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

Cigarette smoke (CS) exposure is causally associated with chronic obstructive pulmonary disease (COPD) development. COPD is a leading cause of death worldwide and is progressive even after smoking cessation (1). Recent advances, including our findings, indicate that accelerated cellular senescence is pathologically involved in the mechanisms for COPD progression via impaired cell repopulation and prolonged inflammation partly conferred by senescence-associated secretory phenotype (SASP) (1–5). Hence, preventing CS-induced cellular senescence can be a promising therapeutic modality for COPD (1, 6). Cellular senescence has been recognized to be a complicated and heterogeneous biological process in a cell type– and stimulus-dependent manner, without an absolute molecular marker, at least in terms of COPD pathogenesis.

Nuclear lamina, a meshwork structure composed of lamins at the nuclear periphery, provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing (7, 8). Lamins are nuclear intermediate filament proteins and are comprised of A-type and B-type lamins. Mutations in the Lamin A gene have been widely implicated in human disorders collectively termed laminopathies, including accelerated aging syndromes represented by Hutchinson Gilford progeria syndrome (9, 10). Lamin B1 duplication is responsible for development of adult-onset autosomal dominant leukodystrophy, and increased lamin B1 mRNA and protein levels have been demonstrated in brain tissues, resulting in central myelin breakdown. However, no mutations of Lamin B1 associated with functional loss and dominant-acting missense have been reported (8, 11).

Lamin B1 is involved in the processes of DNA replication, cell cycle progression, and gene silencing through binding to lamina-associated domains of chromatin (12, 13). Recent advances suggest that cellular senescence is a dynamic multistep evolving process, and downregulation of lamin B1 has an essential role in the progression to full senescence (11, 14–16). Although reduced lamin B1 expression levels have been detected in senescent cells induced by various stimuli, it remains elusive whether lamin B1 reduction is a cause or a consequence of cellular senescence (10, 11, 14). A variety of mechanisms for lamin B1 reduction have been demonstrated (14), and a recent report elucidated the involvement of autophagy-mediated degradation of lamin B1 (17).

Mitochondria have a pivotal role in regulating cellular senescence, partially through generating intrinsic reactive oxygen species (ROS) (18). Proper turnover of damaged mitochondria is cytoprotective and attenuates age-associated detrimental processes (19, 20). We have recently reported accumulation of damaged mitochondria with structural alterations with respect to COPD pathogenesis (4, 5). Mechanistic target of rapamycin kinase (MTOR) is recognized to be an essential molecule for regulating cell growth, and aberrant MTOR activation has been implicated in COPD pathogenesis, at least partly through accelerating the aging process (1). Recent papers also showed that MTOR promotes mitochondrial biogenesis via peroxisome proliferator-activated receptor γ coactivator-1β (PGC-1β) expression, indicating the critical role of MTOR in regulating mitochondrial integrity in association with cellular senescence (18, 21). However, not only the molecular mechanisms for aberrant MTOR activation but also the connection between MTOR and cellular senescence progression in terms of regulating mitochondrial integrity remain largely unknown in COPD (1, 22).

In this study, we hypothesized that lamin B1 protein levels are reduced and causally associated with progression of CS-induced cellular senescence through dysregulating MTOR and mitochondrial integrity as a part of COPD pathogenesis.

Normal and COPD airways were collected from first through fourth order bronchi from pneumonectomy and lobectomy specimens from resections performed for primary lung cancer. Informed consent was obtained from all surgical participants as part of an approved ongoing research protocol by the ethical committee of Jikei University School of Medicine (23-153 [5443]). Human bronchial epithelial cells (HBEC) were isolated with protease treatment, and freshly isolated HBEC were plated onto rat tail collagen type I–coated (10 μg/ml) dishes, incubated overnight, and then the medium was changed to bronchial epithelial growth medium (Clonetics, San Diego, CA). Cultures were characterized immunohistochemically using anti-cytokeratin Abs (Lu-5; Biocare Medical, Concord, CA) and anti-vimentin Ab (Sigma-Adrich, Tokyo, Japan). HBEC showed >95% positive staining with anti-cytokeratin and <5% positive staining with anti-vimentin Ab (data not shown). HBEC were serially passed and used for experiments until passage 3. Most experiments were performed with HBEC from non-COPD patients. The bronchial epithelial cell line BEAS-2B was cultured in RPMI 1640 (no. 11875-093; Gibco Life Technologies) with 10% FCS (no. 26140-079; Life Technologies) and penicillin–streptomycin (no. 15070-063; Life Technologies).

Abs used were rabbit anti–lamin B1 (no. PM064, Medical & Biological Laboratories; no. ab16048, Abcam), rabbit anti-ATG5 (no. 2630S; Cell Signaling Technology), rabbit anti phospho-histone H2A.X (Ser139) (no. 2577S; Cell Signaling Technology), rabbit anti–cyclin-dependent kinase inhibitor 2A (CDKN2A; no. 4824; Cell Signaling Technology, and no. 10883-1-AP; Proteintech), mouse anti-CDKN2A (no. 51-1325GR; BD Biosciences), rabbit anti–cyclin-dependent kinase inhibitor 1A (CDKN1A; no. 2947; Cell Signaling Technology), rabbit anti-phospho-p70 S6Kinase (no. 9205; Cell Signaling Technology, no. SC8416; Santa Cruz), rabbit anti-p70 S6Kinase (no. 9202; Cell Signaling Technology), rabbit anti-phospho-4E-BP-1 (S65) (no. 9451S; Cell Signaling Technology), rabbit anti-4E-BP-1 (S65) (no. 9452S; Cell Signaling Technology), rabbit anti-MAP1LC3B (no. NB600-1384; Novus Biologicals), mouse anti-MAP1LC3B (no. M152-3; Medical & Biological Laboratories), rabbit anti–DEP domain–containing MTOR-interacting protein (DEPTOR)/DEPDC6 (no. NBP1-49674; Novus Biologicals), mouse anti-SDHA (no. ab14715; Abcam), mouse anti-TOM20 (no. sc-17764; Santa Cruz Biotechnology), rabbit anti-PGC-1β (no. ab176328; Abcam), and mouse anti-ACTB (no. A5316; Sigma-Aldrich). MG-132 (BML-P102; Enzo Life Sciences), Torin1 (no. 4247; Tocris Bioscience), bafilomycin A1 (BafA1) from Streptomyces griseus (no. B1793; Sigma-Aldrich), Hoechst 33258 (no. B2883; Sigma-Aldrich), MitoSOX Red (no. M36008; Molecular Probes Life Technologies), pepstatin A (no. 4397; Peptide Institute), E-64-d (no. 4321-v; Peptide Institute), CM-H2DCFDA (no. C6827; Molecular Probes Life Technologies), carbonyl cyanide 3-chlorophenylhydrazone (no. C2759; Sigma-Aldrich), and collagen, type I solution from rat tail (no. C3867; Sigma-Aldrich) were purchased. Metformin was provided from Sumitomo Dainippon Pharma (Tokyo, Japan).

PARK2 expression vector (pRK5-HA-Parkin, no. 17613; Addgene) and Lamin B1 expression vector (mCherry-Lamin B1-10, no. 55069; Addgene) were obtained from Addgene. The LC3B cDNA was the kind gift of Dr. Mizushima (Tokyo University, Tokyo, Japan) and Dr. Yoshimori (Osaka University, Osaka, Japan) and was cloned into the pEGFP-C1 vector (4). Small interfering RNA (siRNA) targeting ATG5 (no. s18159, s18160; Applied Biosystems Life Technologies), Lamin B1 (no. s8224, s8225; Applied Biosystems Life Technologies), DEPTOR (no. s34968, s34970; Applied Biosystems Life Technologies), PGC-1β (no. s43784, s43785; Applied Biosystems Life Technologies), and negative control siRNAs (#AM4635, AM4641; Applied Biosystems Life Technologies) were purchased from Life Technologies. Specific knockdowns of ATG5, Lamin B1, DEPTOR, and PGC-1β were validated using two different siRNA, respectively. Transfections of HBEC and BEAS-2B cells were performed using the Neon Transfection System (no. MPK5000; Invitrogen Life Technologies), using matched optimized transfection kits (no. MPK10096; Invitrogen Life Technologies).

CS extract (CSE) was prepared as previously described (4). Forty milliliters of CS was drawn into the syringe and slowly bubbled into sterile serum-free cell culture media in 15-ml Becton Dickinson Falcon tubes. One cigarette was used for the preparation of 10 ml of solution. CSE solution was filtered (0.22 μm) (no. SLGS033SS; Merck Millipore) to remove insoluble particles and was designated as a 100% CSE solution.

RNA isolation, RT-PCR, and real-time PCR were performed using the SYBR green method as previously described (23, 24). The primers used were LMNB1 sense primer, 5′-AAGCATGAAACGCGCTTGG-3′; LMNB1 antisense primer, 5′-AGTTTGGCATGGTAAGTCTGC-3′; DEPTOR sense primer, 5′-CTCAGGCTGCACGAAGAAAAG-3′; DEPTOR antisense primer, 5′-TTGCGACAAAACAGTTTGGGT-3′; ACTB sense primer, 5′-CATGTACGTTGCTATCCAGGC-3′; and ACTB antisense primer, 5′-CTCCTTAATGTCACGCACGAT-3′. These primer sets yielded PCR products of 152, 76, and 250 bp for LMNB1, DEPTOR, and ACTB, respectively. PCRs of LMNB1 and DEPTOR were validated using two different primers. Primer sequences were from Primer Bank (http://pga.mgh.harvard.edu/primerbank.).

Lung tissues from pneumonectomy and lobectomy specimens were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) and dehydrated with a graded series of ethanol. Fixed tissues were then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with the Hitachi H-7500 transmission electron microscope (Hitachi, Tokyo, Japan). Mitochondria number per 1 μm² of cell area in small airway epithelial cells was evaluated by counting 10 image fields (10,000×) for each sample.

Immunohistochemical staining was performed as previously described with minor modification on the paraffin-embedded lung tissues (24). DEPTOR immunostaining was assessed by measuring areas of total and positively staining cells in small airways at a maginification of ×400 using ImageJ, an open-source image processing program. Immunofluorescence staining was also performed as previously described (24). BEAS-2B were transfected with pEGFP-LC3, and CSE treatment was started 48 h after transfection. Baf A1 (200 nM) treatment was started 6 h before fixation to clearly demonstrate the autophagosome formation of GFP-LC3 “dots,” which result from BafA1 prevention of lysosomal degradation. After 24 h treatment with CSE, BEAS-2B were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.03% Triton X (no. 160-24751; Wako Chemicals USA) for 60 min. After blocking with 1% BSA (no. A2153; Sigma-Aldrich) for 60 min, the primary and secondary Abs were applied according to the manufacturer’s instructions. Confocal laser scanning microscopic analysis (LSM800; Carl Zeiss, Tokyo, Japan) of nuclear lamina and autophagosome was performed. Fluorescence microscopy analysis of phospho-histone H2A.X was performed in HBEC (BX60; Olympus, Tokyo, Japan, and BZ-X700; Keyence, Tokyo, Japan).

Senescence-associated β-galactosidase (SA-β-gal) staining was performed using HBEC grown on 12-well culture plates according to the manufacturer’s instructions (no. CS0030; Sigma-Aldrich).

HBEC, at a density of 3 × 10⁴ per well, were seeded in a 96-well microplate (no. 237105; Thermo Fisher Scientific). CM-H2DCFDA was used to measure total cellular ROS according to the manufacturer’s instructions. After incubation with CM-H2DCFDA (10 μM) 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; Tecan Japan, Kanagawa, Japan). Mitochondrial ROS production was analyzed by MitoSOX Red staining according to the manufacturer’s instructions, which was evaluated by fluorescence microscopy (Olympus and BZ-X700; Keyence).

HBEC grown on 6–12-well culture plates were lysed in RIPA buffer (no. 89900; Thermo Fisher Scientific) with protease inhibitor mixture (no. 11697498001; Roche Diagnostics) and 1 mM sodium orthovanadate, or lysed with Laemmli sample buffer. Western blotting was performed as previously described (24). For each experiment, equal amounts of total protein were resolved by 7.5–15% SDS-PAGE. After SDS-PAGE, proteins were transferred to polyvinylidene difluoride membrane (no. ISEQ00010; Merck Millipore), and incubation with specific primary Ab was performed for 2 h at 37°C, or 24 h at 4°C. After washing several times with PBS–Tween 20, the membrane was incubated with anti-rabbit IgG, HRP-linked secondary Ab (no. 7074; Cell Signaling Technology) or anti-mouse IgG, HRP-linked secondary Ab (no. 7076; Cell Signaling Technology), followed by chemiluminescence detection (no. 34080; Thermo Fisher Scientific, and no. 1705061; Bio-Rad Laboratories) with the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

HBEC were incubated with 1.0% CSE for 48 h, and washed three times with PBS. CSE treatment was started 48 h post-siRNA transfection. To collect the condition medium, HBEC were incubated in serum-free DMEM for 48 h. Human lung homogenates from nonsmokers and COPD patients were also assessed. CXCL8 was measured with a Human IL-8/CXCL8 Quantikine ELISA kit (no. D8000C; R&D Systems).

BEAS-2B were transfected with pEGFP-LC3 and mCherry–Lamin B1, and 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 to prevent degradation. Immunoprecipitation was performed as previously described (17). Cells were lysed in immunoprecipitation buffer containing 20 mM Tris (pH 7.5), 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 1% NP-40, 10% glycerol, supplemented with 1:100 Halt protease and phosphatase 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. The supernatant was incubated with Ab-conjugated beads (no. 28944006; GE Healthcare) and rotated at 4°C overnight. The immunoprecipitation was washed and collected by magnet five times with immunoprecipitation buffer, and boiled with SDS. Samples were analyzed by Western blotting.

C57BL/6J (CLEA Japan, Tokyo, Japan) mice were purchased and were maintained in the animal facility at the Jikei University School of Medicine (no. 25031). All experimental procedures are approved by the Jikei University School of Medicine Animal Care Committee. Mice were exposed to control air (male = 3 and female = 3) or CS (male = 3 and female = 3) 5 d a week over a 6-mo period (inExpose; SCIREQ Scientific Respiratory Equipment). The lungs were removed at 6 mo and were used for histological examination and CXCL1 ELISA (Mouse CXCLI/KC Quantikine ELISA kit, no. MKC008; R&D Systems). For histological examination, the lungs were fixed overnight in 10% buffered formalin, embedded in paraffin, and sections stained with H&E according to conventional protocols for histopathological evaluation. Immunohistochemistry was performed as previously described (24). Quantitative measure of mean linear intercept and wall thickness was performed by using Image J, an open-source image processing program.

Data are shown as the average (±SEM) taken from at least three independent experiments. Comparisons between two different groups were determined by Student t test for parametric data or Mann–Whitney U test for nonparametric data. One-way ANOVA was used for multiple comparisons and Tukey or Bonferroni post hoc tests were used to test for statistical significance. Linear regression analysis was used to compare lamin B1 expression levels in HBEC to age, smoking index (SI), and pulmonary function tests. Significance was defined as p < 0.05. Statistical software used was Prism v.5 (GraphPad Software, San Diego, CA).

CSE treatment for 48 h significantly reduced lamin B1 protein levels in a concentration-dependent manner (Fig. 1A), and 1% CSE was sufficient to see significant changes in HBEC. In our previous paper, accelerated cellular senescence was also demonstrated by 1% of CSE (22); thus, 1% of CSE was selected for further experiments. No apparent alteration of lamin B1 mRNA levels at 48 h CSE treatment was detected by means of quantitative RT-PCR, indicating that lamin B1 is regulated at the protein level during CSE exposure (Fig. 1B). MG132, a proteasome inhibitor, failed to recover lamin B1 reduction (Fig. 1C). Hence, involvement of autophagy was examined by siRNA-mediated ATG5 knockdown for autophagy inhibition. ATG5 knockdown clearly recovered CSE-induced lamin B1 reduction (Fig. 1D). Involvement of autophagic degradation was also demonstrated by efficient inhibition of lamin B1 reduction by the treatment with BafA1, an inhibitor of autolysosomal maturation (data not shown). Immunoprecipitation revealed increased protein association between lamin B1 and LC3B, an essential component for autophagosome formation, in response to CSE exposure (Fig. 1E). Autophagic degradation of lamin B1 was further confirmed by detecting the cytosolic colocalization between lamin B1 and LC3B dot by means of confocal laser scanning microscopic evaluation (Fig. 1F: arrow). Intriguingly, lamin B1 reduction was not observed by treatment with torin1, an autophagy inducer via MTOR inhibition, suggesting that CSE-induced autophagy is specifically responsible for lamin B1 degradation (Fig. 1G).

To clarify the role of lamin B1 reduction, siRNA-mediated lamin B1 knockdown experiments were performed. Efficient lamin B1 knockdown without apparent effect on lamin A/C protein levels was confirmed by Western blotting (Fig. 2G, Supplemental Fig. 1). Lamin B1 knockdown was sufficient to induce cellular senescence by means of SA-β-gal staining, phospho-histone H2A.X (Ser139) staining of DNA damage, and Western blotting of CDKN2A/p16 and CDKN1A/p21, which was significantly enhanced by CSE treatment in HBEC (Fig. 2A–C). To determine SASP status, CXCL8 secretion was examined. Significant increase in CXCL8 secretion was observed in conditioned medium only from CSE-treated HBEC with lamin B1 knockdown, indicating that both CSE and lamin B1 reduction to some extent are necessary for progression to full senescence with SASP (Fig. 2D). Increased ROS reflecting accumulation of mitochondrial damage has been widely implicated in the regulation of cellular senescence (18, 21). Thus, to evaluate ROS production in the setting of lamin B1 knockdown, we performed CM-H2DCFDA assay for total ROS and MitoSOX Red staining for mitochondrial ROS. Increase in ROS was detected by lamin B1 knockdown (Fig. 2E, 2F). CSE induced both total and mitochondrial ROS, which were clearly enhanced by lamin B1 knockdown (Fig. 2E, 2F). Consistent with mitochondrial ROS, increased mitochondrial mass shown by TOM20 and SDHA protein levels were demonstrated by lamin B1 knockdown, especially in the setting of CSE treatment (Fig. 2G, Supplemental Fig. 2A).

To elucidate the mechanistic insight for lamin B1–mediated regulation of mitochondrial mass, MTOR activation and PGC-1β, a downstream transcriptional coactivator for mitochondrial biogenesis, were examined (18). Significantly enhanced phosphorylated S6K and eIF-4E–binding protein (4E-BP1), reflecting MTOR activation, and increased PGC-1β protein levels were detected by lamin B1 knockdown, which were enhanced by CSE treatment (Fig. 2G). Rapamycin, an MTOR inhibitor treatment, clearly abrogated the increase in PGC-1β, TOM20, and SDHA expression levels by lamin B1 knockdown and CSE treatment (Fig. 2H). Involvement of PGC-1β in increased mitochondrial mass, cellular senescence, and ROS were examined by siRNA-mediated PGC-1β knockdown experiments. PGC-1β depletion abrogated both CSE and lamin B1 knockdown–induced mitochondrial accumulation and enhancement of cellular senescence (Fig. 2I, Supplemental Fig. 2A). Mitochondrial ROS production was also clearly reduced by PGC-1β knockdown (Fig. 2J).

DEPTOR constitutes a part of MTOR complexes and has an essential inhibitory role in the kinase activity of MTOR (25). We examined the involvement of DEPTOR in lamin B1 knockdown–mediated MTOR activation. CSE treatment significantly reduced DEPTOR protein levels (Fig. 3A). Lamin B1 knockdown further reduced DEPTOR protein levels, and quantitative RT-PCR showed that DEPTOR was regulated at the mRNA levels (Fig. 3A, 3B). To clarify the involvement of DEPTOR in modulating MTOR activation, siRNA-mediated DEPTOR knockdown experiments were performed (Fig. 3C). DEPTOR knockdown clearly activated MTOR signaling of p-S6K and p-4E-BP1 (Fig. 3C). Consistent with lamin B1 knockdown experiments, DEPTOR knockdown increased PGC-1β and mitochondrial mass accompanied by enhanced mitochondrial ROS production of MitoSOX Red staining in response to CSE exposure (Fig. 3C, 3D, Supplemental Fig. 2B). Increased cellular senescence was also demonstrated in the setting of DEPTOR knockdown, which was further enhanced by CSE exposure (Fig. 3E–G). Rapamycin abrogated increases in PGC-1β, TOM20, and SDHA expression by DEPTOR knockdown (Fig. 3H, Supplemental Fig. 2B), suggesting the functional association between lamin B1–mediated DEPTOR downregulation and MTOR activation with respect to increased mitochondrial mass and enhanced HBEC senescence during CSE exposure.

Next, the involvement of increased mitochondrial mass in regulating cellular senescence in the case of lamin B1 depletion was examined. PARK2 overexpression with subsequent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment clearly eliminated mitochondria in both control and lamin B1 siRNA transfected HBEC, as previously described (18) (Fig. 3I). No increase in mitochondrial ROS was detected by CSE exposure in mitochondria-eliminated HBEC with lamin B1 knockdown (Fig. 3J). Importantly, mitochondria-eliminated HBEC showed clear resistance to cellular senescence induced by CSE exposure even in the setting of lamin B1 knockdown in HBEC (Fig. 3K, 3L).

To elucidate the causal association between physiological long-term CS exposure and lamin B1 reduction of accelerated cellular senescence, a CS exposure mouse model for 6 mo was used. CS exposure induced the COPD phenotypes of emphysematous change (Supplemental Fig. 3). Reduced lamin B1 protein levels were demonstrated in airway epithelial cells in CS-exposed mouse lungs by means of immunohistochemical evaluation and Western blotting of lung homogenates (Fig. 4A, 4B). DEPTOR reduction was also demonstrated in airway epithelial cells in CS-exposed lungs (Fig. 4C, 4D). Immunohistochemical examination showed increased expression of p-S6K in airway epithelial cells (Fig. 4E). Accelerated cellular senescence of enhanced CDKN1A and phospho-histone H2A.X (Ser139) staining in airway epithelial cells accompanied by increased murine CXCL8 homolog CXCL1 were detected in CS-exposed lungs (Fig. 4E, 4F). CS-exposed lung homogenates also demonstrated increased PGC-1β, TOM20, and SDHA protein levels (Fig. 4G). However, no clear reduction of lamin B1 and DEPTOR and no increase in p-S6K were detected in alveolar epithelial cells in CS-exposed mouse compared with control exposed mouse lungs (Supplemental Fig. 4A).

To further confirm the pathological implication of lamin B1 in COPD progression, lamin B1 protein levels in COPD lungs were evaluated by immunohistochemistry. Small airway epithelial cells in normal lungs showed clear lamin B1 staining (Fig. 5A). In contrast, diminished and indistinct lamin B1 staining was detected in COPD lungs (Fig. 5A). Compared with nonsmoker lungs, lamin B1 protein levels tended to be lower in lung homogenates from non-COPD smokers, but a significant decrease was demonstrated in lung homogenates from COPD lungs (Fig. 5B). Reduced lamin B1 protein levels in airway epithelial cells were further demonstrated by using cultured HBEC of COPD patients (Fig. 5C). Next, to illuminate the causal link between reduced lamin B1 protein levels and COPD progression, correlation with age, SI, and pulmonary function tests were examined. Patient characteristics are presented in Table I. Although no significant correlation between lamin B1 protein levels in HBEC and age was detected, clear positive correlations with SI and pulmonary function tests of percentage of forced expiratory volume 1 s (FEV1.0)/forced vital capacity and percent predicted FEV1.0 (%FEV1.0) were demonstrated, suggesting that lamin B1 protein levels are not simply associated with aging but are more directly linked to airway obstruction of COPD pathogenesis (Fig. 5D).

Next, DEPTOR protein levels, MTOR activation, cellular senescence, and mitochondrial mass were examined. Immunohistochemistry and Western blotting using HBEC clearly showed DEPTOR reduction and increased p-S6K expression in COPD lungs (Fig. 5A, 5E). In line with experimental results using lamin B1 knockdown in HBEC, accelerated cellular senescence was also demonstrated by increased phospho-histone H2A.X (Ser139) staining and CXCL8 protein levels in lung homogenates in COPD lungs (Fig. 5F, 5H). Increased PGC-1β, TOM20, and SDHA protein levels were also detected in HBEC of COPD patients (Fig. 5E). In comparison with non-smoker lungs, electron microscopic evaluations showed a significant increase in mitochondrial counts in airway epithelial cells in COPD lungs (Fig. 5G). In comparison with nonsmokers and non-COPD smokers, lamin B1 and DEPTOR mRNA were significantly reduced in HBEC of COPD patients (Fig. 5I). Consistent with CS-exposed mouse models, no apparent reduction of lamin B1 and DEPTOR expression levels and no increase in p-S6K were demonstrated in alveolar lesions in COPD lungs (Supplemental Fig. 4B). These results suggest the existence of pathogenic link between reduced lamin B1–mediated MTOR signaling and enhanced mitochondrial accumulation associated with accelerated cellular senescence in airway epithelial cells during COPD development.

Metformin is a commonly prescribed biguanide antidiabetic medication used to lower blood glucose in type II diabetes patients and also exhibits pleiotropic effects on cellular biology (26). Metformin has been demonstrated to increase DEPTOR protein levels, resulting in suppression of MTOR signaling (26, 27). In line with previous findings, metformin clearly induced DEPTOR protein levels in a dose-dependent manner in HBEC (Fig. 6A). Next, we examined the antisenescence property of metformin during CSE-induced HBEC senescence (Fig. 7). CSE-induced DEPTOR reduction and p-S6K and PGC-1β increase were inhibited by metformin (Fig. 6B). HBEC senescence and mitochondrial accumulation by CSE exposure were also prevented by metformin treatment (Fig. 6B–D). CSE-induced mitochondrial ROS production reflecting mitochondrial damage was also reduced by metformin (Fig. 6E).

In the current study, CSE treatment reduces lamin B1 protein levels, and CSE-induced autophagy is responsible for lamin B1 degradation in HBEC. Knockdown experiments elucidate that lamin B1 is involved in the regulation of CSE-induced intracellular ROS production and cellular senescence associated with increased mitochondrial mass in HBEC. Increased PGC-1β–mediated mitochondrial biogenesis by lamin B1 reduction appears to be conferred by aberrant MTOR activation resulting from reduced DEPTOR. A CS-exposed mouse model also demonstrates enhanced cellular senescence in airway epithelial cells with concomitantly decreased lamin B1 and DEPTOR protein levels accompanied by increased mitochondrial mass. Lamin B1 expression levels are clearly reduced in COPD lungs, especially in small airway epithelial cells, and lamin B1 protein levels in HBEC positively correlate with pulmonary function tests. COPD lungs also show decreased DEPTOR and aberrant MTOR activation accompanied by increased mitochondrial mass and cellular senescence. Antidiabetic metformin induces DEPTOR expression and reduces CSE-induced HBEC senescence. Taken together, it is likely that lamin B1 depletion is not only a simple hallmark of lung aging but is also involved in the mechanisms for progression of cellular senescence through aberrant MTOR signaling in COPD pathogenesis (Fig. 7).

Although it remains elusive whether lamin B1 depletion is a cause or a simple consequence of cellular senescence, recent reports elucidated the participation of lamin B1 reduction in a range of cellular senescence induced by cell replication, ionizing radiation, and oncogene activation (14). Furthermore, lamin B1 reduction in keratinocytes has been demonstrated to be a prognostic biomarker for not only skin aging but also other skin pathology, suggesting that lamin B1 reduction can be mechanistically involved in the progression of aging-associated disorders (10). Our in vitro experiments clarified that lamin B1 reduction accompanied by cellular senescence was evoked by CSE treatment, and lamin B1 knockdown further enhanced CSE-induced cellular senescence in HBEC (Fig. 2A–C). Intriguingly, lamin B1 knockdown also slightly induced HBEC senescence in the absence of CSE, suggesting a pivotal role for lamin B1 in regulating cellular senescence in HBEC (Fig. 2A–C). COPD lungs also showed decreased lamin B1 protein levels in airway epithelial cells (Fig. 5A–C). Lamin B1 protein levels in HBEC were positively correlated with pulmonary function tests (Fig. 5D), indicating that accelerated cellular senescence conferred by lamin B1 reduction can be causally associated with airway obstruction, delineating disease severity of COPD. It has been reported that not only lamin B1 expression levels but also ratio of LaminA/C can be a determinant for cellular senescence (10, 28). However, we observed no alteration of LaminA/C protein levels nor lamin B1 knockdown in CSE-exposed HBEC (Supplemental Fig. 1). Although the participation of lamins in regulating cell function and cell fate appears to be different in a tissue-type–specific manner (29), we speculate that lamin B1 reduction is at least partly responsible for acceleration of cellular senescence in airway epithelial cells, especially during CS exposure with respect to COPD progression.

CSE treatment reduced lamin B1 protein levels in a concentration-dependent manner, which can be attributed to autophagy-mediated degradation (Fig. 1). A recent paper showed that autophagy activation induced by oncogene, oxidative stress, and DNA damage, but not by starvation nor rapamycin, was responsible for lamin B1 depletion (17). In line with those findings, we observed no alteration of lamin B1 protein levels by torin1-mediated autophagy activation, indicating that genotoxic stress-mediated autophagy is specifically involved in lamin B1 degradation. In general, autophagy is recognized to be a cytoprotective mechanisms for turnover of damaged cellular components and attenuating aging-associated detrimental processes (20, 30). It has been speculated that driving cellular senescence resulting from downregulation of lamin B1 can be a mechanism for restricting tumorigenesis by inhibiting cell proliferation in the setting of oncogenic and tumorigenic insults (17). CS has been known to be a major risk factor for carcinogenesis; hence, it is likely that autophagic lamin B1 degradation accompanied by enhanced cellular senescence in response to CS exposure can also be a protective mechanism for preventing lung cancer development. As another mechanism for lamin B1 depletion, reduction of lamin B1 mRNA levels has been reported during replicative senescence in human lung fibroblasts of WI-38 (11). Decrease in mRNA stability was also responsible for lamin B1 mRNA reduction in the setting of cellular senescence induced by ionizing radiation in normal human fibroblasts (14). Furthermore, it has been reported that lamin B1 mRNA levels can be regulated by miR23a in senescent human dermal fibroblasts and keratinocytes, suggesting that lamin B1 is also regulated at the mRNA levels (10). Although no significant decrease in lamin B1 mRNA during CSE-induced HBEC senescence was demonstrated in our vitro models (Fig. 1B), we observed significant reduction of lamin B1 mRNA levels in COPD lung tissues (Fig. 5I). Accordingly, we consider that lamin B1 depletion during relatively short-term CS exposure can be mainly attributed to autophagic degradation, but reduction of lamin B1 mRNA is also responsible for lamin B1 depletion in COPD lungs caused by long-term CS exposure (31).

Aberrant MTOR activation has been implicated in COPD pathogenesis in terms of regulating cellular senescence and aging phenotype (1). It is likely that MTOR activation by lamin B1 knockdown was attributed to DEPTOR reduction (Fig. 3), which is an important component of MTOR complexes and is a natural negative regulator of MTOR kinase activity. Dysregulation of MTOR activity associated with DEPTOR reduction has been widely implicated in disease pathogenesis, including cancer and Alzheimer's disease development (25, 32). DEPTOR reduction at the mRNA level was observed by lamin B1 knockdown (Fig. 3B) and reduced DEPTOR mRNA was demonstrated in COPD lungs (Fig. 5I), suggesting that lamin B1 regulates DEPTOR expression at the mRNA level. Lamin B1 is thought to regulate gene expression through binding to lamina-associated domains of chromatin (15). In general, nuclear lamina-associated genes are transcriptionally inactive and contain repressive histone markers, including H3K27me3 (33), suggesting that lamin B1 depletion–mediated histone modifications may not be directly related to DEPTOR gene repression. Hence, to clarify the mechanism for DEPTOR regulation by lamin B1, further experiments should be performed.

Mitochondria have a pivotal role in regulating phenotypes associated with aging. Among a variety of mechanisms, mitochondrial free radical theory of aging has been widely addressed in mitochondria-linked aging processes (18, 34). A recent study showed clear connection between DNA damage response-induced cellular senescence and PGC-1β–mediated mitochondria biogenesis (18). DDR-mediated MTOR activation was responsible for PGC-1β expression with concomitant mitochondrial biogenesis accompanied by enhanced ROS, resulting in cellular senescence (18). In the current study, lamin B1 depletion–mediated PGC-1β expression was responsible for increased mitochondrial mass and ROS production in response to CSE exposure (Fig. 2I, 2J). Hence, it is likely that PGC-1β–mediated mitochondrial biogenesis is involved in the mechanisms for accumulation of mitochondria conferred by MTOR activation in the setting of lamin B1 reduction (Fig. 7). The participation of increased mitochondrial mass in regulating CSE-induced HBEC senescence in the case of lamin B1 depletion was confirmed by mitochondria elimination experiments by using PARK2 overexpression with concomitant CCCP treatment (Fig. 3) (18). Increased MTOR activation, PGC-1β expression, and mitochondrial mass were also demonstrated in CS-exposed mouse lungs and COPD lungs, suggesting the crucial role of MTOR-regulated mitochondrial mass in modulating cellular senescence in terms of COPD pathogenesis.

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 demonstrating 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 inflammatory 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 conferred by lamin B1 depletion is involved in the mechanisms for aberrant MTOR activation with concomitantly increased mitochondrial mass via PGC-1β–mediated mitochondrial biogenesis. Accordingly, modalities to prevent reduction of DEPTOR to attenuate MTOR signaling may be a promising therapeutic option to suppress accelerated cellular senescence with lamin B1 depletion in the aging-associated pathological condition in COPD.

We thank Stephanie Cambier (University of Washington, Seattle, WA), Emi Kikuchi, and Dr. Toshiaki Tachibana (Jikei University School of Medicine, Tokyo, Japan) for technical support.

The authors have no financial conflicts of interest.