Articles in PresS. Am J Physiol Lung Cell Mol Physiol (October 17, 2014). doi:10.1152/ajplung.00180.2014

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Cigarette smoke extract affects mitochondrial function in alveolar epithelial cells

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Korbinian Ballweg1, Kathrin Mutze1, Melanie Königshoff1, Oliver Eickelberg1, Silke

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Meiners1

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University, Helmholtz Zentrum München, Munich, Member of the German Center for

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Lung Research (DZL), Germany.

Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians

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Running Head: Cigarette smoke extract affects mitochondria in alveolar epithelial cells

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Correspondence to:

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Silke Meiners, PhD

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Comprehensive

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Germany

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Phone:

0049 89 3187 4673

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Fax:

0049 89 3187 194673

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Email:

[email protected]

Pneumology

Center,

Max-Lebsche-Platz

31,

81377

München,

19 20 1 Copyright © 2014 by the American Physiological Society.

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Authors contributions:

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KB and SM designed the experiments, analyzed the data and wrote the manuscript. KB

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performed the experiments. KM and MK isolated pmATII cells. KB, KM, MK, OE and SM

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edited and revised the manuscript.

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Abstract

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Cigarette smoke is the main risk factor for chronic obstructive pulmonary disease

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(COPD). Exposure of cells to cigarette smoke induces an initial adaptive cellular stress

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response involving increased oxidative stress and induction of inflammatory signaling

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pathways. Exposure of mitochondria to cellular stress alters their fusion/fission

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dynamics. While mild stress induces a pro-survival response termed stress induced

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mitochondrial hyperfusion, severe stress results in mitochondrial fragmentation and

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mitophagy.

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In the present study, we analyzed the mitochondrial response to mild and non-toxic

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doses of cigarette smoke extract (CSE) in alveolar epithelial cells. We characterized

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mitochondrial morphology, expression of mitochondrial fusion and fission genes,

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markers of mitochondrial proteostasis as well as mitochondrial functions such as

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membrane potential and oxygen consumption.

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Murine lung epithelial (MLE)12, as well as primary mouse alveolar epithelial cells

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revealed pronounced mitochondrial hyperfusion upon treatment with CSE, accompanied

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by increased expression of the mitochondrial fusion protein mitofusin (MFN) 2 and

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increased metabolic activity. We did not observe any alterations in mitochondrial

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proteostasis, i.e. induction of the mitochondrial unfolded protein response or mitophagy.

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Therefore, our data indicate an adaptive pro-survival response of mitochondria of

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alveolar epithelial cells to non-toxic concentrations of CSE. A hyperfused mitochondrial

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network, however, renders the cell more vulnerable to additional stress such as

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sustained cigarette smoke exposure. As such cigarette smoke induced mitochondrial

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hyperfusion - although being part of a beneficial adaptive stress response in the first

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place - may contribute to the pathogenesis of COPD.

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Key words

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COPD, emphysema, proteostasis, stress-induced-mitochondrial-hyperfusion

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Introduction

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Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death

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worldwide. It is characterized by progressive and irreversible airflow limitation.(4, 44) In

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the western world the main risk factor for development of COPD is cigarette smoke (CS)

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(4, 44), which is a complex mixture of thousands of injurious agents and reactive

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oxidants.(7, 33, 44) Exposure of lung epithelial cells to CS initiates an adaptive cell

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response such as induction of autophagy, impaired proteasome function, and

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proinflammatory and oxidative responses.(33, 7, 35)

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Mitochondria are crucial cellular organelles for cellular energetics, signaling and

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apoptosis. Differences in cellular energy demands or cellular stress rapidly alter

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mitochondrial

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dynamics.(5, 9, 26) Mitochondrial hyperfusion provides a stress resolving mechanism

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for the cell as elongated mitochondria are protected from degradation via autophagy

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and show increased efficiency in ATP synthesis(15, 40), thereby contributing to the

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cells’ ability to repair cellular damage. Furthermore mitochondrial fusion is part of the

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mitochondrial quality control as increased fusion allows enhanced mixing of

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mitochondrial components and thus complementation and dilution of mitochondrial

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damage.(49) In contrast, mitochondrial fragmentation is induced upon severe stress and

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is an early event upon induction of apoptosis. (13, 25) Damaged and fragmented

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mitochondria are then removed by mitochondrial autophagy to prevent spreading of

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damage in the mitochondrial network.(39, 45, 49) Hence, mitochondrial morphology and

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dynamics represent an important adaptive mechanism in response to cellular stress.

behavior

mainly

by

changing

mitochondrial

fusion

and

fission

5

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Recently, cigarette smoke-induced alterations in mitochondrial morphology were

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described in bronchial epithelial cells and asthmatic smooth muscle cells. Hara et al.

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reported increased mitochondrial fragmentation.(18) In contrast, Hoffmann et al.,

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observed elongated mitochondria with increased branching in bronchial epithelial cells

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from COPD patients.(19) In airway smooth muscle cells, CSE induces mitochondrial

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fragmentation and upregulation of the mitochondrial fission protein DRP1 and

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downregulation of the fusion protein MFN2.(3) Taken together, these potentially

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conflicting data suggest cell-type specific differences of mitochondrial morphology in

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response to cigarette smoke, and emphasize the need for further studies.

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In this report, we show, for the first time, that cigarette smoke affects mitochondrial

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morphology of alveolar epithelial cells. Treatment with non-toxic doses of cigarette

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smoke extract results in hyperfusion of mitochondria and increased expression levels of

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the mitochondrial fusion protein MFN2. This effect is accompanied by an increase in

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metabolic activity and mitochondrial membrane potential.

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Materials and Methods

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Reagents:

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Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was dissolved in DMSO to obtain a

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25 mM stock solution. Antimycin A and rotenone were dissolved in DMSO to a 50 mM

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stock solution, respectively. Oligomycin A was dissolved to a 5 mM stock solution in

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DMSO. Tetramethylrhodamin-methylester perchlorate (TMRM) was diluted in DMSO to

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a 1 mM stock solution. Stock solutions were stored at -20°C and diluted to the

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appropriate final concentration directly before use. 2,5-diphenyltetrazolium bromide

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(MTT) was stored at 4°C and dissolved in PBS to a working solution of 5 mg/ml directly

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before use. All reagents except TMRM were obtained from Sigma-Aldrich (St. Louis,

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MO). TMRM was purchased from Life Technologies (Carlsbad, CA).

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Antibodies used were: anti-MFN2 (Abcam, Cambridge, UK), anti-OPA1 (GeneTex,

101

Irvine, CA), anti-DRP1 (Cell Signaling, Cambridge, UK), anti-Calreticulin (Abcam), anti-

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ATP5A (Abcam), anti-α-Tubulin (GeneTex), anti-PINK1 (Novus Biologicals, Littleton,

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CO), anti-MFN1 (Novus Biologicals), anti-β-Actin (Sigma-Aldrich), anti-HSP60 (Cell

104

Signaling) and anti-cytochrome C (BD Bioscience, San Jose, CA). Secondary

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antibodies used were HRP conjugated goat anti-mouse IgG, HRP conjugated goat anti-

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rabbit IgG (GE Healthcare, Chalfont St Giles UK), and AlexaFluor488 conjugated goat

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anti-rabbit IgG (Life Technologies).

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Cell culture

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The mouse lung epithelial cell line (MLE12) was cultured in RPMI Medium (Life

110

Technologies) containing 10% FCS (PAN Biotech) and 1% Penicillin/Streptomycin (Life

7

111

Technologies). Cells were grown at 37°C in a humidified atmosphere containing 5%

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CO2. Treatment medium for MLE12 cells consisted of FCS-free RPMI Medium

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supplemented with 1% Penicillin/Streptomycin.

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Primary murine alveolar epithelial type II (pmATII) cell isolation and culture

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pmATII cells were isolated from C57BL/6N mice (Charles River GmbH, Sulzfeld,

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Germany) at 8-14 weeks of age as describes before. (10, 23) Briefly, mouse lungs were

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lavaged with 500 µl of sterile PBS twice and flushed through the right heart using 0.9%

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NaCl solution (B. Braun Melsungen AG, Melsungen, Germany). Lungs were

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subsequently inflated with 1.5 ml dispase (BD Bioscience, San Jose, CA) and 300 µl of

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1% low melting point agarose (Sigma-Aldrich) and incubated for 45 min at room

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temperature (RT). Lungs were minced and consecutively filtered through 100 µm,

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20 µm, and 10 μm nylon meshes (Sefar, Heiden, Switzerland). Single cell suspension

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was centrifuged at 200 g for 10 min and the cell pellet was resuspended in DMEM cell

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culture medium (Sigma Aldrich). Incubation of the single cell suspension on petri dishes

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coated with antibodies against CD45 and CD16/32 (both BD Bioscience) for 30 min at

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37°C was performed for depletion of macrophages and lymphocytes. Non-adherent

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cells were collected and negative selection for fibroblasts was performed by adherence

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for 25 minutes on cell culture dishes. Again, non-adherent cells were collected and cell

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viability was determined by trypan blue exclusion. Cell purity was assessed by

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immunofluorescence staining of cytospin preparations using antibodies for proSFTPC

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(Merck Millipore, Darmstadt, Germany), panCK (Dako, Hamburg, Germany), CD45 (BD

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Bioscience), and αSMA (Sigma Aldrich). pmATII cells were resuspended in DMEM

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supplemented with 10% FCS (PAA Laboratories, Pasching, Austria), 2 mM l-glutamine, 8

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1% penicillin/streptomycin (both Life Technologies), 3.6 mg/ml glucose (Applichem

135

GmbH, Darmstadt, Germany), and 10 mM HEPES (PAA Laboratories) and cells were

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cultured for 24 h to allow attachment. Medium was changed and cells were cultured up

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to 5 days in a humidified atmosphere of 5% CO2 at 37°C. Treatment medium for pmATII

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cells consisted of FCS free DMEM supplemented with 1% penicillin/streptomycin.

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Cigarette smoke extract preparation

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Cigarette smoke extract (CSE) was prepared by bubbling smoke from 6 cigarettes

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(Research-grade cigarettes (3R4F); Kentucky Tobacco Research and Development

142

Center at the University of Kentucky) through 100 ml of FCS-free cell culture medium at

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a constant airflow. Smoked medium was then sterile filtered through a 0.20 µm filter

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(Minisart; Satorius Stedim Biotech, Göttingen, Germany), split into aliquots, stored at

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-20°C and served as the 100% CSE stock solution. For treatment, CSE was

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supplemented with 1% penicillin/streptomycin and diluted with treatment medium to the

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indicated concentrations directly before use. To assure comparable potency of CSE,

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each stock solution was tested by MTT assays.

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Cytotoxicity testing

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Cytotoxicity was assessed using the cytotoxicity detection kit (Roche, Basel,

151

Switzerland) according to the manufacturer’s instructions. For MLE12 cells, 450,000

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cells/well were seeded into 6-well plates and grown to approx. 80% confluency and

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treated for the indicated times. 15 minutes before the end of the treatment, one well was

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treated with 2% Triton X-100 to serve as the control for maximal cellular release of LDH.

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Supernatants were collected, cleared by centrifugation, and LDH content was measured

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in technical triplicates according to the manufacturer’s instructions using a Tristar LB 9

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941 plate reader (Berthold Technologies, Bad Wildbad, Germany). Data were

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normalized as follows: Untreated cells were set to be 0% cytotoxicity and 2% Triton X-

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100 treated cells were set to be 100% cytotoxicity.

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Metabolic activity assay

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Metabolic activity was measured using the 2,5-diphenyltetrazolium bromide (MTT)

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assay. Briefly, 100,000 cells/well were seeded into 24-well plates, grown to approx. 80%

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confluency, and treated for the indicated times. Each concentration was treated in at

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least six technical replicates. After treatment, 100 ml of freshly prepared thiazolyl blue

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tetrazolium bromide (Sigma) solution (5 mg/ml in PBS) was added to each well and

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incubated for 30 min at 37°C. The supernatant was aspirated, and blue crystals were

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dissolved in 500 ml isopropanol + 0.1% Triton X-100. Absorbance was measured at

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570 nm using a Tristar LB 941 plate reader (Berthold Technologies).

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Mitochondrial membrane potential assessment

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Mitochondrial membrane potential was measured by TMRM fluorescence. 450,000

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cells/well were seeded in 6-well plates and grown to approx. 80% confluency before

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treatment for the indicated times. 10 µM CCCP treatment was used as a positive

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control. After the treatment, cells were stained for 30 minutes in medium containing

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5 nM TMRM (Life Technologies). Cells were washed with PBS, trypsinized, and

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resuspended in 500 µl FACS Buffer (2% FCS + 20 µM EDTA in PBS). Samples were

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then analyzed by FACS analysis and mitochondrial membrane potential was measured

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as mean TMRM fluorescence intensity using the Becton-Dickinson-LSRII. To ensure

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total depolarization, CCCP was added to a final concentration of 100 µM to the samples

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and measurements were repeated after 5, 30, and 60 minutes. 10

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Analysis of mitochondrial superoxide production

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Mitochondrial superoxide generation was analyzed using MitoSOX Red. 450,000

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cells/well were seeded in 6-well plates, grown to approx. 80% confluency, and treated

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for the indicated times. Antimycin A was used as a positive control to induce

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mitochondrial superoxide generation. After the treatment, cells were stained for

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30 minutes in medium containing 5 µM MitoSOX Red (Life Technologies). Cells were

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washed with PBS, trypsinized, and resuspended in 500 µl FACS Buffer. Samples were

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then analyzed by FACS analysis and superoxide production was measured as mean

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MitoSOX fluorescence intensity (BD LSRII).

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Measurement of cellular ATP levels

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Cellular ATP levels were measured using CellTiter-Glo assay kit (Promega, Madiso,

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WI). Briefly, 450,000 cells were seeded in 6 well plates and grown to approx. 80%

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confluency. Cells were treated with control or CSE containing medium for 24h. After the

193

treatment, cells were trypsinized and 40,000 cells/well were transferred to a 96 well

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plate. CellTiter-Glo reagent was added and the luminescent signal was measured after

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shaking the plate thoroughly in a Tristar LB 941 plate reader (Berthold Technologies)

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Analysis of mitochondrial morphology

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Analysis of mitochondrial morphology was performed as described elsewhere.(27)

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Briefly, cells were grown on 15 mm glass coverslips to approx. 50% confluency and

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treated for the indicated times with CSE or with CCCP as a positive control. After the

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treatment, cells were fixed with 4% PFA for 10 minutes, permeabilized with 0.1% Triton

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X-100 in PBS and unspecific binding sites were blocked with Roti-Immunoblock (Roth,

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Karlsruhe, Germany) for 1 hour at room temperature. After blocking, cells were 11

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incubated with anti-cytochrome C antibody for 2 hours at room temperature, washed,

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and incubated with an Alexa Fluor488 coupled secondary antibody for 1 hour at room

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temperature. All subsequent steps were performed with minimal light exposure. Cells

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were washed twice for 10 minutes with PBS and nuclei were stained with DAPI (300 nM

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in PBS for 5 minutes). Finally, cells were mounted on microscopic slides using

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fluorescent mounting medium (Dako, Hamburg, Germany) and imaged using confocal

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laser-scanning microscopy (Zeiss LSM710, Oberkochen, Germany).

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Cells were categorized into three classes according to their mitochondrial morphology:

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Cells displaying an intact network of tubular mitochondria were classified as normal.

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When this network was disrupted and mitochondria appeared predominantly spherical

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or rod-like, they were classified as fragmented. Cells with considerably elongated

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mitochondria, which were more interconnected, were classified as hyperfused. The

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mitochondrial morphology of on average 100 cells was determined by a blinded

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observer.

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Preparation of cell lysates and mitochondrial fractions

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For protein lysate preparation, 450,000 cells/well were seeded in 6-well plates and

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grown to approx. 80% confluency. Cells were treated for the indicated times with CSE

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or control medium. After treatment, cells were trypsinized, washed with PBS, and lysed

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in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium

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deoxycholate, and 0.1% SDS) supplemented with protease inhibitor cocktail (Complete,

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Roche).

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Mitochondria were isolated by ultracentrifugation after cellular lysis using a cell

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disruption bomb (Parr Instruments, Moline, IL). In short, cells were plated in 15 cm 12

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dishes, grown to 80% confluency, and treated for 24h with 25% CSE. After the

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treatment, cells were scraped, pelleted and resuspended in mitochondrial isolation

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buffer (220 mM mannitol, 70 mM sucrose, 5 mM HEPES-KOH, 1 mM EGTA-KOH)

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supplemented with protease inhibitor cocktail (Complete; Roche). Cells were lysed by

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stirring 15 min in a cell disruption bomb at 800 psi nitrogen pressure and subsequent

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rapid depressurizing. Cell lysates were then centrifuged at 600 g to remove nuclei. The

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supernatant was centrifuged at 8000 g to obtain a crude mitochondrial pellet. The

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mitochondrial pellet was resuspended in mitochondrial isolation buffer to serve as the

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mitochondrial fraction, while the supernatant contained the cytosolic fraction. Protein

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content was determined using the Pierce BCA protein assay kit (Thermo Fisher

236

Scientific, Waltham, MA).

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Western blot analysis

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For Western blot analysis, 10-20 µg of protein were subjected to electrophoresis on

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7.5% SDS-PAGE gels and blotted onto polyvinylidenedifluoride (PVDF) membranes.

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Membranes were treated with antibodies using standard Western blot techniques. The

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ECL Plus Detection Reagent (GE Healthcare) and Super Signal West Femto (Thermo

242

Fisher Scientific) were used for chemiluminescent detection, and membranes were

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analyzed using X-Omat LS films (Carestream, Rochester, NY) in a Curix 60 developer

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(Agfa, Mortsel, Belgium). Densitometry analysis was performed using the ImageLab

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Software (Biorad, Hercules, CA).

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Quantitative real-time RT-PCR

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Total RNA from cells was isolated using Roti-Quick-Kit (Carl Roth, Karlsruhe,

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Germany). 100-1000 ng per sample of total RNA were reverse-transcribed using 13

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random hexamers (Life Technologies) and M-MLV reverse transcriptase (Sigma-

250

Aldrich). Quantitative PCR was performed using the SYBR Green LC480 System

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(Roche Diagnostics, Mannheim, Germany). The following gene-specific primer were

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used

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Fw-MFN1: 5’-TGTGTTCGGATTTTCAAGAGGACA-3’

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Rv-MFN1: 5’-CTCCTGGGCTGCATTATCCG-3’

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Fw-MFN2: 5’-CATGTCCACGATGCCCAAC-3’

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Rv-MFN2: 5’-GACAAAGTGCTTGAGAGGGG-3’

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Fw-DRP1: 5’-AGGAGATGCAGAGGATCATTCAG-3’

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Rv-DRP1: 5’-ATCAGCAAAGTCGGGGTGTT-3’

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Fw-OPA1: 5’-ATGATTGGGCCAGACTGGAA-3’

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Rv-OPA1: 5’-AGGTAAGCTGGGTGCTCATC-3’

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Measurement of oxygen consumption

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Cells were seeded in 10 cm dishes, grown to approx. 80% confluency and treated with

263

control medium or 25% CSE containing medium. After 24h of treatment, cells were

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washed with PBS, trypsinized, and 5x106 cells were transferred in an Oxygraph

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chamber (Oroboros Instruments, Innsbruck, Austria) in respiration buffer (0.5 mM

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EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM Taurine, 10 mM KH2PO4, 20 mM

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HEPES-KOH, 110 mM Mannitol and 1 g/l BSA). Oxygen consumption was measured at

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basal conditions (basal respiration), after addition of 2.5 µM Oligomycin (blocking proton

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backflow through complex V to measure residual proton leak), after addition of 1 µM

14

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CCCP (uncoupling the respiratory chain to enable unlimited proton flow through the

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mitochondrial membrane to induce maximum respiration) and after addition of 2.5 µM

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Antimycin A/Rotenone (blocking Complex I and III to inhibit mitochondrial respiration).

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These concentrations were also previously shown to be suitable for measuring oxygen

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consumption rates in MLE12 cells.(11)

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Statistical analysis

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One-way ANOVA with Dunnett’s multiple comparison test was used for statistical

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analysis of mitochondrial membrane potential and mitochondrial superoxide production.

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Differences in protein expression data were evaluated using t-test or one-way ANOVA

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with Dunnett’s multiple comparison test. Alterations in mitochondrial morphology were

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analyzed using the chi-square test. Statistical analysis was performed using GraphPad

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Prism software (version 5.00).

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Results

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Cigarette smoke induces mitochondrial elongation in MLE12 mouse alveolar epithelial

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cells

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In order to analyze the effect of cigarette smoke on mitochondrial function of alveolar

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epithelial cells, we treated the mouse alveolar epithelial cell line MLE12 with non-toxic

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doses of cigarette smoke extract (CSE) and stained mitochondria with cytochrome-C

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antibody. CSE doses up to 25% CSE were non-toxic as assessed by LDH release

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assays and absence of caspase 3 cleavage (Figures 1A&B) and shown before (35). Of

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note, non-toxic doses of CSE induced a striking elongation of mitochondria (Figure 2A,

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Panel B). In order to quantify CSE-induced changes in mitochondrial morphology, we

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classified mitochondrial morphology into three types: “fragmented”, “normal” or

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“hyperfused” morphology (for details see materials & methods part). CSE significantly

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increased the fraction of cells with hyperfused mitochondria compared to untreated

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control cells after 6, 24, or 48h of treatment (Figure 2B). As a positive control for altered

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mitochondrial morphology, we treated cells with CCCP, a mitochondrial uncoupling

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reagent that is well-known to induce fragmentation of mitochondria.(12, 39) Indeed,

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CCCP treatment led to pronounced and time-dependent mitochondrial fragmentation,

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confirming that our classification strategy is a valid tool for semiquantitative analysis of

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mitochondrial morphology (Figures 2A&B).

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The observed changes in mitochondrial morphology were further analyzed by

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expression analysis for the mitochondrial fusion and fission proteins MFN1 and MFN2

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(involved in fusion of outer mitochondrial membranes), OPA1 (inner mitochondrial

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membrane fusion), and DRP1 (involved in mitochondrial fission). MFN2 levels 16

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significantly increased within 24h of treatment with 10% or 25% CSE (Figures 3A&B)

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and returned to baseline after 48h. In contrast, no significant changes in response to

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CSE were observed for MFN1, OPA1, and DRP1 expression (Figures 3A&B). Again,

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CCCP served as a positive control to detect changes in mitochondrial morphology on

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the level of fusion and fission proteins. We observed 5 bands for OPA1 and 2 bands for

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DRP1 according to different isoforms of the proteins. In accordance with published data

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(12, 39), we observed reduction in MFN2 levels and proteolytic cleavage of long OPA1

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isoforms (a,b) at early time points of CCCP treatment (Figures 3A&B). Protein levels of

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the fission protein DRP1, which translocates from the cytosol to mitochondria for

315

triggering mitochondrial fission (16), were analyzed in isolated mitochondrial fractions of

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CSE treated cells. We found no consistent change in DRP1 levels in our enriched

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mitochondrial fractions (Figures 3C&D). Similar results were obtained by analysis of

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mRNA levels of mitochondrial fusion and fission proteins. Only MFN2 mRNA showed a

319

transient increase after 24h of 25% CSE treatment (Figure 3E). Taken together, our

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data reveal increased mitochondrial fusion as an adaptive response to treatment with

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low and non-toxic doses of cigarette smoke extract.

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CSE-mediated mitochondrial elongation is associated with increased mitochondrial

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function

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As mitochondrial hyperfusion has previously been described as a mechanism to

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increase mitochondrial ATP production (15, 40), we comprehensively investigated

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mitochondrial function in CSE treated MLE12 cells by analyzing several hallmarks of

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mitochondrial function. Metabolic activity was significantly increased at non-toxic doses

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of CSE, as analyzed by the MTT assay while increasing doses of CSE up to 75% dose17

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dependently decreased metabolic activity and survival of alveolar epithelial cells

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(Figure 4A). Concordantly, ATP levels were increased in MLE12 cells after 24h of CSE

331

exposure (Figure 4B). Furthermore, CSE treatment resulted in a significant increase in

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mitochondrial membrane potential after 6h, 24h, and 48h of treatment while the

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uncoupling agent CCCP efficiently decreased the mitochondrial membrane potential as

334

assessed by measuring TMRM fluorescence (Figure 5A). Specificity of TMRM

335

dependent assessment of mitochondrial membrane potential was confirmed by

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uncoupling of the respiratory chain and concomitant loss of TMRM fluorescence with

337

high concentrations of CCCP (data not shown).

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Elevation of the mitochondrial membrane potential is often associated with increased

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formation of mitochondrial ROS.(37) Hence, we measured mitochondria-derived

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superoxides using the mitochondria specific probe MitoSOX Red. Remarkably,

341

mitochondrial superoxide production was not altered after CSE treatment (Figure 5B)

342

indicating that the increase in mitochondrial membrane potential does not induce

343

enhanced mitochondrial superoxide formation. The function of our probe was confirmed

344

by adding Antimycin A, a commonly used inducer of mitochondrial superoxide (31),

345

which markedly increased MitoSOX-specific fluorescence (data not shown).

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To assess the underlying mechanism for the increase in mitochondrial membrane

347

potential, we further analyzed mitochondrial oxygen consumption in CSE-treated cells.

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An increase in mitochondrial membrane potential can be caused by increased flow of

349

protons into the intermembrane space and enhanced rate of oxygen consumption

350

through increased activity of the respiratory chain. Alternatively, reducing proton

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backflow into the mitochondrial matrix due to inhibition of complex V of the respiratory 18

352

chain can also increase mitochondrial membrane potential. The rate of basal oxygen

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consumption and respiration was slightly increased in CSE treated alveolar epithelial

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cells. Proton leakage through the mitochondrial inner membrane was not affected by

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CSE treatment as revealed by inhibition of proton backflow by Oligomycin-induced

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inhibition of complex V. Maximal mitochondrial oxygen consumption can be assessed

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by triggering unlimited proton flow through the membrane by CCCP. Maximum

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respiration was also slightly elevated in CSE treated cells. Finally, blocking complex I

359

and III by Rotenone and Antimycin A demonstrated that oxygen consumption was

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indeed due to respiratory chain activity (Figures 5C&E). Importantly, mitochondrial ATP

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production measured as Oligomycin sensitive respiration was also increased in CSE

362

treated cells (Figure 5D).

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Altogether, these data indicate an increase in mitochondrial activity in alveolar epithelial

364

cells in response to CSE in the absence of elevated levels of mitochondrial superoxides.

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CSE treatment of MLE12 cells does not alter mitochondrial proteostasis

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Damage of mitochondria was reported to affect mitochondrial proteostasis as indicated

367

by upregulation of the mitochondrial chaperone HSP60 as part of the mitochondrial

368

unfolded protein response (mitoUPR) (52) and to trigger disposal of defective

369

mitochondria

370

macroautophagy.(50) To analyze whether treatment of alveolar epithelial cells with low

371

doses of CSE affects mitochondrial proteostasis, we analyzed expression levels of the

372

mitochondrial chaperone HSP60 and of ATP5A as a surrogate marker for mitochondrial

373

mass in cell lysates. We furthermore analyzed PINK1 expression in isolated

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mitochondrial fractions. CSE treatment did not affect expression of HSP60 or ATP5A in

by

PINK1/Parkin-mediated

mitophagy,

a

specialized

form

of

19

375

MLE12 cells after 6, 24, or 48h of treatment (Figures 6A&B). Accumulation of full length

376

PINK1 on the mitochondrial outer membrane leads to the initiation of mitophagy. (20) In

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MLE12 cells, we detected a double band for full length PINK1 with a molecular weight of

378

67 kDA and one band for cleaved PINK1 (55 kDA) which was not altered in

379

mitochondrial fractions of CSE or control treated cells (Figures 6C&D). These results

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suggest that CSE treatment with low doses induces an adaptive mitochondrial response

381

in alveolar epithelial cells that does not involve pronounced alterations in mitochondrial

382

proteostasis.

383

Cigarette smoke induces mitochondrial elongation in primary mouse alveolar epithelial

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type II cells

385

To shed light on the effects of CSE on mitochondrial morphology in primary alveolar

386

epithelial cells, we treated primary mouse alveolar epithelial type II (pmATII) cells with

387

CSE for 24h. The pmATII cells showed increased resistance to CSE compared to

388

MLE12 cells as analyzed by LDH release assays (data not shown). Cells treated with

389

50% CSE exhibited pronounced mitochondrial hyperfusion, similar to the hyperfused

390

mitochondria observed for MLE12 cells (Figures 7A&B). Additionally, as observed in

391

MLE12 cells, expression of MFN2 was slightly elevated in CSE treated pmATII. No

392

change in different OPA1 isoforms or in MFN1 and DRP1 levels was detected (Figure

393

8A&B). These data confirm that mitochondrial hyperfusion is a conserved feature of

394

alveolar epithelial cells in the adaptive response to low and non-toxic doses of cigarette

395

smoke.

396

20

397

Discussion

398

In the present study, we demonstrate that MLE12 alveolar epithelial cells respond to

399

non-toxic doses of CSE with mitochondrial hyperfusion involving increased levels of the

400

mitochondrial fusion protein MFN2. Elongation is accompanied by functional

401

mitochondrial changes such as increased metabolic activity and membrane potential

402

upon CSE treatment. Mitochondrial elongation and upregulation of MFN2 were

403

confirmed in primary mouse ATII cells.

404

Non-toxic doses of cigarette smoke extract do not impair mitochondrial proteostasis of

405

alveolar epithelial cells

406

Cigarette smoke exposes lung cells to a plethora of harmful and highly reactive

407

chemicals and is a potent inducer of cellular damage.(7, 33, 44) We exposed cells to

408

cigarette smoke extract which is a commonly used model to assess the effect of

409

cigarette smoke in a cell culture system. In this model system, we found the applied

410

cigarette smoke doses to be non-toxic as assessed by the absence of apoptotic and

411

necrotic cell death markers. Non-toxic doses of cigarette smoke have been shown to

412

affect function and activity of numerous cellular proteins and impair overall protein

413

homeostasis in lung alveolar and bronchial cells (6, 29, 35). Furthermore, cigarette

414

smoke affects organelle-specific proteostasis as shown for activation of the unfolded

415

protein response in the ER in lung tissue homogenates of chronic smokers and in CSE

416

treated cells.(22, 47) Damaged and misfolded proteins in the mitochondria activate the

417

mitochondrial unfolded protein response (mitoUPR). Upregulation of the mitochondrial

418

matrix chaperone HSP60 is a hallmark for mitoUPR induction.(21, 34, 52) Severely

419

damaged mitochondria depolarize and are degraded via mitophagy involving 21

420

PINK1/Parkin activation.(20, 39, 45) We therefore assessed mitochondrial quality

421

control in response to cigarette smoke extract. However, we found no signs for impaired

422

mitochondrial function or induction of mitochondrial damage in alveolar epithelial cells

423

upon non-toxic CSE treatment. HSP60 levels did not increase in CSE treated lung

424

epithelial cells, suggesting the absence of mitoUPR activation. Furthermore, we did not

425

observe any signs of mitochondrial autophagy as levels of the mitophagy marker PINK1

426

and the mitochondrial mass (measured by ATP5A expression) remained unaltered by

427

non-toxic CSE treatment. We thus conclude that our applied non-toxic doses of

428

cigarette smoke extract do not harm mitochondria of alveolar epithelial cells to such an

429

extent that seriously challenges mitochondrial proteostasis. Indeed, our CSE treatment

430

regimen induced only mild oxidative stress to mitochondria as indicated by our finding

431

that mitochondrial superoxide production was not increased by CSE treatment. These

432

mild and probably physiologically more relevant treatment conditions are clearly

433

different from those used in another study that observed induction of necrotic cell death

434

by cigarette smoke extract in primary bronchial epithelial cells.(41) During the

435

preparation of this manuscript another study was published showing increased PINK1

436

expression and mitophagy in bronchial epithelial cells and CS exposed mice. (30) In this

437

study, mitophagy was an upstream event of cigarette smoke induced necroptosis. (30)

438

This seems plausible given the fact that mitochondria are an integral component of

439

apoptosis and necroptosis pathways. (25, 46) It also indicates that the effect of cigarette

440

smoke extract on pulmonary epithelial cells is strongly dependent on the dose and the

441

effects on cell survival. Additionally, there might be not only a dose- but also cell type

442

dependent responsiveness between alveolar and bronchial epithelial cells to cigarette

22

443

smoke extract. Furthermore, a time dependency was observed in wood smoke treated

444

guinea pigs showing early decrease of mitochondrial function, but full recovery and

445

overcompensation later on. (17) In our study, we cannot rule out that mitochondrial

446

function is transiently decreased at earlier time points than 6 hours and improves later.

447

Non-toxic doses of cigarette smoke extract induce adaptive mitochondrial hyperfusion

448

CSE treatment of alveolar epithelial cells induced an adaptive mitochondrial response

449

involving mitochondrial elongation and fusion. Mitochondrial hyperfusion was associated

450

with increased levels of the mitochondrial fusion protein MFN2. Expression of the

451

mitochondrial mass marker ATP5A, the fusion protein MFN1, the inner membrane

452

fusion protein OPA1, and the mitochondrial fission protein DRP1 were not altered in the

453

respiratory epithelial cells. Mitochondrial hyperfusion was initially described to be

454

dependent on OPA1 and MFN1, but not MFN2, however, without expressional changes

455

in any of these proteins. (40) In contrast, starvation induced hyperfusion was dependent

456

on DRP1 phosphorylation and its decreased translocation to mitochondria but

457

expression levels of fusion and fission protein remained unchanged. (15) Of note, we

458

did not observe any mitochondrial translocation of DRP1 in our study indicating that the

459

mechanism of CSE induced hyperfusion is different to starvation induced elongation

460

and probably not mediated by decreased fission activity. Lastly, increased

461

oligomerization of fusion proteins was observed in oxidative stress mediated

462

hyperfusion of mitochondria without changes of absolute fusion and fission protein

463

levels. (38) This clearly shows that expressional changes are not necessary to trigger

464

stress induced hyperfusion but rather altered activity of the fusion and fission machinery

465

contributes to mitochondrial hyperfusion. Concordantly, we find hyperfusion as early as 23

466

6h of treatment where no changes in Mitofusin levels are present and hyperfusion lasts

467

up to 48h were MFN2 levels are already declining to baseline arguing that changes in

468

activity are the critical factor for the observed hyperfusion. As all mitochondrial fusion

469

and fission proteins are present in the cells we cannot determine which proteins actually

470

are responsible for the observed morphology. However, overexpression of MFN2 in

471

neurons or in pulmonary artery smooth muscle cells induced mitochondrial elongation

472

(8, 36) hence, increased fusion and especially increased levels of MFN2 may

473

functionally influence mitochondrial hyperfusion. Thus, upregulation of MFN2 levels

474

might be an adaptive response which is additional to the described stress induced

475

hyperfusion pathways. However, as levels and activity of mitochondrial fusion and

476

fission proteins need to be tightly balanced elevated MFN2 levels are probably not

477

maintained over longer periods.

478

Our results differ from data obtained for CSE exposure of bronchial epithelial cells: One

479

study observed mitochondrial fragmentation in primary human bronchial epithelial cells

480

treated with CSE for 48h. Mitochondrial fragmentation was associated with increased

481

mitochondrial ROS production and localization of the fission protein DRP1 to

482

mitochondria. This study did not observe any changes for MFN2 or other mitochondrial

483

fusion proteins.(18) Another study, however described elongated and branched

484

mitochondria in long term (6 months) cigarette smoke exposure of bronchial epithelial

485

cells and in primary bronchial epithelial cells isolated from COPD patients.(19) We

486

analyzed mitochondrial morphology in 16HBE cells, a human bronchial epithelial cell

487

line, and did not detect alterations in mitochondrial fusion and fission dynamics in

488

control vs. CSE treated cells (data not shown). Thus, we conclude that there is a strong

24

489

dependency on cell type for triggering an adaptive mitochondrial hyperfusion response

490

of lung epithelial cells to cigarette smoke extract.

491

Functional consequences of mitochondrial elongation

492

Mitochondrial hyperfusion was reported to be connected with an increased efficiency of

493

ATP production.(15, 40) Accordingly, we observed that hyperfused mitochondria in

494

MLE12 cells were associated with enhanced metabolic activity, increased cellular ATP

495

levels,

496

consumption rates. These observations are well in agreement with the concept of stress

497

induced mitochondrial hyperfusion as a protective response to increase the cell’s ability

498

to repair damage. This was previously shown for different stressors such as starvation,

499

UV, actinomycin, or cycloheximide treatment. (15, 40) As mitochondrial hyperfusion can

500

also be induced by oxidative stress (38, 48), it seems plausible that this survival

501

response is also part of the oxidative stress response to cigarette smoke extract.

502

We observed mitochondrial hyperfusion in the ATI-like MLE12 alveolar epithelial cells

503

and in primary ATII alveolar epithelial cells. Although MLE12 and pmATII cells probably

504

differ in their metabolic phenotype we speculate that the mitochondrial hyperfusion

505

observed in pmATII cells also affects mitochondrial metabolism in these cells. Increased

506

respiration rates have previously been described for primary ATII cells of smoke-

507

exposed mice but depended on the available substrates: Agarwal et al. observed

508

significantly increased mitochondrial activity and oxygen consumption when applying

509

pyruvate or palmitate as a substrate, while respiration decreased when using

510

glucose.(1, 2) Thus, although we didn’t measure mitochondrial activity in pmATII cells

511

ourselves it is well feasible that mitochondrial hyperfusion results in increased oxygen

augmented

mitochondrial

membrane

potential,

and

elevated

oxygen

25

512

consumption in CS exposed pmATII when respiring on pyruvate. Similar to our results,

513

Hoffmann et al. showed increased ATP levels in CSE treated bronchial epithelial cells

514

with elongated mitochondria (19), further supporting the notion of increased

515

mitochondrial respiratory chain activity in response to cigarette smoke. In summary,

516

cigarette-smoke induced mitochondrial hyperfusion might serve as an adaptive

517

response to increase mitochondrial ATP production in order to cope with CSE induced

518

cell damage. A hyperfused mitochondrial network, however, may render the cell more

519

vulnerable to additional stress as it attenuates mitochondrial quality control.(14)

520

Moreover, because elongated mitochondria are spared from removal by autophagy

521

(16), sustained mitochondrial hyperfusion i.e. due to continuous and chronic exposure

522

to stress might further increase susceptibility upon additional stress of the mitochondrial

523

network. This mechanism was suggested to account for the occurrence of hyperfused

524

dysfunctional mitochondria in aging muscles.(28) Of note, sustained mitochondrial

525

elongation has also been associated with increased cellular senescence.(24) Vice

526

versa, induction of senescence is accompanied by mitochondrial elongation in

527

senescent cells.(48) Alveolar epithelial cell senescence is known to be induced by

528

cigarette smoke treatment (42) and senescence of pulmonary cells has been proposed

529

as a pathogenetic mechanisms for progression of COPD.(32, 43) Furthermore,

530

mitochondrial hyperfusion was recently shown to be accompanied by NF-κb

531

activation.(51) It is thus tempting to speculate that cigarette smoke extract induced

532

mitochondrial hyperfusion - although being part of a beneficial adaptive stress response

533

in the first place - may contribute to age-related COPD pathogenesis via promoting

26

534

diminished mitochondrial quality control, impaired cellular stress resistance, and cellular

535

senescence.

536

Acknowledgement

537

We are very grateful to Otmar Schmid, Fabiana Perocchi, Jennifer Wettmarshausen,

538

Konstanze Winklhofer, and Anne Kathrin Müller-Rischart for their support and helpful

539

advice during the preparation of the manuscript. The authors would also like to thank

540

Christina Lukas, Julia Kipp, and Anastasia van den Berg for excellent technical

541

assistance.

542

Disclosure

543

The authors declare that they have no conflicts of interests.

27

544

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32

708

Figure Legends

709

Figure 1: Low-doses of CSE are nontoxic in MLE12 cells.

710

(A)

711

dose CSE for the indicated times. Each band corresponds to an independent

712

experiment. Positive control: 24h 75% CSE and 6h 75% CSE treated MLE12 cells.

713

(B)

Western blot analysis of Caspase 3 cleavage in MLE12 cells treated with low

LDH assay of CSE treated MLE12 cells after 6, 24, and 48h. n=3 + SEM

714 715

Figure 2: Mitochondria of MLE12 alveolar epithelial cells hyperfuse in response to CSE

716

treatment.

717

(A)

718

medium, 25% CSE, or 10 µM CCCP for 48h. Scale bars: 10 µm

719

(B)

720

of treatment. n=3 + SEM; Differences between the groups were analyzed using the

721

chi2-test: ***:p