cells when JNK signaling was inhibited by SP600125 or JNK siRNA. The results showed that both SP600125 and JNK siRNA inhibited hyperoxia-induced Atg7 expression in MLE-12 cells (Fig. 5d). Taken together, these data provide evidence to demonstrate that
Hyperoxia induces the upregulation of Atg7 through the activation of JNK signaling in MLE-12 cells To elucidate the signaling mechanisms underlying hyperoxia-induced SP-C accumulation in mouse alveolar epithelial type II cells, we focused on MAPK signaling pathways because previous studies have demonstrated that hydrogen peroxide induced ‘‘non-canonical autophagy’’ dependent on MAPK-mediated activation of Atg7 [16, 17]. Therefore, we pretreated MLE-12 cells with specific inhibitors for MAPK signaling U0126, SP600125, and SB203580 prior to hyperoxia stimulation. We found that JNK inhibitor SP600125 blocked hyperoxia-induced
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Fig. 5 Hyperoxia induces the upregulation of Atg7 through the activation of JNK in MLE12 cells. MLE-12 cells were pretreated with specific inhibitors U0126 (5 lM), SP600125 (5 lM), and SB203580 (5 lM) for 1 h, followed by exposure to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The levels of SPC in MLE-12 cells were examined by Western blot analysis (a, n = 3); and the levels of SP-C in cell culture supernatant were examined by ELISA (b, n = 3). MLE-12 cells were transfected with JNK siRNA or treated with SP600125 (5 lM), and then exposed to hyperoxia (80 % oxygen ? 5 % CO2) for 24 h. The distribution of SP-C was detected by immunofluorescence (c). Cell nuclei were stained with DAPI (blue). Scale bar 20 lm; and the levels of Atg7 were determined by Western blot analysis (d, n = 3). *P \ 0.05,**P \ 0.01. (Color figure online)
hyperoxia induces the upregulation of Atg7 through the activation of JNK, leading to hyperoxia-induced SP-C accumulation in mouse alveolar epithelial type II cells.
Discussion In this study, we reported that hyperoxia induced the accumulation of SP-C in neonatal rat lung tissues and mouse lung epithelial type II cells. Inhibition of autophagy signaling by specific inhibitor or the deletion of Atg7 resulted in impaired accumulation of SP-C in lung epithelial type II cells. In addition, we demonstrated that hyperoxia-induced autophagy in lung epithelial type II cells via JNK-mediated upregulation of Atg7. Surfactant is a developmentally and hormonally regulated, phospholipid-rich lipoprotein, and it is synthesized mainly by alveolar epithelial type II cells. A number of hormones, growth factors, and cytokines could regulate surfactant synthesis and modulate fetal lung development and maturation [18–21]. Previous studies demonstrated that chronic
exposure to hyperoxia caused structural changes in type II pneumocyte morphology, resulting in increased phospholipid synthesis, storage, and secreted phospholipid pools [22, 23]. In addition, endotoxin induced a rather uniform response of type II pneumocytes, with increased lamellar body sizes after short-term injury followed by increased surfactant storage [24, 25]. Similar to previous observations, by histological, immunohistochemical, and cytological analysis and the detection of SP-C levels in BALF and supernatants of MEL12 cells, we confirmed SP-C accumulation in alveolar epithelial type II cells. Therefore, our in vitro and in vivo data demonstrate that hyperoxia induces the accumulation of SPC in alveolar epithelial type II cells. Autophagy, a cellular homeostatic process responsible for lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues [26, 27]. In this study, we found lamellar bodies and autophagosomes within alveolar epithelial type II cells. Moreover, the contents of autophagosomes revealed the presence of lamellar bodies. These findings indicate the possibility of the involvement of autophagy in hyperoxia-
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induced SP-C accumulation. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states. During autophagy, cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded and the breakdown products are released for reutilization [27]. In addition to its known role in intracellular recycling, autophagy participates in protein secretion in certain specialized cell types. For example, conditional deletion of Atg7 in pancreatic beta cells led to the impairment of insulin secretion [28, 29]. Similarly, impaired granular exocytosis was observed after the disruption of autophagy in mast cells and osteoclasts [30, 31]. Although we found lamellar bodies within autophagosomes, the mechanisms by which autophagy potentially plays a role during high oxygen stress remain elusive. To better understand the mechanisms by which hyperoxia affects SP-C production in mouse lung epithelial cells, autophagy-related gene Atg7 was inhibited by siRNA. The results indicate that Atg7 knockdown led to the accumulation of SP-C within alveolar epithelial type II cells. Furthermore, the upregulation of Atg7 occurred in alveolar epithelial type II cells during hyperoxia. These data suggest that Atg7 mediates hyperoxia-induced SP-C accumulation. Mitogen-activated protein kinases (MAPKs) have been extensively connected with reactive oxygen species (ROS)mediated macroautophagy [16, 17]. In this study, we found that hyperoxia strongly induced the phosphorylation of JNK in alveolar epithelial type II cells, and specific inhibitor of JNK, SP600125, obviously abolished hyperoxiainduced upregulation of Atg7. Simultaneously, hyperoxiainduced accumulation of SP-C was abrogated by SP600125. These data indicate that hyperoxia induces the upregulation of autophagy-related gene Atg7 via the activation of JNK signaling, which promotes the accumulation of SP-C within alveolar epithelial type II cells. In summary, our findings suggest that hyperoxia triggers autophagy via JNK signaling mediated upregulation of Atg7, leading to the accumulation of SP-C in alveolar epithelial type II cells. Our data provide a new approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81170605).
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