Involvement of Lamin B1 Reduction in Accelerated Cellular Senescence During Chronic Obstructive Pulmonary Disease Pathogenesis

Published January 28, 2019, doi:10.4049/jimmunol.1801293 The Journal of Immunology

Involvement of Lamin B1 Reduction in Accelerated Cellular Senescence during Chronic Obstructive Pulmonary Disease Pathogenesis

Nayuta Saito,*,1 Jun Araya,*,1 Saburo Ito,* Kazuya Tsubouchi,*,† Shunsuke Minagawa,* Hiromichi Hara,* Akihiko Ito,* Takayuki Nakano,*,‡ Yusuke Hosaka,* Akihiro Ichikawa,* Tsukasa Kadota,* Masahiro Yoshida,* Yu Fujita,* Hirofumi Utsumi,* Yusuke Kurita,* Kenji Kobayashi,* Mitsuo Hashimoto,* Hiroshi Wakui,* Takanori Numata,* Yumi Kaneko,* Hisatoshi Asano,x Makoto Odaka,x Takashi Ohtsuka,x Toshiaki Morikawa,x Katsutoshi Nakayama,* and Kazuyoshi Kuwano*

Downregulation of lamin B1 has been recognized as a crucial step for development of full senescence. Accelerated cellular senescence linked to mechanistic target of rapamycin kinase (MTOR) signaling and accumulation of mitochondrial damage has been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. We hypothesized that lamin B1 protein levels are reduced in COPD lungs, contributing to the process of cigarette smoke (CS)–induced cellular senescence via dysregulation of MTOR and mitochondrial integrity. To illuminate the role of lamin B1 in COPD pathogenesis, lamin B1 protein levels, MTOR activation, mitochondrial mass, and cellular senescence were evaluated in CS extract (CSE)–treated human bronchial epithelial cells (HBEC), CS-exposed mice, and COPD lungs. We showed that lamin B1 was reduced by exposure to CSE and that autophagy was responsible for lamin B1 degradation in HBEC. Lamin B1 reduction was linked to MTOR activation through DEP domain– containing MTOR-interacting protein (DEPTOR) downregulation, resulting in accelerated cellular senescence. Aberrant MTOR activation was associated with increased mitochondrial mass, which can be attributed to peroxisome proliferator-activated receptor g coactivator-1b–mediated mitochondrial biogenesis. CS-exposed mouse lungs and COPD lungs also showed reduced lamin B1 and DEPTOR protein levels, along with MTOR activation accompanied by increased mitochondrial mass and cellular senescence. Antidiabetic metformin prevented CSE-induced HBEC senescence and mitochondrial accumulation via increased DEPTOR expression. These findings suggest that lamin B1 reduction is not only a hallmark of lung aging but is also involved in the progression of cellular senescence during COPD pathogenesis through aberrant MTOR signaling. The Journal of Immunology, 2019, 202: 000–000. igarette smoke (CS) exposure is causally associated with complicated and heterogeneous biological process in a cell chronic obstructive pulmonary disease (COPD) devel- type– and stimulus-dependent manner, without an absolute mo- C opment. COPD is a leading cause of death worldwide lecular marker, at least in terms of COPD pathogenesis. and is progressive even after smoking cessation (1). Recent ad- Nuclear lamina, a meshwork structure composed of lamins at the vances, including our findings, indicate that accelerated cellular nuclear periphery, provides the nucleus with mechanical strength for senescence is pathologically involved in the mechanisms for maintaining structure and regulates chromatin organization for COPD progression via impaired cell repopulation and prolonged modulating gene expression and silencing (7, 8). Lamins are nuclear inflammation partly conferred by senescence-associated secre- intermediate filament proteins and are comprised of A-type and tory phenotype (SASP) (1–5). Hence, preventing CS-induced B-type lamins. Mutations in the Lamin A gene have been widely cellular senescence can be a promising therapeutic modality implicated in human disorders collectively termed laminopathies, for COPD (1, 6). Cellular senescence has been recognized to be a including accelerated aging syndromes represented by Hutchinson

*Division of Respiratory Diseases, Department of Internal Medicine, Jikei University Address correspondence and reprint requests to Dr. Jun Araya, Division of Re- School of Medicine, Tokyo 105-8461, Japan; †Research Institute for Diseases of the spiratory Diseases, Department of Internal Medicine, Jikei University School of Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan. E-mail Japan; ‡Department of Pulmonary Medicine, Kyoto Prefectural University of Medicine, address: [email protected] Kyoto 602-8566, Japan; and xDivision of Chest Diseases, Department of Surgery, Jikei The online version of this article contains supplemental material. University School of Medicine, Tokyo 105-8461, Japan Abbreviations used in this article: BafA1, baﬁlomycin A1; CCCP, carbonyl cyanide m- 1N.S. and J.A. contributed equally to this work. chlorophenyl hydrazone; CDKN1A, cyclin-dependent kinase inhibitor 1A; CDKN2A, ORCIDs: 0000-0002-9314-4745 (N.S.); 0000-0001-5783-5998 (A.Ito); 0000-0003- cyclin-dependent kinase inhibitor 2A; COPD, chronic obstructive pulmonary disease; 1114-4838 (T. Nakano); 0000-0002-9533-7241 (T.K.); 0000-0002-8916-7303 (Y.F.); CS, cigarette smoke; CSE, CS extract; DEPTOR, DEP domain–containing MTOR- 0000-0003-2784-2331 (T. Numata); 0000-0002-8521-0175 (H.A.); 0000-0002-8039- interacting protein; 4E-BP1, eIF-4E–binding protein; FEV1.0, forced expiratory volume 9159 (T.O.); 0000-0002-0141-7081 (K.N.). 1 s; %FEV1.0, percent predicted FEV1.0; HBEC, human bronchial epithelial cell; MTOR, mechanistic target of rapamycin kinase; PGC-1b, proliferator-activated recep- Received for publication September 24, 2018. Accepted for publication December tor g coactivator-1b; ROS, reactive oxygen species; SA-b-gal, senescence-associated 22, 2018. b-galactosidase; SASP, senescence-associated secretory phenotype; SI, smoking index; This work was supported by grants from the Japan Society for the Promotion of siRNA, small interfering RNA. Science KAKENHI (JP15K09231 and JP18K08158 to J.A., JP17K09673 to S.M., JP17K09672 to T. Numata, JP15K09233 to K.N., and JP15K09232 to K. Kuwano). Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801293 2 REDUCED LAMIN B1 IN COPD

Gilford progeria syndrome (9, 10). Lamin B1 duplication is re- Cell Signaling Technology), rabbit anti–cyclin-dependent kinase inhibitor sponsible for development of adult-onset autosomal dominant 2A (CDKN2A; no. 4824; Cell Signaling Technology, and no. 10883-1-AP; leukodystrophy, and increased lamin B1 mRNA and protein levels Proteintech), mouse anti-CDKN2A (no. 51-1325GR; BD Biosciences), rabbit anti–cyclin-dependent kinase inhibitor 1A (CDKN1A; no. 2947; have been demonstrated in brain tissues, resulting in central myelin Cell Signaling Technology), rabbit anti-phospho-p70 S6Kinase (no. 9205; breakdown. However, no mutations of Lamin B1 associated with Cell Signaling Technology, no. SC8416; Santa Cruz), rabbit anti-p70 functional loss and dominant-acting missense have been reported S6Kinase (no. 9202; Cell Signaling Technology), rabbit anti-phospho-4E- (8, 11). BP-1 (S65) (no. 9451S; Cell Signaling Technology), rabbit anti-4E-BP-1 (S65) (no. 9452S; Cell Signaling Technology), rabbit anti-MAP1LC3B (no. Lamin B1 is involved in the processes of DNA replication, cell NB600-1384; Novus Biologicals), mouse anti-MAP1LC3B (no. M152-3; cycle progression, and gene silencing through binding to lamina- Medical & Biological Laboratories), rabbit anti–DEP domain–containing associated domains of chromatin (12, 13). Recent advances sug- MTOR-interacting protein (DEPTOR)/DEPDC6 (no. NBP1-49674; Novus gest that cellular senescence is a dynamic multistep evolving Biologicals), mouse anti-SDHA (no. ab14715; Abcam), mouse anti-TOM20 process, and downregulation of lamin B1 has an essential role in (no. sc-17764; Santa Cruz Biotechnology), rabbit anti-PGC-1b (no. ab176328; Abcam), and mouse anti-ACTB (no. A5316; Sigma-Aldrich). MG-132 (BML- the progression to full senescence (11, 14–16). Although reduced P102; Enzo Life Sciences), Torin1 (no. 4247; Tocris Bioscience), bafilomycin lamin B1 expression levels have been detected in senescent cells A1 (BafA1) from Streptomyces griseus (no. B1793; Sigma-Aldrich), Hoechst induced by various stimuli, it remains elusive whether lamin B1 33258 (no. B2883; Sigma-Aldrich), MitoSOX Red (no. M36008; Molecular reduction is a cause or a consequence of cellular senescence Probes Life Technologies), pepstatin A (no. 4397; Peptide Institute), E-64-d (no. 4321-v; Peptide Institute), CM-H2DCFDA (no. C6827; Molecular Probes (10, 11, 14). A variety of mechanisms for lamin B1 reduction have Life Technologies), carbonyl cyanide 3-chlorophenylhydrazone (no. C2759; been demonstrated (14), and a recent report elucidated the in- Sigma-Aldrich), and collagen, type I solution from rat tail (no. C3867; Sigma- volvement of autophagy-mediated degradation of lamin B1 (17). Aldrich) were purchased. Metformin was provided from Sumitomo Dainippon Mitochondria have a pivotal role in regulating cellular senes- Pharma (Tokyo, Japan). cence, partially through generating intrinsic reactive oxygen spe- Plasmids, small interfering RNA, and transfection cies (ROS) (18). Proper turnover of damaged mitochondria is cytoprotective and attenuates age-associated detrimental processes PARK2 expression vector (pRK5-HA-Parkin, no. 17613; Addgene) and Lamin B1 expression vector (mCherry-Lamin B1-10, no. 55069; Addgene) were obtained (19, 20). We have recently reported accumulation of damaged from Addgene. The LC3B cDNA was the kind gift of Dr. Mizushima (Tokyo mitochondria with structural alterations with respect to COPD University, Tokyo, Japan) and Dr. Yoshimori (Osaka University, Osaka, Japan) and pathogenesis (4, 5). Mechanistic target of rapamycin kinase was cloned into the pEGFP-C1 vector (4). Small interfering RNA (siRNA) (MTOR) is recognized to be an essential molecule for regulating targeting ATG5 (no. s18159, s18160; Applied Biosystems Life Technolo- gies), Lamin B1 (no. s8224, s8225; Applied Biosystems Life Technologies), cell growth, and aberrant MTOR activation has been implicated in DEPTOR (no. s34968, s34970; Applied Biosystems Life Technologies), COPD pathogenesis, at least partly through accelerating the aging PGC-1b (no. s43784, s43785; Applied Biosystems Life Technolo- process (1). Recent papers also showed that MTOR promotes gies), and negative control siRNAs (#AM4635, AM4641; Applied mitochondrial biogenesis via peroxisome proliferator-activated Biosystems Life Technologies) were purchased from Life Technologies. receptor g coactivator-1b (PGC-1b) expression, indicating the Specific knockdowns of ATG5, Lamin B1, DEPTOR, and PGC-1b were validated using two different siRNA, respectively. Transfections of critical role of MTOR in regulating mitochondrial integrity in HBEC and BEAS-2B cells were performed using the Neon Transfec- association with cellular senescence (18, 21). However, not only tion System (no. MPK5000; Invitrogen Life Technologies), using the molecular mechanisms for aberrant MTOR activation but also matched optimized transfection kits (no. MPK10096; Invitrogen Life the connection between MTOR and cellular senescence progres- Technologies). sion in terms of regulating mitochondrial integrity remain largely Preparation of CS extract unknown in COPD (1, 22). In this study, we hypothesized that lamin B1 protein levels are CS extract (CSE) was prepared as previously described (4). Forty milliliters of CS was drawn into the syringe and slowly bubbled into reduced and causally associated with progression of CS-induced sterile serum-free cell culture media in 15-ml Becton Dickinson Falcon cellular senescence through dysregulating MTOR and mitochon- tubes. One cigarette was used for the preparation of 10 ml of solution. drial integrity as a part of COPD pathogenesis. CSE solution was filtered (0.22 mm) (no. SLGS033SS; Merck Milli- pore) to remove insoluble particles and was designated as a 100% CSE solution. Materials and Methods RNA isolation, PCR Cell culture, Abs, and reagents RNA isolation, RT-PCR, and real-time PCR were performed using the Normal and COPD airways were collected from first through fourth order SYBR green method as previously described (23, 24). The primers used bronchi from pneumonectomy and lobectomy specimens from resections were LMNB1 sense primer, 59-AAGCATGAAACGCGCTTGG-39; performed for primary lung cancer. Informed consent was obtained from all LMNB1 antisense primer, 59-AGTTTGGCATGGTAAGTCTGC-39; surgical participants as part of an approved ongoing research protocol by the DEPTOR sense primer, 59-CTCAGGCTGCACGAAGAAAAG-39; DEP- ethical committee of Jikei University School of Medicine (23-153 [5443]). TOR antisense primer, 59-TTGCGACAAAACAGTTTGGGT-39; ACTB Human bronchial epithelial cells (HBEC) were isolated with protease sense primer, 59-CATGTACGTTGCTATCCAGGC-39; and ACTB anti- treatment, and freshly isolated HBEC were plated onto rat tail collagen type sense primer, 59-CTCCTTAATGTCACGCACGAT-39. These primer sets m I–coated (10 g/ml) dishes, incubated overnight, and then the medium was yielded PCR products of 152, 76, and 250 bp for LMNB1, DEPTOR, and changed to bronchial epithelial growth medium (Clonetics, San Diego, ACTB, respectively. PCRs of LMNB1 and DEPTOR were validated using CA). Cultures were characterized immunohistochemically using anti- two different primers. Primer sequences were from Primer Bank (http:// cytokeratin Abs (Lu-5; Biocare Medical, Concord, CA) and anti-vimentin pga.mgh.harvard.edu/primerbank.). Ab (Sigma-Adrich, Tokyo, Japan). HBEC showed .95% positive staining with anti-cytokeratin and ,5% positive staining with anti-vimentin Ab Electron microscopy (data not shown). HBEC were serially passed and used for experiments until passage 3. Most experiments were performed with HBEC from non- Lung tissues from pneumonectomy and lobectomy specimens were fixed COPD patients. The bronchial epithelial cell line BEAS-2B was cultured with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) and dehydrated in RPMI 1640 (no. 11875-093; Gibco Life Technologies) with 10% FCS with a graded series of ethanol. Fixed tissues were then embedded in epoxy (no. 26140-079; Life Technologies) and penicillin–streptomycin (no. resin. Ultrathin sections were stained with uranyl acetate and lead citrate 15070-063; Life Technologies). and observed with the Hitachi H-7500 transmission electron microscope Abs used were rabbit anti–lamin B1 (no. PM064, Medical & Biological (Hitachi, Tokyo, Japan). Mitochondria number per 1 mm2 of cell area in Laboratories; no. ab16048, Abcam), rabbit anti-ATG5 (no. 2630S; Cell Sig- small airway epithelial cells was evaluated by counting 10 image fields naling Technology), rabbit anti phospho-histone H2A.X (Ser139) (no. 2577S; (10,0003) for each sample. The Journal of Immunology 3

Immunohistochemistry and immunofluorescence staining washed and collected by magnet five times with immunoprecipitation buffer, and boiled with SDS. Samples were analyzed by Western blotting. Immunohistochemical staining was performed as previously described with minor modification on the paraffin-embedded lung tissues (24). DEPTOR Mouse models immunostaining was assessed by measuring areas of total and positively staining cells in small airways at a maginification of 3400 using ImageJ, C57BL/6J (CLEA Japan, Tokyo, Japan) mice were purchased and were an open-source image processing program. Immunofluorescence staining maintained in the animal facility at the Jikei University School of Medicine was also performed as previously described (24). BEAS-2B were trans- (no. 25031). All experimental procedures are approved by the Jikei Uni- fected with pEGFP-LC3, and CSE treatment was started 48 h after versity School of Medicine Animal Care Committee. Mice were exposed to transfection. Baf A1 (200 nM) treatment was started 6 h before fixation to control air (male = 3 and female = 3) or CS (male = 3 and female = 3) 5 d a clearly demonstrate the autophagosome formation of GFP-LC3 “dots,” week over a 6-mo period (inExpose; SCIREQ Scientific Respiratory which result from BafA1 prevention of lysosomal degradation. After 24 h Equipment). The lungs were removed at 6 mo and were used for histological treatment with CSE, BEAS-2B were fixed with 4% paraformaldehyde examination and CXCL1 ELISA (Mouse CXCLI/KC Quantikine ELISA for 15 min, followed by permeabilization with 0.03% Triton X (no. kit, no. MKC008; R&D Systems). For histological examination, the lungs 160-24751; Wako Chemicals USA) for 60 min. After blocking with 1% were fixed overnight in 10% buffered formalin, embedded in paraffin, and BSA (no. A2153; Sigma-Aldrich) for 60 min, the primary and secondary sections stained with H&E according to conventional protocols for histo- Abs were applied according to the manufacturer’s instructions. Confocal pathological evaluation. Immunohistochemistry was performed as previ- laser scanning microscopic analysis (LSM800; Carl Zeiss, Tokyo, Japan) ously described (24). Quantitative measure of mean linear intercept and of nuclear lamina and autophagosome was performed. Fluorescence mi- wall thickness was performed by using Image J, an open-source image croscopy analysis of phospho-histone H2A.X was performed in HBEC processing program. (BX60; Olympus, Tokyo, Japan, and BZ-X700; Keyence, Tokyo, Japan). Statistics Senescence-associated b-galactosidase staining Data are shown as the average (6SEM) taken from at least three in- Senescence-associated b-galactosidase (SA-b-gal) staining was performed dependent experiments. Comparisons between two different groups using HBEC grown on 12-well culture plates according to the manufacturer’s were determined by Student t test for parametric data or Mann– instructions (no. CS0030; Sigma-Aldrich). Whitney U test for nonparametric data. One-way ANOVAwas used for multiple comparisons and Tukey or Bonferroni post hoc tests were Measurement of ROS production used to test for statistical significance. Linear regression analysis was used to compare lamin B1 expression levels in HBEC to age, smoking HBEC, at a density of 3 3 104 per well, were seeded in a 96-well microplate index (SI), and pulmonary function tests. Significance was defined as (no. 237105; Thermo Fisher Scientific). CM-H2DCFDA was used to measure p , 0.05. Statistical software used was Prism v.5 (GraphPad Software, total cellular ROS according to the manufacturer’s instructions. After incu- San Diego, CA). bation with CM-H2DCFDA (10 mM) for 30 min at 37˚C, fluorescence of DCF was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm by a fluorescence microplate reader (Infinite F 200; Results Tecan Japan, Kanagawa, Japan). Mitochondrial ROS production was analyzed by MitoSOX Red staining according to the manufacturer’s instructions, Autophagic degradation is responsible for lamin B1 reduction which was evaluated by fluorescence microscopy (Olympus and BZ-X700; by CSE treatment in HBEC Keyence). CSE treatment for 48 h significantly reduced lamin B1 protein levels Western blotting in a concentration-dependent manner (Fig. 1A), and 1% CSE was sufficient to see significant changes in HBEC. In our previous paper, HBEC grown on 6–12-well culture plates were lysed in RIPA buffer (no. 89900; Thermo Fisher Scientific) with protease inhibitor mixture accelerated cellular senescence was also demonstrated by 1% of (no. 11697498001; Roche Diagnostics) and 1 mM sodium orthovanadate, CSE (22); thus, 1% of CSE was selected for further experiments. or lysed with Laemmli sample buffer. Western blotting was performed No apparent alteration of lamin B1 mRNA levels at 48 h CSE as previously described (24). For each experiment, equal amounts of treatment was detected by means of quantitative RT-PCR, indicat- total protein were resolved by 7.5–15% SDS-PAGE. After SDS-PAGE, proteins were transferred to polyvinylidene difluoride membrane ing that lamin B1 is regulated at the protein level during CSE ex- (no. ISEQ00010; Merck Millipore), and incubation with specific primary posure (Fig. 1B). MG132, a proteasome inhibitor, failed to recover Ab was performed for 2 h at 37˚C, or 24 h at 4˚C. After washing several lamin B1 reduction (Fig. 1C). Hence, involvement of autophagy times with PBS–Tween 20, the membrane was incubated with anti-rabbit was examined by siRNA-mediated ATG5 knockdown for autophagy IgG, HRP-linked secondary Ab (no. 7074; Cell Signaling Technology) or inhibition. ATG5 knockdown clearly recovered CSE-induced lamin anti-mouse IgG, HRP-linked secondary Ab (no. 7076; Cell Signaling Technology), followed by chemiluminescence detection (no. 34080; B1 reduction (Fig. 1D). Involvement of autophagic degradation was Thermo Fisher Scientific, and no. 1705061; Bio-Rad Laboratories) with also demonstrated by efficient inhibition of lamin B1 reduction by the ChemiDoc Touch Imaging System (Bio-Rad Laboratories). the treatment with BafA1, an inhibitor of autolysosomal maturation CXCL8 ELISA (data not shown). Immunoprecipitation revealed increased protein association between lamin B1 and LC3B, an essential component HBEC were incubated with 1.0% CSE for 48 h, and washed three times with for autophagosome formation, in response to CSE exposure (Fig. 1E). PBS. CSE treatment was started 48 h post-siRNA transfection. To collect the Autophagic degradation of lamin B1 was further confirmed by condition medium, HBEC were incubated in serum-free DMEM for 48 h. Human lung homogenates from nonsmokers and COPD patients were also detecting the cytosolic colocalization between lamin B1 and LC3B assessed. CXCL8 was measured with a Human IL-8/CXCL8 Quantikine dot by means of confocal laser scanning microscopic evaluation ELISA kit (no. D8000C; R&D Systems). (Fig. 1F: arrow). Intriguingly, lamin B1 reduction was not observed Immunoprecipitation by treatment with torin1, an autophagy inducer via MTOR inhibition, suggesting that CSE-induced autophagy is specifically responsible for BEAS-2B were transfected with pEGFP-LC3 and mCherry–Lamin B1, and laminB1degradation(Fig.1G). CSE treatment (1% for 24 h) was started 48 h after transfection. Protease inhibitors (E64d and pepstatin A) treatment was started 6 h before collection Effect of lamin B1 reduction on cellular senescence, MTOR to prevent degradation. Immunoprecipitation was performed as previously activation, and mitochondrial integrity during CSE treatment described (17). Cells were lysed in immunoprecipitation buffer containing in HBEC 20 mM Tris (pH 7.5), 137 mM NaCl, 1 mM MgCl2,1mMCaCl2,1%NP- 40, 10% glycerol, supplemented with 1:100 Halt protease and phosphatase To clarify the role of lamin B1 reduction, siRNA-mediated lamin B1 inhibitor mixture (no. 78440; Thermo Fisher Scientific) and benzonase (no. 70746-4; Novagen) at 12.5 U/ml. The lysates were rotated at 4˚C for 30 min. knockdown experiments were performed. Efficient lamin B1 The supernatant was incubated with Ab-conjugated beads (no. 28944006; knockdown without apparent effect on lamin A/C protein levels was GE Healthcare) and rotated at 4˚C overnight. The immunoprecipitation was confirmed by Western blotting (Fig. 2G, Supplemental Fig. 1). 4 REDUCED LAMIN B1 IN COPD

FIGURE 1. Autophagic degradation of lamin B1 in HBEC. (A) Western blotting (WB) using anti–lamin B1 and anti-ACTB of HBEC lysates treated with indicated percentage of CSE for 48 h. The lower panel shows the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB (n =3).(B) HBEC were treated with CSE (1%), and mRNA samples were collected after treatment of indicated time. Real-time PCR was performed using primers to lamin B1 or ACTB as a control. Lamin B1 mRNA expression was normalized to ACTB. Shown is the fold increase (6SEM) relative to 0 h treated cells (n =3).(C) WB using anti–lamin B1 and anti-ACTB of HBEC lysates treated with CSE (1%) for 48 h in the presence or absence of MG132 (10 mM), a proteasome inhibitor. The lower panel shows the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB (n =3).(D) WB using anti–lamin B1, ATG5, and ACTB. HBEC were transfected with control siRNA or ATG5 siRNA. CSE (1%) treatment was started 48 h after transfection, and protein samples were collected after 48 h treatment. The right panel shows the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB (n =3).(E) WB using anti–lamin B1, anti- MAP1LC3B, and anti-ACTB of BEAS-2B lysates treated with CSE (1%) for 48 h. Protease inhibitor (E64d 10 mg/ml, pepstatin A 10 mg/ml) treatment was started 6 h before collecting cell lysates. The right panels are immunoprecipitation by using anti-MAP1LC3B. Shown is a representative experiment of four, showing similar results. (F) Colocalization analysis of confocal laser scanning microscopic images of lamin B1 staining in BEAS-2B transfected pEGFP-MAP1LC3. BEAS-2B were treated with CSE (1%) for 24 h. Baf A1 (200 nM) treatment was started 6 h before ﬁxation. The images are high magniﬁcation (10003). Scale bar, 10 mm. (G) WB using anti–lamin B1 and anti-ACTB of HBEC lysates treated with CSE (1%) or Torin1 (250 nM) for 48 h. The lower panel shows the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB (n =4).*p , 0.05, by ANOVA and Bonferroni post hoc test.

Lamin B1 knockdown was sufficient to induce cellular senescence by secretion was observed in conditioned medium only from CSE- means of SA-b-gal staining, phospho-histone H2A.X (Ser139) treated HBEC with lamin B1 knockdown, indicating that both staining of DNA damage, and Western blotting of CDKN2A/p16 CSE and lamin B1 reduction to some extent are necessary for and CDKN1A/p21, which was significantly enhanced by CSE progression to full senescence with SASP (Fig. 2D). Increased ROS treatment in HBEC (Fig. 2A–C). To determine SASP status, reflecting accumulation of mitochondrial damage has been widely CXCL8 secretion was examined. Significant increase in CXCL8 implicated in the regulation of cellular senescence (18, 21). Thus, to The Journal of Immunology 5

FIGURE 2. Lamin B1 knockdown enhances cellular senescence, MTOR activation, and mitochondria accumulation in response to CSE exposure in HBEC. HBEC were transfected with control siRNA or Lamin B1 siRNA, and CSE (1.0%) treatment was started 48 h after transfection. (A) Photomi- crographs of SA-b-gal staining of HBEC. HBEC were stained after CSE (1.0% for 48 h) treatment. Shown in lower panel is the percentage (6SEM) of SA-b-gal–positive cells from four independent experiments. (B) Photographs of immunofluorescent staining of phospho-histone H2A.X (Ser139). HBEC were treated with CSE (1.0%) for 48 h. (C) WB using anti-CDKN2A, anti-CDKN1A, and anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment. Shown is a representative experiment of four, showing similar results. (D) ELISA showing CXCL8 in conditioned media from HBEC. Shown are the arbitrary units 6 SEM. (E) Fluorescence intensity of CM-H2DCFDA staining for intracellular ROS production. After treatment with CSE (1.0% for 24 h), incubation with CM-H2DCFDA (10 mM) was performed for 30 min, and fluorescence of DCF was measured by a fluorescence microplate reader. The fluorescence level in the control siRNA without CSE was designated as 1.0. Shown panels are the average (6SEM) taken from six independent experiments. (F) Photographs of Hoechst 33258 and MitoSOX Red fluorescence staining. (G) WB using anti–lamin B1, p-S6K, S6K, p-4E-BP1, 4E-BP1, PGC- 1b, SDHA, TOM20, and anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment. The right panels show the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB from at least three independent experiments. (H) WB using anti-PGC-1b, SDHA, TOM20, and anti-ACTB of HBEC lysates. CSE (1.0% for 48 h) and rapamycin (1 mM for 48 h) treatment was started 48 h after transfection. The panels show the average (6SEM) of relative expressions, which were taken from densitometric analysis of WB from three independent experiments. (I) WB using anti-TOM20, SDHA, CDKN1A, CDKN2A, and anti-ACTB of HBEC lysates. CSE (1.0% for 48 h) treatment was started 48 h after transfection. The right panels show the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB from five independent experiments. (J) Photographs of Hoechst 33258 and MitoSOX Red fluorescence staining. HBEC were transfected with control siRNA or Lamin B1 siRNA and CSE (1.0% for 24 h) treatment was started 48 h after transfection. Scale bars, 100 mm. *p , 0.05, **p , 0.001, by ANOVA and Bonferroni post hoc test. WB, Western blotting. 6 REDUCED LAMIN B1 IN COPD

FIGURE 3. Involvement of DEPTOR in lamin B1 reduction–mediated MTOR activation, increased mitochondrial mass, and cellular senescence in HBEC. (A) WB using anti-DEPTOR, anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment. HBEC were transfected with control siRNA or Lamin B1 siRNA and CSE (1%) treatment was started 48 h after transfection. Shown is a representative experiment of ﬁve, showing similar results. (B) HBEC were transfected with control siRNA or Lamin B1 siRNA and mRNA samples were collected after 48 h after transfection (Figure legend continues) The Journal of Immunology 7

FIGURE 4. CS exposure–induced lamin B1 and DEPTOR reduction, MTOR activation, increased mitochondrial mass, and enhanced cellular senescence in mouse lungs. (A) Immunohistochemical staining of lamin B1 in control air and CS-exposed mouse lungs. (B) WB using anti–lamin B1 and anti-ACTB of lung homogenates from control air (n = 6) and CS-exposed mouse (n = 6) lungs. The lower panel is the average (6SEM) taken from densitometric analysis of WB. (C) Immunohistochemical staining of DEPTOR in control air and CS-exposed mouse lungs. (D) WB using anti-DEPTOR and anti-ACTB of lung homogenates from control air (n = 6) and CS-exposed mouse (n = 6) lungs. The lower panel is the average (6SEM) taken from densitometric analysis of WB. (E) Immunohistochemical staining of p-S6K and CDKN1A, and immunofluorescent staining of phospho-histone H2A.X (Ser139) in control air and CS-exposed mouse lungs. (F) ELISA showing CXCL1 in lung homogenates from control air (n = 6) and CS-exposed mouse (n = 6) lungs. Shown is the average 6 SEM (pg/ml). (G) WB using anti-PGC-1b, TOM20, SDHA, and anti-ACTB of lung homogenates from control air (n = 6) and CS-exposed mouse (n = 6) lungs. The right panels are the average (6SEM) taken from densitometric analysis of WB. Scale bars, 100 mm. *p , 0.05, by unpaired Student t test. WB, Western blotting. evaluate ROS production in the setting of lamin B1 knockdown, we To elucidate the mechanistic insight for lamin B1–mediated performed CM-H2DCFDA assay for total ROS and MitoSOX Red regulation of mitochondrial mass, MTOR activation and PGC-1b, staining for mitochondrial ROS. Increase in ROS was detected by a downstream transcriptional coactivator for mitochondrial bio- lamin B1 knockdown (Fig. 2E, 2F). CSE induced both total and genesis, were examined (18). Significantly enhanced phosphory- mitochondrial ROS, which were clearly enhanced by lamin B1 lated S6K and eIF-4E–binding protein (4E-BP1), reflecting knockdown (Fig. 2E, 2F). Consistent with mitochondrial ROS, in- MTOR activation, and increased PGC-1b protein levels were creased mitochondrial mass shown by TOM20 and SDHA protein detected by lamin B1 knockdown, which were enhanced by CSE levels were demonstrated by lamin B1 knockdown, especially in the treatment (Fig. 2G). Rapamycin, an MTOR inhibitor treatment, setting of CSE treatment (Fig. 2G, Supplemental Fig. 2A). clearly abrogated the increase in PGC-1b, TOM20, and SDHA

(n = 5). Real-time PCR was performed and DEPTOR mRNA expression was normalized to ACTB. Shown is the fold increase (6SEM) relative to control treated cells. *p , 0.05, by paired Student t test. (C) WB using anti-DEPTOR, p-S6K, S6K, p-4E-BP1, 4E-BP1, PGC-1b, TOM20, SDHA, and anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment. HBEC were transfected with control siRNA or DEPTOR siRNA and CSE (1.0%) treatment was started 48 h after transfection. The right panels show the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB from three independent experiments. (D) Photographs of Hoechst 33258 and MitoSOX Red fluorescence staining. HBEC were transfected with control siRNA or DEPTOR siRNA and CSE (1.0% for 24 h) treatment was started 48 h after transfection. (E) Photomicrographs of SA-b-gal staining of HBEC. HBEC were stained after CSE (1.0% for 48 h) treatment. Shown in right panel is the percentage (6SEM) of SA-b-gal positive cells from three independent experiments. (F) WB using anti-CDKN2A, anti-CDKN1A, and anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment. Shown is a representative experiment of three, showing similar results. (G) Photographs of immunofluorescent staining of phospho-histone H2A.X (Ser139). HBEC were stained after CSE (1.0% for 48 h) treatment. (H) WB using anti-PGC-1b, TOM20, SDHA, and anti-ACTB of HBEC lysates. CSE (1.0% for 48 h) and rapamycin (1 mM for 48 h) treatment was started 48 h after transfection. The panels show the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB from three independent experiments. (I) WB using anti-TOM20, SDHA, and anti-ACTB of HBEC lysates. HBEC were transfected with PARK2 expression vector. CCCP (6.25 mM for 48 h) treatment was started 24 h after transfection and CSE (1.0% for 48 h) treatment was started 72 h after transfection. Shown is a representative experiment of three, showing similar results. (J) Photographs of Hoechst 33258 and MitoSOX Red fluorescence staining. (K) Photomicrographs of SA-b-gal staining of HBEC. Shown in right panel is the percentage (6SEM) of SA-b-gal positive cells from three independent experiments. *p , 0.05, by ANOVA and Bonferroni post hoc test (C, E, H and K). (L) Photographs of immunofluorescent staining of phospho- histone H2A.X (Ser139). Scale bars, 100 mm. WB, Western blotting. 8 REDUCED LAMIN B1 IN COPD

FIGURE 5. Reduced Lamin B1 and DEPTOR, MTOR activation, and increased mitochondrial mass in COPD lungs. (A) Immunohistochemical staining of lamin B1, DEPTOR, and p-S6K in nonsmoker, non-COPD smoker, and COPD lungs. Shown panels are high magnification view of original magnification 3200. Scale bar, 50 mm for lamin B1, and 100 mm for DEPTOR and p-S6K. (B) Western blotting (WB) using anti–lamin B1 and anti-ACTB of lung homogenates from nonsmoker, non-COPD smoker, and COPD patients. The lower panel is the average (6SEM) taken (Figure legend continues) The Journal of Immunology 9 expression levels by lamin B1 knockdown and CSE treatment CS exposure induces lamin B1 reduction and cellular (Fig. 2H). Involvement of PGC-1b in increased mitochondrial senescence in a mouse model mass, cellular senescence, and ROS were examined by siRNA- To elucidate the causal association between physiological long- b mediated PGC-1 knockdown experiments. PGC-1b depletion term CS exposure and lamin B1 reduction of accelerated cellu- abrogated both CSE and lamin B1 knockdown–induced mito- lar senescence, a CS exposure mouse model for 6 mo was used. CS chondrial accumulation and enhancement of cellular senescence exposure induced the COPD phenotypes of emphysematous change (Fig. 2I, Supplemental Fig. 2A). Mitochondrial ROS production (Supplemental Fig. 3). Reduced lamin B1 protein levels were was also clearly reduced by PGC-1b knockdown (Fig. 2J). demonstrated in airway epithelial cells in CS-exposed mouse Lamin B1 knockdown–mediated DEPTOR reduction is lungs by means of immunohistochemical evaluation and Western responsible for MTOR activation and accelerated cellular blotting of lung homogenates (Fig. 4A, 4B). DEPTOR reduction senescence in HBEC was also demonstrated in airway epithelial cells in CS-exposed lungs (Fig. 4C, 4D). Immunohistochemical examination showed DEPTOR constitutes a part of MTOR complexes and has an es- increased expression of p-S6K in airway epithelial cells (Fig. 4E). sential inhibitory role in the kinase activity of MTOR (25). We Accelerated cellular senescence of enhanced CDKN1A and examined the involvement of DEPTOR in lamin B1 knockdown– phospho-histone H2A.X (Ser139) staining in airway epithe- mediated MTOR activation. CSE treatment significantly reduced lial cells accompanied by increased murine CXCL8 homolog DEPTOR protein levels (Fig. 3A). Lamin B1 knockdown further CXCL1 were detected in CS-exposed lungs (Fig. 4E, 4F). reduced DEPTOR protein levels, and quantitative RT-PCR showed CS-exposed lung homogenates also demonstrated increased that DEPTOR was regulated at the mRNA levels (Fig. 3A, 3B). To PGC-1b, TOM20, and SDHA protein levels (Fig. 4G). How- clarify the involvement of DEPTOR in modulating MTOR acti- ever, no clear reduction of lamin B1 and DEPTOR and no increase vation, siRNA-mediated DEPTOR knockdown experiments were in p-S6K were detected in alveolar epithelial cells in CS- performed (Fig. 3C). DEPTOR knockdown clearly activated exposed mouse compared with control exposed mouse lungs MTOR signaling of p-S6K and p-4E-BP1 (Fig. 3C). Consistent (Supplemental Fig. 4A). with lamin B1 knockdown experiments, DEPTOR knockdown increased PGC-1b and mitochondrial mass accompanied by en- Reduced Lamin B1 and DEPTOR protein levels with hanced mitochondrial ROS production of MitoSOX Red staining concomitantly increased MTOR activation and mitochondrial in response to CSE exposure (Fig. 3C, 3D, Supplemental Fig. 2B). mass in COPD lungs Increased cellular senescence was also demonstrated in the setting To further confirm the pathological implication of lamin B1 in of DEPTOR knockdown, which was further enhanced by CSE COPD progression, lamin B1 protein levels in COPD lungs were exposure (Fig. 3E–G). Rapamycin abrogated increases in PGC- evaluated by immunohistochemistry. Small airway epithelial cells 1b, TOM20, and SDHA expression by DEPTOR knockdown in normal lungs showed clear lamin B1 staining (Fig. 5A). In (Fig. 3H, Supplemental Fig. 2B), suggesting the functional asso- contrast, diminished and indistinct lamin B1 staining was detected ciation between lamin B1–mediated DEPTOR downregulation in COPD lungs (Fig. 5A). Compared with nonsmoker lungs, lamin and MTOR activation with respect to increased mitochondrial B1 protein levels tended to be lower in lung homogenates from mass and enhanced HBEC senescence during CSE exposure. non-COPD smokers, but a significant decrease was demonstrated Next, the involvement of increased mitochondrial mass in in lung homogenates from COPD lungs (Fig. 5B). Reduced lamin regulating cellular senescence in the case of lamin B1 depletion B1 protein levels in airway epithelial cells were further demon- was examined. PARK2 overexpression with subsequent carbonyl strated by using cultured HBEC of COPD patients (Fig. 5C). Next, cyanide m-chlorophenyl hydrazone (CCCP) treatment clearly to illuminate the causal link between reduced lamin B1 protein eliminated mitochondria in both control and lamin B1 siRNA levels and COPD progression, correlation with age, SI, and pul- transfected HBEC, as previously described (18) (Fig. 3I). No in- monary function tests were examined. Patient characteristics are crease in mitochondrial ROS was detected by CSE exposure presented in Table I. Although no significant correlation between in mitochondria-eliminated HBEC with lamin B1 knock- lamin B1 protein levels in HBEC and age was detected, clear down (Fig. 3J). Importantly, mitochondria-eliminated HBEC positive correlations with SI and pulmonary function tests of showed clear resistance to cellular senescence induced by CSE percentage of forced expiratory volume 1 s (FEV1.0)/forced vital exposure even in the setting of lamin B1 knockdown in HBEC capacity and percent predicted FEV1.0 (%FEV1.0) were demon- (Fig. 3K, 3L). strated, suggesting that lamin B1 protein levels are not simply

from densitometric analysis of WB. (C) WB using anti–lamin B1 and anti-ACTB of HBEC lysates isolated from nonsmoker, non-COPD smoker, and COPD patients. The lower panel is the average (6SEM) taken from densitometric analysis of WB. **p , 0.001, by ANOVA and Bonferroni post hoc test (B and C). (D) Shown are the relationship between relative lamin B1 expression normalized to ACTB in HBEC and age, SI, FEV1.0/forced vital capacity, and %FEV1.0, respectively. HBEC were from nonsmoker, non-COPD smoker, and COPD patients. Linear regression analysis was used to compare lamin B1 expression levels in HBEC to age, SI, and pulmonary function tests. (E) WB using anti- DEPTOR, p-S6K, S6K, PGC-1b, TOM20, SDHA, and anti- ACTB of HBEC from nonsmoker (n = 3), non-COPD smoker (n = 3), and COPD (n = 3) patients. The panels show the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB. (F) Photographs of immunoﬂuorescent staining of phospho-histone H2A.X (Ser139). Scale bar, 100 mm. (G) Electron microscopic evaluation of mitochondria in small airway epithelial cells in lungs from nonsmoker (n = 3) and COPD (n = 3). The lower panel is the average (6SEM) count of mitochondria per 1 mm2 of cell area and taken from 10 image ﬁelds (10,0003) for each sample. Scale bar, 1 mm. (H) ELISA showing CXCL8 in lung homogenates from nonsmoker (n = 8) and COPD (n = 9) lungs. Shown is the average 6 SEM (pg/ml). *p , 0.05, by unpaired Student t test (G and H). (I) Lamin B1 and DEPTOR mRNA expression levels in HBEC: nonsmoker (n = 3), non-COPD smoker (n = 3), and COPD (n = 3). mRNA samples were collected from lung tissues. Real-time PCR was performed using primers to lamin B1, DEPTOR, and ACTB as a control. Lamin B1 and DEPTOR expressions were normalized to ACTB. Shown is the fold increase (6SEM) relative to one nonsmoker. *p , 0.05, by ANOVA and Bonferroni post hoc test (E and I). 10 REDUCED LAMIN B1 IN COPD

Table I. Patient characteristics (for HBEC)

Characteristic Nonsmoker (n = 12) Non-COPD (n = 12) Smoker COPD (n = 11) p Value Age, y 70.3 6 9.2 65.0 6 6.7 66.2 6 6.8 NS Male, % of group 66.7 83.3 90.9 ,0.0048a SI (pack-years) 0 44.9 6 37.4 72.1 6 35.5 ,0.0001b FEV1.0/FVC, % 78.9 6 6.6 80.6 6 8.3 63.9 6 6.1 ,0.0001b %FEV1.0 107.7 6 10.7 93.9 6 10.3 80.6 6 17.4 ,0.0001 Values are mean 6 SD. ax2 test for independence. bANOVA and Bonferroni post hoc test. FVC, forced vital capacity. associated with aging but are more directly linked to airway ob- by aberrant MTOR activation resulting from reduced DEPTOR. A struction of COPD pathogenesis (Fig. 5D). CS-exposed mouse model also demonstrates enhanced cellular se- Next, DEPTOR protein levels, MTOR activation, cellular senescence in airway epithelial cells with concomitantly decreased nescence, and mitochondrial mass were examined. Immunohis- lamin B1 and DEPTOR protein levels accompanied by increased tochemistry and Western blotting using HBEC clearly showed mitochondrial mass. Lamin B1 expression levels are clearly reduced DEPTOR reduction and increased p-S6K expression in COPD in COPD lungs, especially in small airway epithelial cells, and lungs (Fig. 5A, 5E). In line with experimental results using lamin lamin B1 protein levels in HBEC positively correlate with pulmo- B1 knockdown in HBEC, accelerated cellular senescence was also nary function tests. COPD lungs also show decreased DEPTOR and demonstrated by increased phospho-histone H2A.X (Ser139) aberrant MTOR activation accompanied by increased mitochondrial staining and CXCL8 protein levels in lung homogenates in COPD mass and cellular senescence. Antidiabetic metformin induces lungs (Fig. 5F, 5H). Increased PGC-1b, TOM20, and SDHA DEPTOR expression and reduces CSE-induced HBEC senescence. protein levels were also detected in HBEC of COPD patients Taken together, it is likely that lamin B1 depletion is not only a (Fig. 5E). In comparison with non-smoker lungs, electron mi- simple hallmark of lung aging but is also involved in the mecha- croscopic evaluations showed a significant increase in mitochon- nisms for progression of cellular senescence through aberrant drial counts in airway epithelial cells in COPD lungs (Fig. 5G). In MTOR signaling in COPD pathogenesis (Fig. 7). comparison with nonsmokers and non-COPD smokers, lamin B1 Although it remains elusive whether lamin B1 depletion is a cause or and DEPTOR mRNA were significantly reduced in HBEC of a simple consequence of cellular senescence, recent reports elucidated COPD patients (Fig. 5I). Consistent with CS-exposed mouse the participation of lamin B1 reduction in a range of cellular senescence models, no apparent reduction of lamin B1 and DEPTOR ex- induced by cell replication, ionizing radiation, and oncogene activation pression levels and no increase in p-S6K were demonstrated in (14). Furthermore, lamin B1 reduction in keratinocytes has been alveolar lesions in COPD lungs (Supplemental Fig. 4B). These demonstrated to be a prognostic biomarker for not only skin aging but results suggest the existence of pathogenic link between reduced also other skin pathology, suggesting that lamin B1 reduction can be lamin B1–mediated MTOR signaling and enhanced mitochondrial mechanistically involved in the progression of aging-associated dis- accumulation associated with accelerated cellular senescence in orders (10). Our in vitro experiments clarified that lamin B1 reduction airway epithelial cells during COPD development. accompanied by cellular senescence was evoked by CSE treatment, and lamin B1 knockdown further enhanced CSE-induced cellular Metformin induces DEPTOR expression and prevents senescence in HBEC (Fig. 2A–C). Intriguingly, lamin B1 knockdown CSE-induced HBEC senescence also slightly induced HBEC senescence in the absence of CSE, sug- Metformin is a commonly prescribed biguanide antidiabetic medi- gestingapivotalroleforlaminB1inregulating cellular senescence in cation used to lower blood glucose in type II diabetes patients and HBEC (Fig. 2A–C). COPD lungs also showed decreased lamin B1 also exhibits pleiotropic effects on cellular biology (26). Metformin protein levels in airway epithelial cells (Fig. 5A–C). Lamin B1 protein has been demonstrated to increase DEPTOR protein levels, result- levels in HBEC were positively correlated with pulmonary function ing in suppression of MTOR signaling (26, 27). In line with pre- tests (Fig. 5D), indicating that accelerated cellular senescence con- vious findings, metformin clearly induced DEPTOR protein levels ferred by lamin B1 reduction can be causally associated with airway in a dose-dependent manner in HBEC (Fig. 6A). Next, we exam- obstruction, delineating disease severity of COPD. It has been reported ined the antisenescence property of metformin during CSE-induced that not only lamin B1 expression levels but also ratio of LaminA/C HBEC senescence (Fig. 7). CSE-induced DEPTOR reduction and can be a determinant for cellular senescence (10, 28). However, we p-S6K and PGC-1b increase were inhibited by metformin (Fig. 6B). observed no alteration of LaminA/C protein levels nor lamin B1 HBEC senescence and mitochondrial accumulation by CSE expo- knockdown in CSE-exposed HBEC (Supplemental Fig. 1). Although sure were also prevented by metformin treatment (Fig. 6B–D). the participation of lamins in regulating cell function and cell fate CSE-induced mitochondrial ROS production reflecting mitochon- appears to be different in a tissue-type–specific manner (29), we drial damage was also reduced by metformin (Fig. 6E). speculate that lamin B1 reduction is at least partly responsible for acceleration of cellular senescence in airway epithelial cells, especially Discussion during CS exposure with respect to COPD progression. In the current study, CSE treatment reduces lamin B1 protein levels, CSE treatment reduced lamin B1 protein levels in a and CSE-induced autophagy is responsible for lamin B1 degradation concentration-dependent manner, which can be attributed to in HBEC. Knockdown experiments elucidate that lamin B1 is autophagy-mediated degradation (Fig. 1). A recent paper showed involved in the regulation of CSE-induced intracellular ROS that autophagy activation induced by oncogene, oxidative stress, production and cellular senescence associated with increased mi- and DNA damage, but not by starvation nor rapamycin, was tochondrial mass in HBEC. Increased PGC-1b–mediated mito- responsible for lamin B1 depletion (17). In line with those find- chondrial biogenesis by lamin B1 reduction appears to be conferred ings, we observed no alteration of lamin B1 protein levels by The Journal of Immunology 11

FIGURE 6. Effect of metformin on DEPTOR expression, MTOR signaling, mitochondrial mass, and HBEC senescence by CSE exposure. (A)WB using anti-DEPTOR anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment in the presence of indicated concentration of metformin. The lower panel shows the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB (n =3).(B) WB using anti–lamin B1, DEPTOR, p-S6K, S6K, PGC-1b, TOM20, SDHA, CDKN2A, CDKN1A, and anti-ACTB of HBEC lysates after CSE (1.0% for 48 h) treatment in the presence or absence of metformin (2 mM). The right panels show the average (6SEM) of relative expressions, which are taken from densitometric analysis of WB (n =4).(C) Photomicrographs of SA-b-gal staining of HBEC. HBEC were stained after CSE (1.0% for 48 h) treatment in the presence or absence of metformin (2 mM). Shown in right panel is the percentage (6SEM) of SA-b-gal–positive cells from three independent experiments. (D) Photographs of immunoﬂuorescent staining of phospho-histone H2A.X (Ser139). Shown in right panel is the percentage (6SEM) of positively stained cells from three independent experiments. (E) Photographs of Hoechst 33258 and MitoSOX Red ﬂuorescence staining. Shown in right panel is the percentage (6SEM) of positively stained cells from three independent experiments. Scale bars, 100 mm. *p , 0.05, **p , 0.001, by ANOVA and Bonferroni post hoc test. WB, Western blotting.

torin1-mediated autophagy activation, indicating that genotoxic HBEC senescence was demonstrated in our vitro models (Fig. 1B), stress-mediated autophagy is specifically involved in lamin B1 we observed significant reduction of lamin B1 mRNA levels in degradation. In general, autophagy is recognized to be a cyto- COPD lung tissues (Fig. 5I). Accordingly, we consider that lamin protective mechanisms for turnover of damaged cellular compo- B1 depletion during relatively short-term CS exposure can be nents and attenuating aging-associated detrimental processes (20, mainly attributed to autophagic degradation, but reduction of lamin 30). It has been speculated that driving cellular senescence B1 mRNA is also responsible for lamin B1 depletion in COPD resulting from downregulation of lamin B1 can be a mechanism lungs caused by long-term CS exposure (31). for restricting tumorigenesis by inhibiting cell proliferation in the Aberrant MTOR activation has been implicated in COPD path- setting of oncogenic and tumorigenic insults (17). CS has been ogenesis in terms of regulating cellular senescence and aging phe- known to be a major risk factor for carcinogenesis; hence, it is notype (1). It is likely that MTOR activation by lamin B1 likely that autophagic lamin B1 degradation accompanied by en- knockdown was attributed to DEPTOR reduction (Fig. 3), which is hanced cellular senescence in response to CS exposure can also be an important component of MTOR complexes and is a natural a protective mechanism for preventing lung cancer development. negative regulator of MTOR kinase activity. Dysregulation of As another mechanism for lamin B1 depletion, reduction of lamin MTOR activity associated with DEPTOR reduction has been B1 mRNA levels has been reported during replicative senescence widely implicated in disease pathogenesis, including cancer and in human lung fibroblasts of WI-38 (11). Decrease in mRNA Alzheimer’s disease development (25, 32). DEPTOR reduction at stability was also responsible for lamin B1 mRNA reduction in the the mRNA level was observed by lamin B1 knockdown (Fig. 3B) setting of cellular senescence induced by ionizing radiation in and reduced DEPTOR mRNA was demonstrated in COPD lungs normal human fibroblasts (14). Furthermore, it has been reported (Fig. 5I), suggesting that lamin B1 regulates DEPTOR expression at that lamin B1 mRNA levels can be regulated by miR23a in se- the mRNA level. Lamin B1 is thought to regulate gene expression nescent human dermal fibroblasts and keratinocytes, suggesting through binding to lamina-associated domains of chromatin (15). In that lamin B1 is also regulated at the mRNA levels (10). Although general, nuclear lamina-associated genes are transcriptionally in- no significant decrease in lamin B1 mRNA during CSE-induced active and contain repressive histone markers, including H3K27me3 12 REDUCED LAMIN B1 IN COPD

Metformin has been proposed to be a potential antisenescent modality of COPD treatment via suppressing MTOR by AMPK activation (1). A recent paper showed metformin can also regulate MTOR by increasing DEPTOR (27). Actually, our in vitro experiments using HBEC elucidated an antisenescent property of metformin during CSE exposure, which was at least partly regulated by DEPTOR expression (Fig. 6). However, unphysiological higher concentrations of metformin were used to show increase in DEPTOR expression in HBEC (Fig. 6). Previous papers demon- strating AMPK activation also used similar higher concentrations of metformin (35, 36), which can be attributed to low expression levels of organic cation transporter 1, a receptor for metformin uptake (37). Accordingly, to clarify the antisenescence property, in vivo experimental models using physiological concentrations of metformin should be performed. Intriguingly, therapeutic potential of metformin for COPD has been recently demonstrated by using elastase-induced emphysema models (38). Metformin reduced elastase-induced airspace enlargement through regulating inﬂam- matory responses and cellular senescence (38). Although participation of DEPTOR-mediated MTOR regulation by metformin with respect to COPD treatment remains uncertain, we speculate that metformin can be a promising geroprotective modality of COPD treatment via DEPTOR upregulation in the setting of reduced lamin B1 with aberrant MTOR activation. In summary, we demonstrated that lamin B1 reduction can be not only a promising hallmark but also responsible for progression to cellular senescence during COPD pathogenesis. Reduced DEPTOR FIGURE 7. Hypothetical model of involvement of lamin B1 reduction in conferred by lamin B1 depletion is involved in the mechanisms for COPD pathogenesis. CS-induced autophagy is responsible for lamin B1 degradation. Reduced lamin B1–mediated MTOR activation is caused by aberrant MTOR activation with concomitantly increased mito- DEPTOR depletion. Aberrant MTOR activation is causally linked to in- chondrial mass via PGC-1b–mediated mitochondrial biogenesis. creased mitochondrial mass, which can be attributed to PGC-1b–induced Accordingly, modalities to prevent reduction of DEPTOR to at- mitochondria biogenesis, resulting in full senescence with SASP for COPD tenuate MTOR signaling may be a promising therapeutic option to progression. DEPTOR expression by metformin may prevent aberrant suppress accelerated cellular senescence with lamin B1 depletion MTOR activation during COPD pathogenesis. in the aging-associated pathological condition in COPD.

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