- Email: [email protected]
GOLGA2 loss causes fibrosis with autophagy in the mouse lung and liver
Accepted Manuscript GOLGA2 loss causes fibrosis with autophagy in the mouse lung and liver Sungjin Park, Sanghwa Kim, Minjung Kim, Youngeun Hong, Ah Young Lee, Hyunji Lee, Quangdon Tran, Minhee Kim, Hyeonjeong Cho, Jisoo Park, Kwang Pyo Kim, Jongsun Park, Myung-Haing Cho PII:
S0006-291X(17)32227-1
DOI:
10.1016/j.bbrc.2017.11.049
Reference:
YBBRC 38842
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 6 November 2017 Accepted Date: 7 November 2017
Please cite this article as: S. Park, S. Kim, M. Kim, Y. Hong, A.Y. Lee, H. Lee, Q. Tran, M. Kim, H. Cho, J. Park, K.P. Kim, J. Park, M.-H. Cho, GOLGA2 loss causes fibrosis with autophagy in the mouse lung and liver, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.11.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: GOLGA2 Loss Causes Fibrosis with Autophagy in the Mouse Lung and Liver
Author information Sungjin Parka,*, Sanghwa Kimd,*, Minjung Kimc,*, Youngeun Honga, Ah Young Leeb, Hyunji Leea,
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Quangdon Trana, Minhee Kima, Hyeonjeong Choa, Jisoo Parka, Kwang Pyo Kimc,§, Jongsun Parka,§ & Myung-Haing Chob,e,f,g,h,§ a
Department of Pharmacology and Medical Science, Metabolic Syndrome and Cell Signaling
Laboratory, Cancer Research Institute, College of Medicine, Chungnam National University, Daejeon
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35015, Republic of Korea. b
Laboratory of Toxicology, Research Institute for Veterinary Science and College of Veterinary
c
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Medicine, Seoul National University, Seoul 08826, Republic of Korea.
Department of Applied Chemistry, College of Applied Science, Kyung Hee University, Yongin 17104,
Republic of Korea. d
Division of Basic Radiation Bioscience, Korea Institute of Radiological & Medical Science, Seoul, Republic of Korea
e
Graduate School of Convergence Science and Technology, Seoul National University, Suwon 16229, Republic of Korea f
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Graduate Group of Tumor Biology, Seoul National University, Seoul 08826, Republic of Korea
g
Advanced Institute of Convergence Technology, Seoul National University, Suwon 16229, Republic of Korea
h
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Institute of GreenBio Science Technology, Seoul National University, Pyeongchang 25354, Republic of Korea
*
These authors contributed equally to this work.
§
Correspondding
author.
E-mail:
[email protected]
(MHC);
[email protected]
(JP);
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[email protected] (KPK);
Abstract
Autophagy is a biological recycling process via the self-digestion of organelles, proteins, and lipids for energy-consuming differentiation and homeostasis. The Golgi serves as a donor of the doublemembraned phagophore for autophagosome assembly. In addition, recent studies have demonstrated that pulmonary and hepatic fibrosis is accompanied by autophagy. However, the relationships among Golgi function, autophagy, and fibrosis are unclear. Here, we show that the deletion of GOLGA2, encoding a cis-Golgi protein, induces autophagy with Golgi disruption. The induction of autophagy
ACCEPTED MANUSCRIPT leads to fibrosis along with the reduction of subcellular lipid storage (lipid droplets and lamellar bodies) by autophagy in the lung and liver. GOLGA2 knockout mice clearly demonstrated prefibrosis features such as autophagy-activated cells, densely packed hepatocytes, increase of alveolar macrophages, and decrease of alveolar surfactant lipids (dipalmitoylphosphatidylcholine). Therefore,
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we confirmed the associations among Golgi function, fibrosis, and autophagy. Moreover, GOLGA2
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knockout mice may be a potentially valuable animal model for studying autophagy-induced fibrosis.
Highlights
GOLGA2/GM130 loss induces autophagy with Golgi disruption in liver cells and tran
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sgenic mice.
GOLGA2/GM130 loss leads to degradation of lipid structures (LBs and LDs) by auto phagy.
GOLGA2/GM130 loss causes liver and lung fibrosis.
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1. Introduction
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Keywords: GOLGA2 knockout mice; Golgi disruption; autophagy; lung fibrosis; liver fibrosis
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Autophagy is a degradation pathway for organelles, proteins, and lipids via lysosomal fusion, resulting in the recycling of these energy sources. Hepatic fibrosis is associated with autophagy. Usually, the damaged hepatocyte region is replaced though the replication of neighboring hepatocytes. However, under a condition of heavy damage, activated hepatic stellate cells (HSCs) transform into myofibroblast-like cells, which ultimately results in hepatic fibrosis. The activation of HSCs consumes an extreme amount of energy. To produce the required energy source, the autophagy process leads to degradation of the abundant lipid droplets (LDs) in the HSCs [1, 2].
ACCEPTED MANUSCRIPT Several studies have shown that pulmonary fibrosis is also accompanied by autophagy. The levels of autophagy proteins were shown to be increased in both chronic obstructive pulmonary disease (COPD) and asthma patients [3, 4]. The fibrotic remodeling in COPD and asthma is associated with the transforming growth factor-beta (TGF-β) pathway [5]. TGF-β induces autophagy to produce
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collagen and fibronectin [6]. A recent study revealed that amiodarone (AD)-induced lung fibrosis led to the induction of autophagy in alveolar type II cells, which secreted lamellar bodies (LBs), a component of the surfactant. Interestingly, LC3B, an autophagy marker, was shown to be recruited to
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the membrane of the LB in AD-induced alveolar type II cells in greater amounts compared with control cells [7]. Before the secretion of surfactants, the LB stores components of the surfactants
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synthesized from the Golgi apparatus and rough endoplasmic reticulum (ER) [8]. The alteration of the surfactant system is a typical feature of lung diseases derived from fibrotic airway remodeling [9]. Autophagosome formation is initiated from the assembly of a double-membraned structure called the phagophore. ER–Golgi traffic contributes the machinery to form the autophagosome [10, 11]. The
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Golgi proteins are essential regulators of autophagy formation [12, 13]. A recent study showed that the down-regulated expression of GOLGA2/GM130, a cis-Golgi matrix protein, induces formation of the autophagosome via the release of GABARAP from the Golgi during mitosis [12]. Furthermore,
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GOLGA2 small hairpin RNA suppressed tumorigenesis through the induction of autophagy a KrasLA1 lung cancer mouse model [14]. However, the relationships among Golgi function, autophagy, and
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fibrosis have thus far remained unclear. Here, we provide new evidence that disruption of Golgi function leads to fibrosis through autophagy in the liver and lung tissues.
2. Materials and Methods
2.1. Cell culture and generation of a stable GOLGA2 knockout cell line Chang liver cells were cultured in Dulbecco's modified Eagle medium/high-glucose (WELGENE Inc., Daegu, South Korea) supplemented with 10% fetal bovine serum (Biowest, Kansas City, MO, USA), and 1% antibiotics-antimycotics (Life Technologies, Inc.) at 37°C in 5% CO2. The cells were transfected with GOLGA2 CRISPR/Cas9 and HDR plasmids using the Neon® transfection system for
ACCEPTED MANUSCRIPT electroporation (Invitrogen, Grand Island, NY, USA). After transfection, the cells were selected with culture medium supplemented with 0.5 µg/ml of puromycin (Sigma-Aldrich).
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2.2. Generation of GOLGA2/GM130 knockout mice The embryonic stem cell line with deletion from exon 1 to exon 26 of GOLGA2 was purchased from the Wellcome Trust Sanger Institute and used to generate the target gene. The embryonic stem cells were injected into C57B/L6J mouse blastocysts for subsequent injection into pseudopregnant female
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mice (Macrogen Inc., Seoul, Korea). The offspring were all heterozygous; thus, to obtain homozygous mice, the offspring were bred to each other. Germ-line transmission was confirmed in the offspring
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with polymerase chain reaction (PCR). We extracted DNA from the tails of the mice using Tissue Genomic DNA Isolation Kit Mini (CosmoGenetech Inc., Seoul, Korea). The primers GOLGA2 KO-F (5′-CTTCGTATAATGTATGCTATACGAAGTTATGCTAGC-3′)
and
Reg-GOLGA2-R
(5′-
ACTGAGCCGGTGAAAACTTAGAAGC-3′) were used to detect GOLGA2-/- (homozygous
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genotype) alleles, and the primers Mus GOLGA2 Exon 27 wt-F (5′-AAGACTGGCGGCCAAAGCC3′) and Reg-GOLGA2-R were used to detect GOLGA2+/+ alleles. All animals used in this study were maintained under the animal protocols of Seoul National University guidelines and the study was
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Seoul, Korea).
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approved by the Animal Care and Use Committee at Seoul National University (SNU-141007-1;
2.3. Chemicals, primers, antibody sources, and plasmids The following antibodies, chemicals, and plasmids were included: GM130 (Alexa 647-conjugated) from BD Biosciences; SQSTM1 and LC3B from Sigma-Aldrich; alpha-smooth muscle actin (αSMA) from Abcam; β-actin and GM130 CRISPR knockout plasmids (sc-400787 and sc-400787 HDR) from Santa Cruz Biotechnology; HCS LipidTOXtm Deep Red neutral lipid stains from Invitrogen; and Alexa Fluor 488-labeled anti-rabbit antibodies from Life Technologies. The free fatty acids (FFAs) included palmitic acid and oleic acid (1:2, respectively). The sequences of primers used for quantitative PCR were mouse GOLGA2, forward (5′-CAGGCAGACAGGTATAACAAG-3′) and
ACCEPTED MANUSCRIPT reverse (5′-CGGAGTTTCTCTTCCAGTTC-3′).
2.4. Immunofluorescence Chang cells were cultured on 8-well chamber slides (SPL Life Sciences), fixed in 3%
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paraformaldehyde (EMD Millipore Corporation) for 10 min at 4°C, permeabilized with 0.25% Triton® X-100 (Sigma-Aldrich) for 5 min, blocked for 30 min, and incubated with primary or secondary antibodies in blocking solution [in phosphate-buffered saline with 0.1% saponin (Sigma-
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Aldrich) and 3% bovine serum albumin (BOVOGEN)].
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2.5. Immunoblot analysis
The cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1% v/v Nonidet P-40, 120 mM NaCl, 25 mM sodium fluoride, 40 mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 2 mM microcystin-LR for 30 min at 4°C. Following centrifugation at 12,000 ×g for 30 min, the protein concentration was measured, and
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equal amounts of lysate were used for immunoblotting. Western blotting was performed with specific antibodies and anti-rabbit antibody conjugated to horseradish peroxidase (KOMA Biotechnology). Visualization was achieved with chemiluminescence through X-ray film exposure (Agfa-Gevaert
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N.V).
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2.6. Histology and immunohistochemistry The liver and lung tissues of male mice were fixed in 10% formalin. After paraffin embedding, tissue blocks were cut to a thickness of 4 µm and transferred to a silane-coated microslide (MUTO Pure Chemicals Co., Ltd.). For histological analysis, deparaffinized tissue sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation or Sirius Red (Polysciences, Inc. for the lung; Sigma for the liver) for detection of collagen accumulation according to the manufacturer’s instructions. For immunohistochemistry, deparaffinized tissue sections were boiled with retrieval buffer for 10 min by microwave, and then the slides were incubated in 5% hydrogen peroxide for 15
ACCEPTED MANUSCRIPT min to quench endogenous peroxidase activity. After washing in Tris-buffered saline plus Tween 20, the sections were blocked for 1 h at room temperature. The indicated primary antibody and secondary horseradish peroxide-conjugated antibody, in turn, were applied onto the tissue slides. After washing, the tissue slides were reacted with DAB substrate solution (DAKO) for the appropriate reaction and
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quickly washed with distilled water. Finally, the tissue slides were counterstained with hematoxylin, dehydrated, and then mounted. The samples were observed using a light microscope (Eclips Ti-S,
2.7. Transmission electron microscope (TEM) analysis
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Nikon, Japan).
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The tissues were fixed with 2.5% glutaraldehyde in 1% osmium tetroxide buffer at 4°C overnight. The fixed tissues were dehydrated with a diluted ethanol series from 50% to 100%, and infiltrated in propylene oxide Epon resin at 70°C overnight. Ultrathin sections of a thickness of 40 mm were placed
2.8. Statistical analysis
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on a copper grid and observed using a JEM1010 TEM (JEOL, Tokyo, Japan).
Data are expressed as the mean ± SEM. Student’s t-test was used for comparisons between two
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Software).
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groups. All statistical analyses were performed using GraphPad Software version 5.01 (GraphPad
3. Results
3.1. Knockout of GOLGA2 induces autophagy in liver Chang cells Consistent with our previous study [14], to determine whether GOLGA2 deletion affects autophagy induction in Chang cells, we first established the stable GOLGA2 knockout cell line by the CRISPR/Cas9 system with a knock-in of a selection marker (puromycin), and confirmed GOLGA2 loss by immunofluorescence. GOLGA2 expression in knockout cells was not observed in the perinuclear region, which localizes at a single Golgi, compared to the control cells (Fig. 1A).
ACCEPTED MANUSCRIPT Immunoblot analysis further revealed that the expression levels of both SQSTM1/p62 and LC3-II, autophagy markers, were elevated in the GOLGA2 knockout cells (Fig. 1B). Next, to determine whether GOLGA2 knockout led to an increase in FFA-dependent autophagy, we performed immunofluorescence of SQSTM and LD staining of FFA (250 µM, palmitic acid:oleic acid = 1:2)-
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treated Chang cells for 24 h. The immunofluorescence revealed that the SQSTM level was increased in the FFA-treated GOLGA2 knockout cells. In contrast, the size of the LDs was decreased in the FFA-treated GOLGA2 knockout cells, indicating that the increased autophagy lipolyzed the LDs [15]
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autophagy in both normal and autophagy-induced conditions.
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in GOLGA2 knockout cells (Fig. 1C). Thus, these findings suggest that GOLGA2 loss induces
3.2. Generation and identification of GOLGA2 knockout mice
To generate a GOLGA2 knockout mouse model, embryonic stem cells with GOLGA2 exon 1 to exon 26 deleted were injected into C57BL/6 blastocysts to transfer into a uterine of pseudopregnant female
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mice (Fig. 2A). Finally, generated GOLGA2 (+/-) mice were inbred, and the offspring were identified by PCR analysis for genotyping (Fig. 2B). The GOLGA2 (-/-) male mice did not show any expression of GOLGA2 mRNA in all organs examined, including the lung, liver, brain, heart, and kidney (Fig. 2C
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and D). Wild type (+/+) and heterozygous (+/-) mice were born at the expected Mendelian ratio, whereas GOLGA2 knockout pups (-/-) were born at only 40% of the expected number, suggesting that
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GOLGA2 knockout might cause perinatal lethality in embryonic development (Fig. 2E). GOLGA2 knockout male mice also showed growth retardation (Fig. 2F). In detail, the body length and body weight of GOLGA2-/- mice were significantly decreased compared with those of the wild type (+/+) and heterozygous (+/-) mice (Fig. 2G and H).
3.3. GOLGA2 loss induces autophagy in the liver and lung tissues of GOLGA2 knockout mice We assessed whether the GOLGA2 deletion induces autophagy in the liver and lung of the GOLGA2 knockout mice. The expression level of LC3B was clearly increased in both the liver and lung tissues
ACCEPTED MANUSCRIPT in GOLGA2 knockout mice (Fig. 3A and B). The TEM images of the liver and lung tissues showed changed phenotypes of the subcellular organelles. The morphological changes showed “autophagosomal-forming” structures [16] that partially wrapped the mitochondria with the doublemembraned phagophore in the liver tissue of GOLGA2 knockout mice. In addition, the TEM images
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revealed that the phenotype of the Golgi stack [17, 18] and LD were not detected in the liver cells of GOLGA2 knockout mice (Fig. 3C). The TEM images of the lungs of GOLGA2 knockout mice showed several smaller-sized LBs, an indicator of alveolar type II cells, suggesting a phenotype of discharged
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LBs [19] (Fig. 3D). Next, to monitor dipalmitoyl-phosphatidylcholine (DPPC), which is known as a major lipid of surfactants [20], we performed lipid-scanning analysis of the lungs of GOLGA2
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knockout and control mice using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The lipid scanning analysis revealed that the DPPC level was decreased in the lungs of GOLGA2 knockout mice compared to that in wild type mice. Consistent with the imaged composition patterns of DPPC in the lungs, the levels of other phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) were also found to be decreased in the lungs of GOLGA2
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knockout mice (Fig. 3E and Supplementary Fig. S1). Taken together, these findings suggest that GOLGA2 loss may impair the pulmonary surfactant system.
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3.4. Effect of GOLGA2 loss on lung and liver fibrosis Histological analyses (H&E staining) in GOLGA2 knockout mice showed several alterations in the
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liver and lungs compared to wild type mice. The densities of hepatic cells were increased in the liver of GOLGA2 knockout mice (Fig. 4A, left panels). The number of alveolar macrophages was increased in the alveoli of GOLGA2 knockout mice, suggesting that GOLGA2 loss stimulated the immune system in the lung (Fig. 4B, left panels). Next, to assess collagen accumulation, Sirius Red staining was performed in the liver and lung of GOLGA2 knockout and wild type mice. These results revealed typical fibrotic alterations such as the accumulation and deposition of extracellular matrix in the liver and lung of GOLGA2 knockout mice (Fig. 4A and B; middle and right panels). In addition, the αSMA level, a marker of myofibroblast-like formation, was elevated around the portal vein of
ACCEPTED MANUSCRIPT GOLGA2 knockout mice (Fig. 4C). Moreover, immunoblot analyses showed that the level of SMAD4, which is downstream of the TGF-β pathway and is known as an important signaling pathway of fibrogenesis [21], was drastically increased in the liver of GOLGA2 knockout mice (Fig. 4D). Thus, these results suggested that GOLGA2 loss induces fibrosis in the liver and lung tissues
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(Fig. 4E)
4. Discussion
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The Golgi plays an important role in the glycosylation, sorting, and transport of proteins within the intracellular system. Golgi disruption has been closely linked to various diseases, including cancer
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[22]. Previous studies have demonstrated that an abnormal Golgi structure derived from various sources of external or internal damage can promote the accumulation of misfolded proteins and Golgi fragmentation, eventually causing autophagy [23, 24]. Our previous study revealed that the downregulation of GOLGA2 expression resulted in an abnormal Golgi structure such as swelling of the Golgi cisternae along with the induction of autophagy in human lung cancer cells and mouse lung
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cancer tissues [25]. Consistently, our present results showed the induction of autophagy in the GOLGA2-deleted stable cell line and GOLGA2 knockout mice (Figs. 1 and 3).
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These results raise the question as to how autophagy is induced in the tissue cells. The autophagy protein LC3 is recruited to the LBs in alveolar type II cells during pulmonary fibrosis [7]. In the
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present study, the TEM images indicated that the morphological alteration of LBs, which was manifested by the discharge and vacuolization of the LBs, may be associated with the induction of autophagy in the lungs of GOLGA2 knockout mice. (Fig. 3). The LBs participate in the secretory function of the pulmonary surfactant [26]. The pulmonary surfactant is synthesized, secreted, and recycled by type II cells, which is modified in the ER, transported through the Golgi, and then stored in the LBs. In addition, the secretion of the pulmonary surfactant maintains the alveolar surface tension by the formation of a surface-active film. The alveolar surfactant pool is degraded by alveolar macrophages [27]. In particular, the alveolar macrophages participate in the catabolism and clearance of DPPC [28], which modulates the inflammatory response [29]. A few of the autophagy-activated
ACCEPTED MANUSCRIPT cells shown in Fig. 3B may be type II cells or alveolar macrophages. These cells may lead to the degradation of lipids through an autophagy process in the lungs of GOLGA2 knockout mice. Consistently, our present results showed that GOLGA2 loss leads to the consumption of lipids along with the induction of autophagy in liver Chang cells (Fig. 1). Moreover, the lipids imaging analysis
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revealed the decrease in the levels of several PCs and LPCs along with the decrease of the DPPC level in the lungs of GOLGA2 knockout mice (Supplementary Fig. S1). Therefore, our results provide the possibility that the discharge of LBs by GOLGA2 loss can impair the balance between biogenesis and
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the degradation of pulmonary surfactants, eventually leading to fibrogenesis in the lungs of GOLGA2 knockout mice by inducing autophagy.
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Interestingly, we also confirmed hepatic fibrosis by inducing autophagy in GOLGA2 knockout mice. The activation of HSCs from liver damage is a major mechanism of fibrogenesis [30, 31]. The abundant LDs of the HSCs supply energy from the degradation of LDs by autophagy [32]. Thus, the excessive induction of the autophagy process may be an important contributor to fibrogenesis, by
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switching HSCs to myofibroblast-like cells [33]. As a result, no LDs were observed in the liver cells of GOLGA2 knockout mice and the hepatocytes were densely packed (Figs. 3 and 4). Consistent with previous studies, these observations represent the typical features of hepatic fibrosis. However, unlike
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chemical-induced hepatic fibrosis such as with CCL4 in which the fibrosis appears strongly in both the perivenular and periportal regions [34], the pattern of hepatic fibrosis caused by GOLGA2 gene loss is
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presented diffusely in the whole liver (Fig. 4). Taken together, our study provides the first demonstration that genetic dysfunction of the Golgi contributes to fibrogenesis. In further studies, it will be important to confirm whether similar fibrotic progression may occur in other tissues of GOLGA2 knockout mice. We expect that the GOLGA2 knockout mice will serve as a good model for fibrosis research once overcoming the problems of embryo/fetal lethality and the shortened lifespan.
Acknowledgments
ACCEPTED MANUSCRIPT This work was financially supported by the National Research Foundation of Korea (NRF) grant funded
by
the
Korea
Government
(MEST)
(NRF-2012M3A9B6055302,
NRF-
2015R1A2A2A01003597, NRF-2017R1A6A3A11031556). The authors declared no conflict of
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interest.
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ACCEPTED MANUSCRIPT questions, Physiology (Bethesda), 25 (2010), pp. 132-141. [28] O. Gurel, M. Ikegami, Z.C. Chroneos, A.H. Jobe, Macrophage and type II cell catabolism of SPA and saturated phosphatidylcholine in mouse lungs, Am. J. Physiol. Lung Cell. Mol. Physiol., 280 (2001), pp. L1266-1272.
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[29] J.R. Wright, Pulmonary surfactant: a front line of lung host defense, J. Clin. Invest., 111 (2003), pp. 1453-1455.
[30] M. Zeisberg, C. Yang, M. Martino, M.B. Duncan, F. Rieder, H. Tanjore, R. Kalluri, Fibroblasts
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derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition, J. Biol. Chem., 282 (2007), pp. 23337-23347.
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[31] R.C. Benyon, J.P. Iredale, Is liver fibrosis reversible?, Gut, 46 (2000), pp. 443-446. [32] T. Fujimoto, Y. Ohsaki, J. Cheng, M. Suzuki, Y. Shinohara, Lipid droplets: a classic organelle with new outfits, Histochem. Cell Biol., 130 (2008), pp. 263-279.
[33] R.K. Moreira, Hepatic stellate cells and liver fibrosis, Arch. Pathol. Lab. Med., 131 (2007), pp. 1728-1734.
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[34] T. Fujii, B.C. Fuchs, S. Yamada, G.Y. Lauwers, Y. Kulu, J.M. Goodwin, M. Lanuti, K.K. Tanabe, Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression
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Figure legends
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of CD133 and epidermal growth factor, BMC Gastroenterol., 10 (2010).
Fig. 1. GOLGA2 deletion increases autophagy in liver Chang cells. (A) Representative images of immunofluorescence staining for GOLGA2 (red) in GOLGA2 knockout Chang cells compared with control Chang cells. Nuclei are labeled with Hoechst. (B) Immunoblot analyses of SQSTM and LC3 in GOLGA2 knockout and control Chang cells. β-actin was used as a loading control. (C) Representative images of SQSTM and of lipid droplets in FFA-treated GOLGA2 knockout and control Chang cells. Scale bar, 10 µm.
ACCEPTED MANUSCRIPT Fig. 2. Generation of GOLGA2 knockout mice. (A) Genomic structure of the mouse GOLGA2 gene (top column), and illustration of recombinant deleted GOLGA2 ES cells (bottom column). (B) PCR genotyping of the GOLGA2 (+/+, wild type), GOLGA2 (+/-, heterozygote), and GOLGA2 (-/-, knockout) mice. (C) GOLGA2 mRNA expression levels determined by qRT-PCR (upper column) and
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semi-quantitative PCR (bottom column) in the indicated organs (n = 2). (D) Representative image of the organs, including the lung, liver, brain, kidney, and spleen, in wild type and knockout mice. (E) Numbers of mice born to reaching embryonic development. (F) Images of wild type, heterozygote,
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and knockout male and female mice. Distribution of body length (G) and body weight (H) in wild type, heterozygote, and knockout mice. Error bars represent standard deviations of at least three
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animals per group. P < 0.05 was considered statistically significant compared with the corresponding control values.
Fig. 3. GOLGA2 deletion induces autophagy in the lung and liver. (A) Immunofluorescence of LC3B
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in the liver of GOLGA2 knockout mice. (B) Immunofluorescence of LC3B in the lung of GOLGA2 knockout mice. Immunostaining of LC3B (green, conjugated with Alexa 488). Scale bar, 100 µm. Representative TEM images of the liver (C) and lung (D) in wild type and knockout mice. White
droplet;
M,
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arrowheads indicate the discharged lamellar bodies. Nu, nucleus; LB, lamellar body; LD, lipid mitochondria;
G,
Golgi.
(E)
MALDI-TOF
lipid-scanning
images
of
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dipamitoylphosphatidylcholine (PC(32:0)+Na) in the lung of GOLGA2 knockout and wild type newborn mice.
Fig. 4. Absence of GOLGA2 promotes fibrosis progression. (A) Histological analysis (left panels) and Sirius Red staining (middle and right panels) of the liver in wild type and knockout mice. Black arrows indicate fibrosis in the liver (right bottom panels). (B) Histological analysis (left panels) and Sirius Red staining (middle and right panels) of the lung in wild type and knockout mice. Red arrows indicate fibrosis in the lung (right bottom panels). Black arrowheads indicate alveolar macrophages
ACCEPTED MANUSCRIPT (left bottom panel). (C) Immunohistochemistry of α-SMA (brown) in the liver. (D) Immunoblot analyses of SMAD4 in the liver of GOLGA2 knockout and wild type mice (n = 2). (E) Role of
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S0006-291X(17)32227-1
DOI:
10.1016/j.bbrc.2017.11.049
Reference:
YBBRC 38842
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 6 November 2017 Accepted Date: 7 November 2017
Please cite this article as: S. Park, S. Kim, M. Kim, Y. Hong, A.Y. Lee, H. Lee, Q. Tran, M. Kim, H. Cho, J. Park, K.P. Kim, J. Park, M.-H. Cho, GOLGA2 loss causes fibrosis with autophagy in the mouse lung and liver, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.11.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: GOLGA2 Loss Causes Fibrosis with Autophagy in the Mouse Lung and Liver
Author information Sungjin Parka,*, Sanghwa Kimd,*, Minjung Kimc,*, Youngeun Honga, Ah Young Leeb, Hyunji Leea,
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Quangdon Trana, Minhee Kima, Hyeonjeong Choa, Jisoo Parka, Kwang Pyo Kimc,§, Jongsun Parka,§ & Myung-Haing Chob,e,f,g,h,§ a
Department of Pharmacology and Medical Science, Metabolic Syndrome and Cell Signaling
Laboratory, Cancer Research Institute, College of Medicine, Chungnam National University, Daejeon
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35015, Republic of Korea. b
Laboratory of Toxicology, Research Institute for Veterinary Science and College of Veterinary
c
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Medicine, Seoul National University, Seoul 08826, Republic of Korea.
Department of Applied Chemistry, College of Applied Science, Kyung Hee University, Yongin 17104,
Republic of Korea. d
Division of Basic Radiation Bioscience, Korea Institute of Radiological & Medical Science, Seoul, Republic of Korea
e
Graduate School of Convergence Science and Technology, Seoul National University, Suwon 16229, Republic of Korea f
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Graduate Group of Tumor Biology, Seoul National University, Seoul 08826, Republic of Korea
g
Advanced Institute of Convergence Technology, Seoul National University, Suwon 16229, Republic of Korea
h
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Institute of GreenBio Science Technology, Seoul National University, Pyeongchang 25354, Republic of Korea
*
These authors contributed equally to this work.
§
Correspondding
author.
E-mail:
[email protected]
(MHC);
[email protected]
(JP);
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[email protected] (KPK);
Abstract
Autophagy is a biological recycling process via the self-digestion of organelles, proteins, and lipids for energy-consuming differentiation and homeostasis. The Golgi serves as a donor of the doublemembraned phagophore for autophagosome assembly. In addition, recent studies have demonstrated that pulmonary and hepatic fibrosis is accompanied by autophagy. However, the relationships among Golgi function, autophagy, and fibrosis are unclear. Here, we show that the deletion of GOLGA2, encoding a cis-Golgi protein, induces autophagy with Golgi disruption. The induction of autophagy
ACCEPTED MANUSCRIPT leads to fibrosis along with the reduction of subcellular lipid storage (lipid droplets and lamellar bodies) by autophagy in the lung and liver. GOLGA2 knockout mice clearly demonstrated prefibrosis features such as autophagy-activated cells, densely packed hepatocytes, increase of alveolar macrophages, and decrease of alveolar surfactant lipids (dipalmitoylphosphatidylcholine). Therefore,
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we confirmed the associations among Golgi function, fibrosis, and autophagy. Moreover, GOLGA2
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knockout mice may be a potentially valuable animal model for studying autophagy-induced fibrosis.
Highlights
GOLGA2/GM130 loss induces autophagy with Golgi disruption in liver cells and tran
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sgenic mice.
GOLGA2/GM130 loss leads to degradation of lipid structures (LBs and LDs) by auto phagy.
GOLGA2/GM130 loss causes liver and lung fibrosis.
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1. Introduction
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Keywords: GOLGA2 knockout mice; Golgi disruption; autophagy; lung fibrosis; liver fibrosis
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Autophagy is a degradation pathway for organelles, proteins, and lipids via lysosomal fusion, resulting in the recycling of these energy sources. Hepatic fibrosis is associated with autophagy. Usually, the damaged hepatocyte region is replaced though the replication of neighboring hepatocytes. However, under a condition of heavy damage, activated hepatic stellate cells (HSCs) transform into myofibroblast-like cells, which ultimately results in hepatic fibrosis. The activation of HSCs consumes an extreme amount of energy. To produce the required energy source, the autophagy process leads to degradation of the abundant lipid droplets (LDs) in the HSCs [1, 2].
ACCEPTED MANUSCRIPT Several studies have shown that pulmonary fibrosis is also accompanied by autophagy. The levels of autophagy proteins were shown to be increased in both chronic obstructive pulmonary disease (COPD) and asthma patients [3, 4]. The fibrotic remodeling in COPD and asthma is associated with the transforming growth factor-beta (TGF-β) pathway [5]. TGF-β induces autophagy to produce
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collagen and fibronectin [6]. A recent study revealed that amiodarone (AD)-induced lung fibrosis led to the induction of autophagy in alveolar type II cells, which secreted lamellar bodies (LBs), a component of the surfactant. Interestingly, LC3B, an autophagy marker, was shown to be recruited to
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the membrane of the LB in AD-induced alveolar type II cells in greater amounts compared with control cells [7]. Before the secretion of surfactants, the LB stores components of the surfactants
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synthesized from the Golgi apparatus and rough endoplasmic reticulum (ER) [8]. The alteration of the surfactant system is a typical feature of lung diseases derived from fibrotic airway remodeling [9]. Autophagosome formation is initiated from the assembly of a double-membraned structure called the phagophore. ER–Golgi traffic contributes the machinery to form the autophagosome [10, 11]. The
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Golgi proteins are essential regulators of autophagy formation [12, 13]. A recent study showed that the down-regulated expression of GOLGA2/GM130, a cis-Golgi matrix protein, induces formation of the autophagosome via the release of GABARAP from the Golgi during mitosis [12]. Furthermore,
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GOLGA2 small hairpin RNA suppressed tumorigenesis through the induction of autophagy a KrasLA1 lung cancer mouse model [14]. However, the relationships among Golgi function, autophagy, and
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fibrosis have thus far remained unclear. Here, we provide new evidence that disruption of Golgi function leads to fibrosis through autophagy in the liver and lung tissues.
2. Materials and Methods
2.1. Cell culture and generation of a stable GOLGA2 knockout cell line Chang liver cells were cultured in Dulbecco's modified Eagle medium/high-glucose (WELGENE Inc., Daegu, South Korea) supplemented with 10% fetal bovine serum (Biowest, Kansas City, MO, USA), and 1% antibiotics-antimycotics (Life Technologies, Inc.) at 37°C in 5% CO2. The cells were transfected with GOLGA2 CRISPR/Cas9 and HDR plasmids using the Neon® transfection system for
ACCEPTED MANUSCRIPT electroporation (Invitrogen, Grand Island, NY, USA). After transfection, the cells were selected with culture medium supplemented with 0.5 µg/ml of puromycin (Sigma-Aldrich).
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2.2. Generation of GOLGA2/GM130 knockout mice The embryonic stem cell line with deletion from exon 1 to exon 26 of GOLGA2 was purchased from the Wellcome Trust Sanger Institute and used to generate the target gene. The embryonic stem cells were injected into C57B/L6J mouse blastocysts for subsequent injection into pseudopregnant female
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mice (Macrogen Inc., Seoul, Korea). The offspring were all heterozygous; thus, to obtain homozygous mice, the offspring were bred to each other. Germ-line transmission was confirmed in the offspring
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with polymerase chain reaction (PCR). We extracted DNA from the tails of the mice using Tissue Genomic DNA Isolation Kit Mini (CosmoGenetech Inc., Seoul, Korea). The primers GOLGA2 KO-F (5′-CTTCGTATAATGTATGCTATACGAAGTTATGCTAGC-3′)
and
Reg-GOLGA2-R
(5′-
ACTGAGCCGGTGAAAACTTAGAAGC-3′) were used to detect GOLGA2-/- (homozygous
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genotype) alleles, and the primers Mus GOLGA2 Exon 27 wt-F (5′-AAGACTGGCGGCCAAAGCC3′) and Reg-GOLGA2-R were used to detect GOLGA2+/+ alleles. All animals used in this study were maintained under the animal protocols of Seoul National University guidelines and the study was
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Seoul, Korea).
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approved by the Animal Care and Use Committee at Seoul National University (SNU-141007-1;
2.3. Chemicals, primers, antibody sources, and plasmids The following antibodies, chemicals, and plasmids were included: GM130 (Alexa 647-conjugated) from BD Biosciences; SQSTM1 and LC3B from Sigma-Aldrich; alpha-smooth muscle actin (αSMA) from Abcam; β-actin and GM130 CRISPR knockout plasmids (sc-400787 and sc-400787 HDR) from Santa Cruz Biotechnology; HCS LipidTOXtm Deep Red neutral lipid stains from Invitrogen; and Alexa Fluor 488-labeled anti-rabbit antibodies from Life Technologies. The free fatty acids (FFAs) included palmitic acid and oleic acid (1:2, respectively). The sequences of primers used for quantitative PCR were mouse GOLGA2, forward (5′-CAGGCAGACAGGTATAACAAG-3′) and
ACCEPTED MANUSCRIPT reverse (5′-CGGAGTTTCTCTTCCAGTTC-3′).
2.4. Immunofluorescence Chang cells were cultured on 8-well chamber slides (SPL Life Sciences), fixed in 3%
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paraformaldehyde (EMD Millipore Corporation) for 10 min at 4°C, permeabilized with 0.25% Triton® X-100 (Sigma-Aldrich) for 5 min, blocked for 30 min, and incubated with primary or secondary antibodies in blocking solution [in phosphate-buffered saline with 0.1% saponin (Sigma-
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Aldrich) and 3% bovine serum albumin (BOVOGEN)].
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2.5. Immunoblot analysis
The cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1% v/v Nonidet P-40, 120 mM NaCl, 25 mM sodium fluoride, 40 mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 2 mM microcystin-LR for 30 min at 4°C. Following centrifugation at 12,000 ×g for 30 min, the protein concentration was measured, and
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equal amounts of lysate were used for immunoblotting. Western blotting was performed with specific antibodies and anti-rabbit antibody conjugated to horseradish peroxidase (KOMA Biotechnology). Visualization was achieved with chemiluminescence through X-ray film exposure (Agfa-Gevaert
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N.V).
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2.6. Histology and immunohistochemistry The liver and lung tissues of male mice were fixed in 10% formalin. After paraffin embedding, tissue blocks were cut to a thickness of 4 µm and transferred to a silane-coated microslide (MUTO Pure Chemicals Co., Ltd.). For histological analysis, deparaffinized tissue sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation or Sirius Red (Polysciences, Inc. for the lung; Sigma for the liver) for detection of collagen accumulation according to the manufacturer’s instructions. For immunohistochemistry, deparaffinized tissue sections were boiled with retrieval buffer for 10 min by microwave, and then the slides were incubated in 5% hydrogen peroxide for 15
ACCEPTED MANUSCRIPT min to quench endogenous peroxidase activity. After washing in Tris-buffered saline plus Tween 20, the sections were blocked for 1 h at room temperature. The indicated primary antibody and secondary horseradish peroxide-conjugated antibody, in turn, were applied onto the tissue slides. After washing, the tissue slides were reacted with DAB substrate solution (DAKO) for the appropriate reaction and
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quickly washed with distilled water. Finally, the tissue slides were counterstained with hematoxylin, dehydrated, and then mounted. The samples were observed using a light microscope (Eclips Ti-S,
2.7. Transmission electron microscope (TEM) analysis
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Nikon, Japan).
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The tissues were fixed with 2.5% glutaraldehyde in 1% osmium tetroxide buffer at 4°C overnight. The fixed tissues were dehydrated with a diluted ethanol series from 50% to 100%, and infiltrated in propylene oxide Epon resin at 70°C overnight. Ultrathin sections of a thickness of 40 mm were placed
2.8. Statistical analysis
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on a copper grid and observed using a JEM1010 TEM (JEOL, Tokyo, Japan).
Data are expressed as the mean ± SEM. Student’s t-test was used for comparisons between two
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Software).
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groups. All statistical analyses were performed using GraphPad Software version 5.01 (GraphPad
3. Results
3.1. Knockout of GOLGA2 induces autophagy in liver Chang cells Consistent with our previous study [14], to determine whether GOLGA2 deletion affects autophagy induction in Chang cells, we first established the stable GOLGA2 knockout cell line by the CRISPR/Cas9 system with a knock-in of a selection marker (puromycin), and confirmed GOLGA2 loss by immunofluorescence. GOLGA2 expression in knockout cells was not observed in the perinuclear region, which localizes at a single Golgi, compared to the control cells (Fig. 1A).
ACCEPTED MANUSCRIPT Immunoblot analysis further revealed that the expression levels of both SQSTM1/p62 and LC3-II, autophagy markers, were elevated in the GOLGA2 knockout cells (Fig. 1B). Next, to determine whether GOLGA2 knockout led to an increase in FFA-dependent autophagy, we performed immunofluorescence of SQSTM and LD staining of FFA (250 µM, palmitic acid:oleic acid = 1:2)-
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treated Chang cells for 24 h. The immunofluorescence revealed that the SQSTM level was increased in the FFA-treated GOLGA2 knockout cells. In contrast, the size of the LDs was decreased in the FFA-treated GOLGA2 knockout cells, indicating that the increased autophagy lipolyzed the LDs [15]
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autophagy in both normal and autophagy-induced conditions.
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in GOLGA2 knockout cells (Fig. 1C). Thus, these findings suggest that GOLGA2 loss induces
3.2. Generation and identification of GOLGA2 knockout mice
To generate a GOLGA2 knockout mouse model, embryonic stem cells with GOLGA2 exon 1 to exon 26 deleted were injected into C57BL/6 blastocysts to transfer into a uterine of pseudopregnant female
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mice (Fig. 2A). Finally, generated GOLGA2 (+/-) mice were inbred, and the offspring were identified by PCR analysis for genotyping (Fig. 2B). The GOLGA2 (-/-) male mice did not show any expression of GOLGA2 mRNA in all organs examined, including the lung, liver, brain, heart, and kidney (Fig. 2C
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and D). Wild type (+/+) and heterozygous (+/-) mice were born at the expected Mendelian ratio, whereas GOLGA2 knockout pups (-/-) were born at only 40% of the expected number, suggesting that
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GOLGA2 knockout might cause perinatal lethality in embryonic development (Fig. 2E). GOLGA2 knockout male mice also showed growth retardation (Fig. 2F). In detail, the body length and body weight of GOLGA2-/- mice were significantly decreased compared with those of the wild type (+/+) and heterozygous (+/-) mice (Fig. 2G and H).
3.3. GOLGA2 loss induces autophagy in the liver and lung tissues of GOLGA2 knockout mice We assessed whether the GOLGA2 deletion induces autophagy in the liver and lung of the GOLGA2 knockout mice. The expression level of LC3B was clearly increased in both the liver and lung tissues
ACCEPTED MANUSCRIPT in GOLGA2 knockout mice (Fig. 3A and B). The TEM images of the liver and lung tissues showed changed phenotypes of the subcellular organelles. The morphological changes showed “autophagosomal-forming” structures [16] that partially wrapped the mitochondria with the doublemembraned phagophore in the liver tissue of GOLGA2 knockout mice. In addition, the TEM images
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revealed that the phenotype of the Golgi stack [17, 18] and LD were not detected in the liver cells of GOLGA2 knockout mice (Fig. 3C). The TEM images of the lungs of GOLGA2 knockout mice showed several smaller-sized LBs, an indicator of alveolar type II cells, suggesting a phenotype of discharged
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LBs [19] (Fig. 3D). Next, to monitor dipalmitoyl-phosphatidylcholine (DPPC), which is known as a major lipid of surfactants [20], we performed lipid-scanning analysis of the lungs of GOLGA2
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knockout and control mice using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The lipid scanning analysis revealed that the DPPC level was decreased in the lungs of GOLGA2 knockout mice compared to that in wild type mice. Consistent with the imaged composition patterns of DPPC in the lungs, the levels of other phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) were also found to be decreased in the lungs of GOLGA2
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knockout mice (Fig. 3E and Supplementary Fig. S1). Taken together, these findings suggest that GOLGA2 loss may impair the pulmonary surfactant system.
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3.4. Effect of GOLGA2 loss on lung and liver fibrosis Histological analyses (H&E staining) in GOLGA2 knockout mice showed several alterations in the
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liver and lungs compared to wild type mice. The densities of hepatic cells were increased in the liver of GOLGA2 knockout mice (Fig. 4A, left panels). The number of alveolar macrophages was increased in the alveoli of GOLGA2 knockout mice, suggesting that GOLGA2 loss stimulated the immune system in the lung (Fig. 4B, left panels). Next, to assess collagen accumulation, Sirius Red staining was performed in the liver and lung of GOLGA2 knockout and wild type mice. These results revealed typical fibrotic alterations such as the accumulation and deposition of extracellular matrix in the liver and lung of GOLGA2 knockout mice (Fig. 4A and B; middle and right panels). In addition, the αSMA level, a marker of myofibroblast-like formation, was elevated around the portal vein of
ACCEPTED MANUSCRIPT GOLGA2 knockout mice (Fig. 4C). Moreover, immunoblot analyses showed that the level of SMAD4, which is downstream of the TGF-β pathway and is known as an important signaling pathway of fibrogenesis [21], was drastically increased in the liver of GOLGA2 knockout mice (Fig. 4D). Thus, these results suggested that GOLGA2 loss induces fibrosis in the liver and lung tissues
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(Fig. 4E)
4. Discussion
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The Golgi plays an important role in the glycosylation, sorting, and transport of proteins within the intracellular system. Golgi disruption has been closely linked to various diseases, including cancer
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[22]. Previous studies have demonstrated that an abnormal Golgi structure derived from various sources of external or internal damage can promote the accumulation of misfolded proteins and Golgi fragmentation, eventually causing autophagy [23, 24]. Our previous study revealed that the downregulation of GOLGA2 expression resulted in an abnormal Golgi structure such as swelling of the Golgi cisternae along with the induction of autophagy in human lung cancer cells and mouse lung
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cancer tissues [25]. Consistently, our present results showed the induction of autophagy in the GOLGA2-deleted stable cell line and GOLGA2 knockout mice (Figs. 1 and 3).
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These results raise the question as to how autophagy is induced in the tissue cells. The autophagy protein LC3 is recruited to the LBs in alveolar type II cells during pulmonary fibrosis [7]. In the
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present study, the TEM images indicated that the morphological alteration of LBs, which was manifested by the discharge and vacuolization of the LBs, may be associated with the induction of autophagy in the lungs of GOLGA2 knockout mice. (Fig. 3). The LBs participate in the secretory function of the pulmonary surfactant [26]. The pulmonary surfactant is synthesized, secreted, and recycled by type II cells, which is modified in the ER, transported through the Golgi, and then stored in the LBs. In addition, the secretion of the pulmonary surfactant maintains the alveolar surface tension by the formation of a surface-active film. The alveolar surfactant pool is degraded by alveolar macrophages [27]. In particular, the alveolar macrophages participate in the catabolism and clearance of DPPC [28], which modulates the inflammatory response [29]. A few of the autophagy-activated
ACCEPTED MANUSCRIPT cells shown in Fig. 3B may be type II cells or alveolar macrophages. These cells may lead to the degradation of lipids through an autophagy process in the lungs of GOLGA2 knockout mice. Consistently, our present results showed that GOLGA2 loss leads to the consumption of lipids along with the induction of autophagy in liver Chang cells (Fig. 1). Moreover, the lipids imaging analysis
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revealed the decrease in the levels of several PCs and LPCs along with the decrease of the DPPC level in the lungs of GOLGA2 knockout mice (Supplementary Fig. S1). Therefore, our results provide the possibility that the discharge of LBs by GOLGA2 loss can impair the balance between biogenesis and
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the degradation of pulmonary surfactants, eventually leading to fibrogenesis in the lungs of GOLGA2 knockout mice by inducing autophagy.
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Interestingly, we also confirmed hepatic fibrosis by inducing autophagy in GOLGA2 knockout mice. The activation of HSCs from liver damage is a major mechanism of fibrogenesis [30, 31]. The abundant LDs of the HSCs supply energy from the degradation of LDs by autophagy [32]. Thus, the excessive induction of the autophagy process may be an important contributor to fibrogenesis, by
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switching HSCs to myofibroblast-like cells [33]. As a result, no LDs were observed in the liver cells of GOLGA2 knockout mice and the hepatocytes were densely packed (Figs. 3 and 4). Consistent with previous studies, these observations represent the typical features of hepatic fibrosis. However, unlike
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chemical-induced hepatic fibrosis such as with CCL4 in which the fibrosis appears strongly in both the perivenular and periportal regions [34], the pattern of hepatic fibrosis caused by GOLGA2 gene loss is
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presented diffusely in the whole liver (Fig. 4). Taken together, our study provides the first demonstration that genetic dysfunction of the Golgi contributes to fibrogenesis. In further studies, it will be important to confirm whether similar fibrotic progression may occur in other tissues of GOLGA2 knockout mice. We expect that the GOLGA2 knockout mice will serve as a good model for fibrosis research once overcoming the problems of embryo/fetal lethality and the shortened lifespan.
Acknowledgments
ACCEPTED MANUSCRIPT This work was financially supported by the National Research Foundation of Korea (NRF) grant funded
by
the
Korea
Government
(MEST)
(NRF-2012M3A9B6055302,
NRF-
2015R1A2A2A01003597, NRF-2017R1A6A3A11031556). The authors declared no conflict of
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interest.
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Figure legends
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of CD133 and epidermal growth factor, BMC Gastroenterol., 10 (2010).
Fig. 1. GOLGA2 deletion increases autophagy in liver Chang cells. (A) Representative images of immunofluorescence staining for GOLGA2 (red) in GOLGA2 knockout Chang cells compared with control Chang cells. Nuclei are labeled with Hoechst. (B) Immunoblot analyses of SQSTM and LC3 in GOLGA2 knockout and control Chang cells. β-actin was used as a loading control. (C) Representative images of SQSTM and of lipid droplets in FFA-treated GOLGA2 knockout and control Chang cells. Scale bar, 10 µm.
ACCEPTED MANUSCRIPT Fig. 2. Generation of GOLGA2 knockout mice. (A) Genomic structure of the mouse GOLGA2 gene (top column), and illustration of recombinant deleted GOLGA2 ES cells (bottom column). (B) PCR genotyping of the GOLGA2 (+/+, wild type), GOLGA2 (+/-, heterozygote), and GOLGA2 (-/-, knockout) mice. (C) GOLGA2 mRNA expression levels determined by qRT-PCR (upper column) and
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semi-quantitative PCR (bottom column) in the indicated organs (n = 2). (D) Representative image of the organs, including the lung, liver, brain, kidney, and spleen, in wild type and knockout mice. (E) Numbers of mice born to reaching embryonic development. (F) Images of wild type, heterozygote,
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and knockout male and female mice. Distribution of body length (G) and body weight (H) in wild type, heterozygote, and knockout mice. Error bars represent standard deviations of at least three
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animals per group. P < 0.05 was considered statistically significant compared with the corresponding control values.
Fig. 3. GOLGA2 deletion induces autophagy in the lung and liver. (A) Immunofluorescence of LC3B
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in the liver of GOLGA2 knockout mice. (B) Immunofluorescence of LC3B in the lung of GOLGA2 knockout mice. Immunostaining of LC3B (green, conjugated with Alexa 488). Scale bar, 100 µm. Representative TEM images of the liver (C) and lung (D) in wild type and knockout mice. White
droplet;
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arrowheads indicate the discharged lamellar bodies. Nu, nucleus; LB, lamellar body; LD, lipid mitochondria;
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Golgi.
(E)
MALDI-TOF
lipid-scanning
images
of
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dipamitoylphosphatidylcholine (PC(32:0)+Na) in the lung of GOLGA2 knockout and wild type newborn mice.
Fig. 4. Absence of GOLGA2 promotes fibrosis progression. (A) Histological analysis (left panels) and Sirius Red staining (middle and right panels) of the liver in wild type and knockout mice. Black arrows indicate fibrosis in the liver (right bottom panels). (B) Histological analysis (left panels) and Sirius Red staining (middle and right panels) of the lung in wild type and knockout mice. Red arrows indicate fibrosis in the lung (right bottom panels). Black arrowheads indicate alveolar macrophages
ACCEPTED MANUSCRIPT (left bottom panel). (C) Immunohistochemistry of α-SMA (brown) in the liver. (D) Immunoblot analyses of SMAD4 in the liver of GOLGA2 knockout and wild type mice (n = 2). (E) Role of
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