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The FASEB Journal article fj.13-232629. Published online September 11, 2013.

The FASEB Journal • Research Communication

Circadian clock function is disrupted by environmental


tobacco/cigarette smoke, leading to lung
inflammation and injury via a SIRT1-BMAL1 pathway
Jae-Woong Hwang,*,1 Isaac K. Sundar,*,1 Hongwei Yao,* Michael T. Sellix,†
and Irfan Rahman*,2
*Department of Environmental Medicine, Lung Biology and Disease Program, and †Department of
Medicine, Division of Endocrinology and Metabolism, University of Rochester Medical Center,
Rochester, NY, USA

ABSTRACT Patients with obstructive lung diseases and injury via a SIRT1-BMAL1 pathway. FASEB J. 28,
display abnormal circadian rhythms in lung function. 000 – 000 (2014). www.fasebj.org
We determined the mechanism whereby environmental
tobacco/cigarette smoke (CS) modulates expression of Key Words: oxidative stress 䡠 cytokines 䡠 chronic obstructive
the core clock gene BMAL1, through Sirtuin1 (SIRT1) pulmonary disease 䡠 locomotor activity 䡠 emphysema 䡠 mouse
deacetylase during lung inflammatory and injurious
responses. Adult C57BL6/J and various mice mutant
for SIRT1 and BMAL1 were exposed to both chronic (6 Circadian rhythms are biological oscillations that
mo) and acute (3 and 10 d) CS, and we measured the occur with a near-24-h period and are synchronized or
rhythmic expression of clock genes, circadian rhythms of entrained to environmental cues, such as the day–night
locomotor activity, lung function, and inflammatory and transition (1). In mammals, the central circadian pace-
emphysematous responses in the lungs. CS exposure maker is localized in the suprachiasmatic nucleus
(100 –300 mg/m3 particulates) altered clock gene expres- (SCN) of the hypothalamus (1). In addition to the
sion and reduced locomotor activity by disrupting the central pacemaker, peripheral tissues, including liver,
central and peripheral clocks and increased lung inflam- heart, and lung, are autonomous circadian oscillators
mation, causing emphysema in mice. BMAL1 was acety- (2). The clock in each of these tissues plays a critical
lated and degraded in the lungs of mice exposed to CS role in optimizing cellular function and responses to
and in patients with chronic obstructive pulmonary environmental stimuli (1, 2). The coordinated activity
disease (COPD), compared with lungs of the nonsmok- of these oscillators can be referred to as the circadian
ing controls, linking it mechanistically to CS-induced timing system (2). Disruption of the circadian timing
reduction of SIRT1. Targeted deletion of Bmal1 in lung system has been shown to cause cellular dysfunction
epithelium augmented inflammation in response to CS, and chronic diseases (3). Circadian rhythms of respira-
which was not attenuated by the selective SIRT1 activa- tory function have been described in both experimen-
tor SRT1720 (EC50ⴝ0.16 ␮M) in these mice. Thus, the tal animals (4, 5) and healthy human subjects (6, 7).
circadian clock, specifically the enhancer BMAL1 in However, the effect of environmental stress on clock
epithelium, plays a pivotal role, mediated by SIRT1- function in the lung and its role in lung pathophysiol-
dependent BMAL1, in the regulation of CS-induced ogy are unknown.
lung inflammatory and injurious responses.— Hwang, The molecular clock drives intrinsic daily rhythms of
J.-W., Sundar, I. K., Yao, H., Sellix, M. T., Rahman, I. physiology and behavior. At the cellular level, the clock
Circadian clock function is disrupted by environmental is defined as a transcriptional and translational feed-
tobacco/cigarette smoke, leading to lung inflammation back loop (TTFL) oscillator (1). The core loop consists
of the CLOCK:BMAL1 heterodimer, which activates
the transcription of Period (PER) and Cryptochrome
Abbreviations: ANOVA, analysis of variance; BAL, bron- (CRY). PER and CRY proteins form heterodimeric
choalveolar lavage; BW, body weight; COG, center of gravity; complexes that inhibit their own transcription by sup-
COPD, chronic obstructive pulmonary disease; CRY, crypto-
chrome; CS, cigarette smoke; CSE, cigarette smoke extract;
1
E, tissue elastance; H&E, hematoxylin and eosin; IP-10, These authors contributed equally to this work.
2
CXCL10/interferon-␥ inducible protein 10; KC, CXCL1/ Correspondence: Department of Environmental Medi-
keratinocyte-derived chemokine; Lm, mean linear intercept; cine, University of Rochester Medical Center, Box 850, 601
MCP-1, CCL2/monocyte chemotactic protein; PER, Period; Elmwood Avenue, Rochester 14642, NY, USA. E-mail:
qPCR, quantitative real-time PCR; QsC, quasi-static compli- irfan_rahman@urmc.rochester.edu
ance; R, lung resistance; SCN, suprachiasmatic nucleus; doi: 10.1096/fj.13-232629
SIRT1, Sirtuin1; Tg, transgenic; TPM, total particulate matter; This article includes supplemental data. Please visit http://
ZT, zeitgeber time www.fasebj.org to obtain this information.

0892-6638/14/0028-0001 © FASEB 1
pressing the activity of CLOCK:BMAL1 (1). Emerging 30, 31). SIRT1 heterozygous knockout mice were used, be-
evidence suggests that the molecular clock is intimately cause SIRT1 homozygous knockout mice have a low perinatal
associated with the response to environmental stress in survival rate (30). The mice were housed under a 12:12
light:dark (L:D) cycle with lights on at 6 AM, and were fed a
pathophysiology (8 –11). However, the role of the circa- regular diet and water ad libitum, unless otherwise indicated.
dian clock gene BMAL1 in oxidative stress–induced in- For acute (10 d) CS exposure, the mice were entrained for a
flammation remains elusive. minimum of 2 wk to an inverted L:D cycle (lights on from 6
It is well known that tobacco and cigarette smoke PM to 6 AM), except for wheel-running experiments, before
(CS) induces oxidative stress and consequently leads to CS exposure during their active phase. For 3 d and chronic (6
excessive pulmonary inflammation in the pathogenesis mo) CS exposure, the mice were kept in a standard 12:12 L:D
cycle with lights on from 6 AM to 6 PM throughout the
of chronic obstructive pulmonary disease (COPD) and
experiment. All of the procedures described in this study
emphysema (12). Daily rhythms of lung function, bron- were approved by the University Committee on Animal
chodilator responses, surfactant protein levels, steroid Research at the University of Rochester.
efficacy, mucus secretion, and chronic cough are hall-
marks of obstructive lung diseases known to be clock Tobacco/CS exposure
dependent (5, 7, 12–17). Patients with COPD display
rhythmic variation in respiratory symptoms, including Eight-week-old mice were used for tobacco/CS exposure (27,
nocturnal breathlessness, insomnia, and an early-morn- 32, 33). We used both acute (3 and 10 d exposure, which
ing decline in lung function associated with increased induces lung inflammatory responses) and chronic (6 mo
cough and mucus hypersecretion (18 –20). These symp- exposure, which causes pulmonary emphysema) mouse mod-
toms may stem from altered clock function in lung els of CS exposure to determine the effect and mechanism of
tissue, and it is reasonable to suspect that disrupted both acute and chronic CS exposure on circadian clock
function and lung inflammation. The mice were exposed to CS
clock function can have a profound influence on lung from a mainstream-delivering Baumgartner-JaegerCSM2082i ciga-
function and lung pathology. However, it is not known rette-smoking machine (CH Technologies, Westwood, NJ,
whether CS directly alters clock function during the USA) for 3 d or an environmental sidestream-delivering
pathogenesis of COPD. Teague TE-10 smoking machine (Teague Enterprises, Davis,
Sirtuin1 (SIRT1), an NAD⫹-dependent deacetylase, CA, USA) for 10 d and 6 mo of CS exposure in the Inhalation
affects clock function by binding with CLOCK:BMAL1 Facility at the University of Rochester Medical Center. For 3 d
complexes and deacetylating BMAL1 and PER2 pro- CS exposure, the mice were placed in individual compart-
ments of a wire cage, which was then placed inside a closed
teins (21–25). CS reduces the levels and activity of plastic box connected to the smoke source. The smoke was
SIRT1, and patients with COPD have reduced lung generated from 3R4F research cigarettes containing 11.0 mg
levels of SIRT1 (26 –28). Hence, it is likely that CS- total particulate matter (TPM), 9.4 mg tar, and 0.73 mg
mediated reduction of SIRT1 leads to increased acety- nicotine/cigarette (University of Kentucky, Lexington, KY,
lation of circadian clock proteins, culminating in ab- USA). The TPM per cubic meter of air in the exposure
normal clock gene expression and proinflammatory chamber was monitored in real time with a MicroDust
Proaerosol monitor (Casella CEL, Bedford, UK) and verified
responses in the lung. We hypothesize that environ-
daily by gravimetric sampling (33). The smoke concentration
mental CS affects molecular clock function in lung was set at ⬃300 mg/m3 TPM by adjusting the flow rate of the
tissue via the reduction of SIRT1, which in turn leads to diluted medical air, and the level of carbon monoxide in the
inflammatory and injurious responses in the pathogen- chamber was 350 ppm (33). The mice received two 1-h
esis of COPD and emphysema. To address this hypoth- exposures per day (1 h apart), daily for 3 consecutive days
esis, we determined whether BMAL1, as a substrate of according to the Federal Trade Commission protocol (1
SIRT1, has a role in the regulation of CS-induced lung puff/min of 2 s duration and 35 ml volume) and were
euthanized 24 h after the last exposure (33, 34). The control
inflammatory and injurious responses. mice were exposed to filtered air in a chamber and protocol
identical to those used for CS exposure. For 10 d and chronic
6 mo CS exposure, 3R4F cigarettes were used to generate a
MATERIALS AND METHODS mixture of sidestream (89%) and mainstream (11%) smoke
at a concentration of ⬃100 mg/m3 TPM, so as to avoid
possible toxicity to the mice at a high concentration of
Animals long-term CS exposure (32). Each smoldering cigarette was
puffed for 2 s, once every minute for a total of 5 puffs, at a
Male C57BL/6J (C57) and Bmal1-floxed mutant mice were flow rate of 1.05 L/min, to provide a standard puff of 35 cm3.
purchased from the Jackson Laboratory (Bar Harbor, ME, Mice received 5 h exposure per day, 5 d/wk for the duration
USA). To generate conditional deletion of Bmal1 in lung of exposure and were euthanized at 6-h intervals 24 h after
epithelial cells (CC10-Cre/Bmal1floxed/floxed, hereafter re- the last CS exposure. The 6 h sampling interval was based on
ferred as BMAL1-CC10 cre), Bmal1-floxed mutant mice were prior studies on circadian gene expression in rodents (35).
crossed with mice expressing a Cre transgene driven by the
CC10 promoter (obtained from Dr. Thomas Mariani, Univer-
sity of Rochester, Rochester, NY, USA; refs. 29, 30). Trans- Locomotor activity recording
genic (Tg) mice overexpressing Sirt1 (SIRT1 Tg mice) were
obtained from Dr. Leonard Guarente (Massachusetts Insti- The mice were individually housed and allowed free access to
tutes of Technology, Cambridge, MA, USA) and Dr.Wei Gu food and water. Locomotor activity was measured with the
(Columbia University, New York, NY, USA), and SIRT1 Photobeam Activity System (San Diego Instruments, San
knockout mice (SIRT1-deficient mice) were from Dr. Michael Diego, CA, USA), a computerized system that measures the
McBurney (University of Ottawa, Ottawa, ON, Canada; refs. frequency of photobeam breaks along the side of the cage.

2 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


For wheel-running behavior, the mice were housed singly in Canada; refs. 27, 32). Quasi-static compliance (QsC), lung
standard mouse cages fitted with a stainless-steel running resistance (R), and tissue elastance (E) were measured in the
wheel (Coulbourn Instruments, Flushing, NY, USA) placed mice, anesthetized by sodium pentobarbital [50 mg/kg body
within a light-tight chamber (Phenome Technologies, Lin- weight (BW), intraperitoneally]]. A tracheotomy was per-
colnshire, IL, USA). Total cage activity (photobeam break) formed, and an 18-gauge cannula was inserted 3 mm into an
and wheel-running activity were recorded in 1 min intervals anterior nick in the exposed trachea and connected to a
and analyzed with ClockLab software (Actimetrics, Evanston, computer-controlled rodent ventilator (FlexiVent; Scireq).
IL, USA). For wheel-running analyses, the mice were exposed Initially, the mice were ventilated with room air (150 breaths/
to CS during their inactive phase for 10 d and then trans- min) at a volume of 10 ml/kg body mass. After 3 min of
ferred to recording chambers. They remained under a 12:12 ventilation, measurement of lung mechanical properties was
L:D cycle for 2 d followed by release into constant darkness. initiated by a computer-generated program to measure QsC,
The free-running period of wheel-running activity was deter- R, and E at 3 cmH2O positive end expiratory pressure
mined during 2 wk of constant darkness with a ␹2 periodo- obtained by fitting a model to each impedance spectrum. The
gram (P⬍0.001), and data were analyzed with an unpaired t calibration procedure removed the impedance of the equip-
test (GraphPad Prism, San Diego, CA, USA). ment and tracheal tube within this system (37, 38). These
measurements were repeated 3 times for each animal.
Real-time luminescence recording
Bronchoalveolar lavage (BAL)
Adult male Period2::luciferase knock-in (PER2::LUC) mice
(36) were euthanized by excess CO2 exposure 3 h before The mice were anesthetized at 24 h after the last exposure by
lights off [zeitgeber time (ZT) 9 –12; lights off⫽ZT12]. Por- an intraperitoneal injection of 100 mg/kg BW pentobarbital
tions of the lung and the brain were removed and collected in sodium (Abbott Laboratories, Abbott Park, IL, USA) and
cold, sterile Hanks’ balanced salt solution (HBSS). After the killed by exsanguination. The heart and lungs were removed
tissue was blocked, the brain was sliced in the coronal plane en bloc, and the lungs were lavaged 3 times with 0.6 ml of
with a vibrating microtome at a thickness of 300 ␮m. A section saline (0.9% sodium chloride) via a cannula inserted into the
containing the bilateral SCN near the midpoint of the trachea (4). The lavaged fluid was centrifuged, and the
rostrocaudal extent was recovered, and a minimum of tissue, cell-free supernatants were frozen at ⫺80°C for later analysis.
including the paired SCNs was removed with fine scalpels. The BAL inflammatory cell pellet was resuspended in 1 ml
The paired SCNs were then separated by a precise midline saline, and the total number of cells was counted with a
cut, producing 2 hemi-SCN tissue explants. Small (5 mm3) hemocytometer. Cytospin slides (Thermo Shandon, Pitts-
fragments of lung tissue were isolated. The tissue specimens burgh, PA, USA) were prepared at 50,000 cells/slide, and
were placed in 35-mm culture dishes with 1.2 ml of culture differential cell counts (⬃500 cells/slide) were performed on
medium [DMEM supplemented with B27 (Life Technologies, cytospin-prepared slides stained with Diff-Quik (Dade Beh-
Inc., Grand Island, NY, USA), 10 mM HEPES, 352.5 ␮g/ml ring, Newark, DE, USA).
NaHCO3, 3.5 mg/ml d-glucose, 25 U/ml penicillin, 25 ␮g/ml
streptomycin, and 0.1 mM luciferin (Promega, Madison, WI,
USA)]. Cultures were prepared with clean medium as de- RNA isolation and quantitative PCR (qPCR)
scribed above (control) or with the same medium containing
cigarette smoke extract (CSE) and sealed with sterile vacuum Total RNA was isolated from brain and nonlavaged lung
grease and a glass coverslip. Sealed cultures were maintained tissue specimens (stored in RNAlater; Ambion, Austin, TX,
at 35°C in a light-tight incubator, and luminescence was USA) with an RNeasy kit (Qiagen, Valencia, CA, USA). RNA
continuously recorded (counts/second) with an automated yields were determined by UV absorbance with a NanoDrop
luminometer (LumiCycle; Actimetrics). Raw luminescence instrument (ND-1000 Spectrophotometer; NanoDrop Tech-
data were detrended (24 h moving average) and smoothed (2 nologies, Wilmington, DE, USA). cDNA was synthesized from
h moving average; Origin Pro 8.5, OriginLabs, Northampton, 0.5 ␮g of total RNA by using the RT2 First Strand Kit
MA, USA). (SABioscience, Frederick, MD, USA). To validate the expres-
sion of diverse genes in brain and lung tissues, qPCR was
Lung morphometry performed with a Bio-Rad iCycler real-time system and SYBR
Green qPCR Master mix from SABioscience. In chronic CS
Mouse lungs (which had not been lavaged) were inflated by exposure, qPCR data were gathered from circadian gene and
1% low-melting-point agarose at a pressure of 25 cmH2O, and proinflammatory cytokine gene expression at the ZT24 time
then fixed with 4% neutral buffered formalin (27, 32). Fixed point (n⫽2/air group) in all datasets. All the specific primers
lung tissues were dehydrated, embedded in paraffin, and were purchased from SABioscience. Gene expression was
sectioned (4 ␮m) with a rotary microtome (Microm Interna- normalized to 18s rRNA levels. Relative RNA abundance was
tional GmbH, Walldorf, Germany). The lung sections were quantified by the comparative 2⫺⌬⌬Ct method. Significant
deparaffinized, rehydrated by passing them through a series rhythms of gene expression were verified with CircWave
of xylene and graded alcohol, and stained with hematoxylin software (version 1.4). In addition, the center of gravity
and eosin (H&E). Alveolar size was estimated from the mean (COG), or peak phase, was determined for each rhythm by
linear intercept (Lm) of the airspace, which is a measure of using CircWave (39).
airspace enlargement or emphysema performed with Meta-
Morph software (Molecular Devices, Sunnyvale, CA, USA; Proinflammatory mediator analysis
refs. 27, 32). Lm was calculated for each sample on the basis
of 10 random fields/slide, observed at ⫻200. The airway and The levels of proinflammatory mediators, such as CCL2/
vascular structures were eliminated from the analysis. monocyte chemotactic protein-1 (MCP-1), CXCL1/keratino-
cyte derived chemokine (KC), and CXCL10/interferon-␥
Measurements of lung mechanical properties inducible protein 10 (IP-10) in lung homogenates were
measured by enzyme-linked immunosorbent assay (ELISA)
The mechanical properties of the mouse lungs were deter- performed with the respective duo-antibody kits (R&D Sys-
mined with the FlexiVent apparatus (Scireq, Montreal, QC, tems, Minneapolis, MN, USA), according to the manufacturer’s

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 3


instructions. The results were expressed in the samples as RESULTS
picograms per milligram protein.

CS exposure differentially affected the amplitude


Immunoblot and immunoprecipitation and phase of circadian clock gene expression in the
lungs and brain
Protein samples from lung homogenates were separated by
6 –10% SDS-PAGE. The separated proteins were electroblot-
We used qPCR to investigate the effect of environmen-
ted onto nitrocellulose membranes (Amersham Biosciences,
Piscataway, NJ, USA). The membranes were blocked for 1 h at tal CS exposure on rhythmic expression of core clock
room temperature with 5% BSA and then probed with genes in mouse lungs. Both chronic (6 mo exposure,
anti-SIRT1, anti-Ac Lys (1:1000; Cell Signaling Technology, which causes pulmonary emphysema) and acute (3 and
Danvers, MA, USA), anti-BMAL1 and anti-CLOCK, (1:1000- 10 d exposure, which induces a lung inflammatory
1:2000; Abcam, Cambridge, MA, USA), and anti-acetyl response) environmental CS exposure models were
BMAL1 (1:1000; Millipore, Billerica, MA, USA) antibodies, to used to determine the effects of CS on rhythmic
determine the respective proteins. After 3 washing steps (10
expression of core clock genes in lung tissue and the
min each), the levels of protein were detected with secondary
antibody (1:5000 dilution) in 2.5% BSA in PBS containing functional relationship between altered clock function
0.1% Tween 20 (v/v) for 1 h linked to horseradish peroxidase and lung inflammation. Most of the core clock genes
(Dako, Carpinteria, CA, USA), and bound complexes were (bmal1, clock, per1, per2, cry1, cry2, rev-erb␣, and rev-erb␤)
detected by ECL (Perkin Elmer, Wellesley, MA, USA). Equal were rhythmically expressed in lung tissue from mice
loading of the samples was determined by quantitation of exposed to air or CS, either acutely (10 d; Fig. 1A, B) or
proteins and by stripping and reprobing the membranes chronically (6 mo; Supplemental Fig. S1A, B). CircWave
for ␤-actin (Oncogene, Cambridge, MA, USA). Immuno- analysis confirmed statistically significant (P⬍0.001)
precipitation was performed to determine the acetylation
of BMAL1 in human whole-lung homogenates from non- rhythms of bmal1, clock, per1, per2, rev-erb␣, and rev-erb␤
smokers, smokers, and patients with COPD, as described gene expression (Fig. 1A, B, Table 1, Supplemental Fig.
previously (26, 27, 34). ImageJ 1.41densitometry software S1A, and Table 2). Of the known core clock genes, only
(U.S. National Institutes of Health, Bethesda, MD, USA) ror␣ mRNA was not rhythmic in the lungs of the mice
was used for gel band quantitative densitometry. exposed to air or CS for 6 mo (Supplemental Fig. S1A).
In both the air- and CS-exposed mice, the expression of
Administration of SRT1720 bmal1, clock, and cry1 displayed nocturnal acrophases,
peaking in the mid to late portions of the dark phase
SRT1720 (100 mg/kg, ⬎95% pure by C-13 NMR and LCMS, (ZT18 –24; ZT0⫽lights on; ZT12⫽lights off; Fig. 1A, B
synthesized by Life Chemicals, Niagara-on-the-Lake, ON, Can- and Supplemental Fig. S1A, B). As expected, in both
ada) was administered to the acute air- and CS-exposed mice groups, the peaks of per1, per2, cry2, rev-erb␣, and rev-erb␤
daily for 3 d through oral gavage 1 h before CS exposure (40). were nearly antiphase to bmal1, which peaked between
ZT6 and ZT12 (Fig. 1A, B and Supplemental Fig. S1A,
Human samples B). Chronic CS exposure caused a modest reduction in
the amplitude of bmal1 and rev-erb␣ expression and
Lung tissue specimens from nonsmokers, smokers, and pa- substantially reduced the amplitude of per1 expression
tients with COPD were obtained as described previously (26, in the lungs (Supplemental Fig. S1A, B). Further,
27, 34). CircWave analyses indicated that chronic CS exposure
altered the phase of per1 and per2 expression in the
Statistical analysis
lungs (Supplemental Fig. S1B).
As with the chronic-exposure model, acute CS expo-
The period of PER2::LUC expression in each tissue explant sure also reduced the amplitude of bmal1, clock, and per2
was determined with a ␹2 periodogram analysis (LumiCycle mRNA expression, but only modestly affected the am-
Analysis Software; Actimetrics). A minimum of 3 d of data plitude of per1 expression in the lungs (Fig. 1A).
were used to calculate the period of PER2::LUC expression in Surprisingly, acute CS exposure appeared to have time-
each explant of lung and SCN tissue. Period data from the dependent suppressive effects on cry1, cry2, and rev-erb␤
lung tissue were analyzed with 1-factor analysis of variance expression (Fig. 1A). Finally, CircWave analyses indi-
(ANOVA) and the Newman-Keuls post hoc test. Period data
cated that acute CS slightly delayed the phase of rev-erb␣
from the SCN tissue (2 groups) were analyzed with an
unpaired t test. Data are presented as the mean ⫾ se. For expression in the lungs (Fig. 1B). Together, data from
statistical analysis of qPCR data, CircWave 1.4 software was both the acute and chronic models reveal that the
used. In addition to CircWave analysis, statistical significance amplitude and phase of clock gene expression in the
between the air- and CS-exposed groups was calculated by lungs were differentially affected by CS exposure, al-
Fisher’s multiple comparison, with 2-way ANOVA. To empha- though the effects of acute exposure were more robust.
size the waveform of the data, representative protein expres- Parallel effects of CS exposure on the phase and
sion was subjected to nonlinear regression analysis with a amplitude of clock gene expression were observed in
sixth- order polynomial (Prism; Graphpad). Statistical analysis
of significance was calculated by StatView software in a 1-way brain tissue of the acutely CS-exposed mice (Supple-
ANOVA followed by Tukey’s post hoc test for multigroup mental Fig. S3), suggesting that the effects of CS on
comparisons. Values of P ⬍ 0.05 represented a significant clock function are not limited to lung tissue. The effect
difference. of acute CS exposure on clock gene expression in the lung

4 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


Figure 1. Acute CS exposure differentially affected clock and sirt1 gene expression in mouse lungs. Acute (10 d) air or CS
exposure was performed as described in Materials and Methods. On the day after the last exposure, lungs were harvested every
6 h for 42 h beginning at ZT12. Total RNA was extracted from lung tissues, and cDNA was prepared for gene expression analysis
by qPCR. A) Expression of core clock genes (bmal1, clock, per1, per2, cry1, cry2, rev-erb␣, rev-erb␤, and ror␣) was examined by qPCR.
CircWave analysis confirmed statistically significant rhythms of each clock gene (P⬍0.05 for bmal1; P⬍0.001 for per1, cry1, cry2,
rev-erb␣, rev-erb␤, and ror␣) in CS-exposed mice. B) COG or peak phase values for each gene expression rhythm were obtained
by using CircWave and plotted on a horizontal phase map. Gray shading indicates the relative dark phase (ZT12–24). C)
Circadian rhythm of sirt1 gene expression in lung tissue was analyzed by qPCR in lung tissue from mice after acute air or CS
exposure. Data from air-exposed (open circle) and CS-exposed (solid circle) mice are shown as the mean ⫾ se (n⫽3– 4
mice/group) for each time point. *P ⬍ 0.05 vs. corresponding air-exposed mice.

(Fig. 2A) and SCN (Fig. 2B) was confirmed by real-time dramatically and dose dependently reduced the ampli-
monitoring of PER2::LUC expression in lung and SCN tude of PER2::LUC expression in the lung tissue ex-
tissue explants. Medium containing CSE (0.1-0.25%) plants (Fig. 2A). CSE treatment dose dependently

TABLE 1. COG and significance for each clock gene expression TABLE 2. COG values and significance for each clock gene
rhythm, as determined by CircWave analysis in acute air- or expression rhythm, as determined by CircWave analysis in chronic
CS-exposed mouse lung air- or CS-exposed mouse lung

Air CS Air CS
Clock Clock
gene COG P COG P gene COG P COG P

Bmal1 11.6 ⫾ 1.5 0.001*** 12.3 ⫾ 3.1 0.05* Bmal1 21.7 ⫾ 1.4 0.001*** 22.6 ⫾ 1.9 0.001***
Clock 10.0 ⫾ 2.8 0.001*** 1.1 ⫾ 3.5 NS Clock 21.4 ⫾ 2.58 0.001*** 21.8 ⫾ 2.6 NS
Per1 23.9 ⫾ 2.3 0.001*** 23.8 ⫾ 2.0 0.001*** Per1 14.2 ⫾ 3.1 0.001*** 3.6 ⫾ 3.3 NS
Per2 0.0 ⫾ 2.18 0.001*** 1.6 ⫾ 3.4 NS Per2 14.6 ⫾ 2.2 0.001*** 11.0 ⫾ 2.3 0.001***
Cry1 8.1 ⫾ 3.1 0.001*** 4.0 ⫾ 2.2 0.001*** Cry1 19.9 ⫾ 2.5 NS 19.0 ⫾ 2.5 0.001***
Cry2 23.5 ⫾ 3.4 NS 1.1 ⫾ 2.2 0.001*** Cry2 16.0 ⫾ 3.4 NS 0.5 ⫾ 3.0 NS
Rev-erb ␣ 19.6 ⫾ 1.6 0.001*** 23.7 ⫾ 1.3 0.001*** Rev-erb ␣ 6.2 ⫾ 2.1 0.001*** 3.3 ⫾ 1.7 0.001***
Rev-erb ␤ 22.1 ⫾ 2.1 0.001*** 23.1 ⫾ 2.2 0.001*** Rev-erb ␤ 11.6 ⫾ 3.1 0.05* 10.8 ⫾ 3.1 NS
Ror ␣ 6.1 ⫾ 3.34 NS 1.4 ⫾ 2.2 0.001*** Ror ␣ 19.1 ⫾ 3.3 NS 18.9 ⫾ 3.4 NS

Data are shown as mean NS ⫾ se (n⫽3– 4/group). NS, not Data are shown as means ⫾ se (n⫽3– 4/group). NS, not signif-
significant. *P ⬍ 0.05, ***P ⬍ 0.001. icant. *P ⬍ 0.05, ***P ⬍ 0.001.

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 5


Figure 2. Effects of CSE on the amplitude and period of PER2::LUC expression in lung and SCN tissue explants. A) PER2::LUC
expression in representative lung tissue explants treated with medium alone (controls). Treatment at the time of culture with
CSE at 0.1 and 0.25% significantly and dose dependently dampened the rhythm of PER2::LUC expression in lung tissue.
Treatment with CSE dose dependently increased the period of PER2::LUC expression in lung explants (control, 24⫾0.25; 0.1%
CSE, 26.21⫾0.37; 0.25% CSE, 27.79⫾1.24). B) PER2::LUC expression rhythms in representative hemi-SCN tissue explants
treated with medium alone (controls). Treatment with 0.1% CSE did not reduce the amplitude of PER2::LUC expression in SCN
explants, but 0.1% CSE significantly shortened the period of PER2::LUC expression in the SCN when compared with controls
(controls, 26⫾0.43 vs. 0.1% CSE, 24.42⫾0.20). Each line represents tissue from different animals. Data from control and CSE
treatment represent means ⫾ se (n⫽4 – 6/group). *P ⬍ 0.05, **P ⬍ 0.01 vs. corresponding untreated control (medium alone).

increased the period of PER2::LUC expression in the tude of the response increased, indicating that the
lung tissue explants (Fig. 2A). Of note, treatment with primary effect of CS exposure was a reduction in
0.1% CSE did not affect the amplitude of PER2::LUC nighttime activity (Supplemental Fig. S2B).
expression in the SCN explants, compared to that in The effect of acute CS exposure on total cage loco-
the lung tissue (Fig. 2B). However, the same treatment motor activity and wheel-running behavior were mea-
significantly shortened the period of PER2::LUC ex- sured to determine whether the reduction in activity
pression in the CS-exposed SCN explants when com- that we observed is initiated at an early stage of smok-
pared with that in the untreated control (Fig. 2B). ing. We used our beam break monitor to record total
Overall, CSE treatment reduced the amplitude and cage activity for 2 wk before CS exposure, during the
differentially affected the period of PER2::LUC expres- active phase for 10 d of CS exposure and during a 2 wk
sion in brain and lung tissue explants ex vivo. period after CS exposure (clean air). The mice were
exposed to CS during their active phase for 5 h/d over
CS exposure reduced total locomotor activity and 10 consecutive days. Locomotor activity was almost
shortened the free-running period of wheel-running immediately attenuated in the CS-exposed mice, and
behavior in mice was persistently lower after CS exposure ended (Fig. 3A).
Although activity increased during recovery, the mice
Seeing that CS exposure altered the amplitude and managed to approach only ⬃70% of their original
period of clock gene expression in lung and brain activity level after 2 wk of breathing clean air (Fig. 3B).
tissue, we investigated its effect on the circadian Activity onset time or phase angle of entrainment was
rhythms of behavior in mice. The total cage activity of estimated with Clocklab software (Actimetrics) and did
individual C57BL/6J mice was recorded by infrared- not appear to be affected by CS (Fig. 3C). Analysis of
beam breaks during the 6 mo of CS exposure (17 d wheel-running behavior revealed a small, but signifi-
before starting and continuing until the end of chronic cant, reduction in the free-running period of activity in
CS exposure). Mice exposed to chronic CS showed a mice after acute exposure to CS (␶⫽23.61⫾0.06) dur-
significant reduction in total cage locomotor activity ing their inactive phase when compared with the air-
(Supplemental Fig. S2A). Analysis of total ambulatory exposed mice (␶⫽23.80⫾0.03; P⬍0.05) (Fig. 4). Short-
counts across the 24 h day revealed that chronic CS ening of the free-running period of wheel-running
exposure significantly reduced activity in the WT mice activity suggests that CS alters clock gene expression in
within 5 d of the first exposure, and the decrease the central clocks that regulate behavioral rhythms, a
persisted until the beginning of the 6th mo of CS finding supported by our analysis of PER2::LUC expres-
exposure (Supplemental Fig. S2B). When daytime and sion in SCN tissue explants (Fig. 2B) and clock gene
nighttime activity were analyzed separately, the ampli- expression in whole brain (Supplemental Fig. S3).

6 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


Figure 3. Acute CS exposure reduced activity, but did not affect the phase angle of entrainment or activity consolidation. Acute
(10 d) air or CS exposure was performed as described in Materials and Methods. Locomotor activity was recorded from
individual mice for a period of 38 d. A) Representative double-plotted actograms showing considerable reduction in activity of
CS-exposed mice (right) relative to air-exposed mice (left). Gray shading indicates the relative dark phase (ZT12-24). During
the periods of CS exposure (ZT3-9), activity was not recorded (stars). B) Nocturnal activity was plotted in ambulatory counts.
CS reduced locomotor activity, which recovered up to 70% of the activity level in air-exposed mice during the postexposure
phase. C) Activity onset relative to lights off (ZT12; phase angle entrainment) during the postexposure. Shading indicates the
dark phase (ZT12-24). Data from air- and CS-exposed groups represent the mean ⫾ se (n⫽3– 4 mice/group) for each time
point. *P ⬍ 0.05 vs. corresponding air-exposed mice.

Disruption of circadian clock function in the lungs rhythmic in the lungs, and is disrupted by both acute and
resulted in increased inflammatory responses chronic CS exposure.
To understand the functional link between CS-medi-
To investigate whether CS alters circadian rhythmicity ated alterations in circadian rhythmicity and proinflam-
of proinflammatory cytokine responses, we determined matory cytokine responses, we determined the abun-
the expression of proinflammatory cytokine genes after dance of proinflammatory cytokines (ccl1/mcp-1, cxcl1/kc,
chronic and acute CS exposure. In air-exposed mice and MIP-2) in mouse lungs after CS exposure. Acute CS
from the acute exposure experiments, lung mRNA exposure appeared to cause a phase shift in the rhythms
expression of both monocyte chemotactic protein–1 of MCP-1 and KC release, such that MCP-1 peaked in the
(ccl1/mcp-1) and keratinocyte chemoattractant (cxcl1/kc) middle of the day (ZT6 vs. ZT18 in air-exposed group),
peaked during the dark phase (Fig. 5A–C and Supple- and KC levels peaked closer to lights off (ZT12; Fig. 5D,
mental Fig. S4A–C). CircWave analysis confirmed signifi- E). Acute CS also appeared to increase the amplitude of
cant circadian rhythms of both genes in air-exposed mice KC release in the mouse lung tissue (Fig. 5E). Chronic CS
(P⬍0.001). In contrast, the expression of interferon-␥- exposure had differential and cytokine-specific effects on
induced protein 10 (cxcl10/IP10) peaked during the light MCP-1, KC, and MIP-2 levels in the tissue (Supplemental
phase, but did not have a significant circadian rhythm of Fig. S4D–F). The levels of MCP-1 and KC were increased
expression in air-exposed mice (Fig. 5C and Supplemen- in the latter portion of the dark phase (ZT18-24) after
tal Fig. S4C). Acute and chronic CS exposure disrupted chronic CS exposure.
rhythms of ccl1/mcp-1 and cxcl1/kc expression in the lungs The increased level of cytokines was associated with a
(Fig. 5A, B and Supplemental Fig. S4A, B). These results rhythmic influx of inflammatory cells in the lung in
suggest that proinflammatory cytokine gene expression is response to acute (Fig. 6A) and chronic (Fig. 6B) CS

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 7


Figure 4. Effect of acute CS exposure
on circadian rhythms of wheel-running
activity. A) Representative actograms
of mice that were exposed to 10 d of
CS (see Materials and Methods) and
then placed in cages with attached
running wheels. Mice were entrained
to a 12:12 L:D cycle for 2 d and then
released into constant darkness (D:D)
for 14 d. Gray shaded region indicates
the light phase. Arrow: first full day in
D:D. B) Free-running period of run-
ning activity in D:D was calculated
across the 2 wk period with a ␹2 peri-
odogram. Period was significantly shorter in the CS group (␶⫽23.61⫾0.06) compared with that in the air-exposed
group (␶⫽23.80⫾0.03). *P ⬍ 0.05 vs. corresponding air-exposed mice.

exposure. Neutrophil influx into BAL fluid increased from day (ZT0 and ZT6) and night (ZT12 and ZT18),
significantly during the late night (ZT24), whereas E was significantly reduced, whereas lung compliance
there was no significant difference in the number of increased in the chronic CS-exposed mice (Table 3).
macrophages and total cells in BAL fluid (Fig. 6B). There was no statistically significant difference in BW
Thus, our data show that the disruption of circadian or mortality between the chronic CS- and air-exposed
clock function in the lung was associated with aug- mice.
mented proinflammatory responses during both acute
and chronic CS exposure.
Rhythmic expression of SIRT1 was disrupted by CS
Disruption of circadian clock function was associated exposure, resulting in increased acetylation of
with airspace enlargement and emphysema along with BMAL1 in lungs
alteration in lung mechanical properties and function
To investigate the mechanisms of CS-induced disruption
To investigate the potential effect of circadian clock of the lung inflammatory response, we determined the
dysfunction due to CS-induced airspace enlargement and levels of BMAL1 acetylation and the abundance of SIRT1
emphysema, we subjected mice to chronic CS exposure in mouse lung after an acute (10 d) CS exposure. In the
and determined their susceptibility to CS-induced air- lungs of the control air-exposed mice, the protein abun-
space enlargement and emphysema by lung histopatho- dance of SIRT1 and BMAL1 showed clear rhythmic
logic and functional measurements. There was a sig- oscillations that peaked in the night (Fig. 7A, B). How-
nificant difference in lung histopathology (H&E) ever, CS reduced the abundance of SIRT1 and BMAL1
between air- and CS-exposed mice after 6 mo, which across the entire day, which was consistent with gene
was assessed by determining the Lm (air 49.56⫾3.25 expression data (Fig. 1A and Supplemental Fig. S1A). We
vs. CS 61.40⫾1.60; P⬍0.01, n⫽4 – 6 mice/group). Sim- observed a similar effect of CS on the rhythm of sirt1 gene
ilarly, the overall lung E was significantly decreased and expression in the lung and brain tissues (Fig. 1C and
R was decreased, but not significantly, in the mice Supplemental Fig. S3C). Furthermore, the mice with CS
exposed to chronic CS. However, lung QsC was in- exposure showed a reduction in BMAL1 levels (Fig. 7A).
creased significantly in chronic CS-exposed mice as Similarly, the BMAL1 levels were modestly reduced in the
compared to that in air-exposed mice (Table 3). We smokers and significantly reduced in lungs of the patients
also found a significant decrease in lung E and R in the with COPD, compared with levels in lungs of the non-
chronic CS-exposed mice at ZT18, but not at other smokers (Fig. 8A, B). CS caused increased BMAL1 acety-
times of the day (ZT0, ZT6, ZT12, and ZT24), when lation in the mouse lungs (Fig. 7A, B), and a similar
compared to those parameters in air-exposed mice increase in BMAL1 acetylation was observed in the lungs
(Tables 3 and 4). However, when we combined the data of the patients with COPD and the smokers, compared to

8 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


Figure 5. Acute CS exposure
affected circadian rhythms of
proinflammatory cytokine gene
expression and abundance in
mouse lungs. Acute (10 d) air or
CS exposure was performed as
described in Materials and Meth-
ods. At the end of CS or air
exposure, lungs were harvested
every 6 h for 42 h beginning
at ZT12 on the day following
the last CS exposure. Total
RNA was extracted from lung
tissue, and cDNA was pre-
pared for gene expression analysis by qPCR. Expression of proinflammatory cytokine genes including ccl1/mcp-1 (A),
cxcl1/kc (B), and cxcl10/ip-10 (C) was determined by qPCR. Levels of the proinflammatory mediators MCP-1 (D) and
KC (E) were also measured in lung homogenates obtained from acute air- or CS-exposed mice. Significant circadian
rhythms of cytokine gene expression were detected by CircWave analysis. Data from air-exposed (open circle)
and CS-exposed (solid circle) mice represent means ⫾ se (n⫽3 mice/group) for each time point. *P ⬍ 0.05,
**P ⬍ 0.01, ***P ⬍ 0.001 vs. corresponding air-exposed mice.

those of the nonsmokers (Fig. 8C, D). Increased BMAL1 Lung epithelial cell–specific BMAL1 deletion
acetylation in the CS-exposed mice was associated with increased lung inflammation, which was not
reduced locomotor activity (Figs. 9 and 10). Given that attenuated by treatment with a selective
SIRT1 modulates the circadian clock mechanism by con- pharmacological SIRT1 activator
trolling BMAL1 acetylation, we hypothesize that acetyla-
tion of BMAL1 is increased by the reduction of SIRT1 To determine the role of the SIRT1-BMAL1 pathway in
levels after CS exposure. CS-induced circadian disruption and inflammatory re-
To determine whether SIRT1 reduction is responsi- sponses, we established lung epithelial cell–specific
BMAL1⫺/⫺ (BMAL1-CC10 cre) mice and exposed
ble for increased BMAL1 acetylation, SIRT1-deficient
them to an acute period (3 d) of CS. The BMAL1-CC10
(SIRT1⫹/⫺) and SIRT1-overexpressing (Tg) mice were
cre mice exhibited normal rhythms of locomotor activ-
exposed to acute CS for 3 d, and the level of BMAL1
ity (ambulatory count and onset time; Fig. 11) and, as
acetylation was measured in lung tissue. Acute CS exposure in the WT mice, the activity in these mice was signifi-
significantly increased the acetylation of BMAL1 in the cantly reduced by CS exposure (Fig. 11A, B). As in the
SIRT1-deficient mice relative to both the air-exposed WT mice, we detected rhythmic expression of proin-
SIRT1-deficient mice and the CS-exposed WT mice flammatory cytokine genes, including ccl1/mcp-1 and
(Fig. 7C). This response was attenuated in the SIRT1 Tg cxcl1/kc, in the BMAL1-CC10 cre mice that were af-
mice, most likely because of the persistently elevated fected by CS, such that the expression of ccl1/mcp-1 was
level of SIRT1 deacetylase activity (Fig. 7C). Moreover, increased at ZT0 but remained unchanged at ZT12
locomotor activity levels were significantly reduced (Fig. 12A, B). Cxcl1/kc gene expression was elevated at
during acute (3 d) CS exposure in SIRT1-deficient but both times in the CS-exposed animals. We found simi-
not in SIRT1-overexpressing mice (Figs. 9 and 10). lar effects of lung-specific BMAL1 deletion on rhythms
These results suggest that SIRT1 reduction in response of proinflammatory cytokine release (Fig. 12B). Over-
to CS exposure leads to an increase in BMAL1 acetyla- all, lung epithelial cell–specific clock disruption altered
tion and reduced levels of BMAL1, causing altered the inflammatory response to CS. To further examine
molecular clock function and increased proinflamma- the involvement of SIRT1 and BMAL1 in these proin-
tory responses in the lungs. flammatory responses, we exposed BMAL1-CC10 cre

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 9


Figure 6. A) Acute CS had time-dependent
effects on rhythms of inflammatory cell influx
in mouse lungs. Acute (10 d) air or CS expo-
sure was performed as described in Materials
and Methods. At the end of CS or air exposure,
lungs were harvested every 6 h for 42 h, begin-
ning at ZT12 on the day following the last CS
exposure. The number of total cells in BAL
fluid from acute air- or CS-exposed mice was
determined. At least 500 cells in the BAL fluid
were counted with a hemocytometer to deter-
mine the number of neutrophils (i), macro-
phages (ii), and total cells (iii) on cytospin
slides stained with Diff-Quik. Data from air- and
CS-exposed lungs are shown as means ⫾ se
(n⫽3–4/group) for each time point. B) Chronic
CS had time-dependent effects on rhythms of in-
flammatory cell influx in CS-exposed mouse
lung. Environmental tobacco/chronic CS ex-
posure (6 mo air or CS) was performed as
described in Materials and Methods. On the
day after the last exposure, lungs were har-
vested every 6 h for 24 h beginning at ZT0, and
then at ZT6, ZT12, ZT18, and ZT24. The
number of total cells in BAL fluid from chronic
air- or CS-exposed mice was determined. At
least 500 cells in the BAL fluid were counted
with a hemocytometer, to determine the num-
ber of neutrophils (i), macrophages (ii), and
total cells (iii) on cytospin slides stained with
Diff-Quik. Data from air- and CS-exposed are shown as means ⫾ se (n⫽3– 4/group) for each time point. *P ⬍ 0.05 vs.
corresponding air-exposed mice.

mice to CS (3 d), followed by administration of the injurious responses. We examined the putative mecha-
selective pharmacologic SIRT1 activator SRT1720. The nisms of clock disruption in response to chronic envi-
BMAL1-CC10 cre, global SIRT1-deficient, and SIRT1 ronmental CS exposure in lung and brain tissues in a
Tg mice exhibited normal diurnal patterns of locomo- mouse model of COPD and emphysema. We deter-
tor activity (Figs. 9 –11). As expected, locomotor activity mined the effect of COPD and emphysema on molec-
during the night was reduced in these mice during the ular clock function in the lung. We show for the first
acute CS exposure and returned to normal levels after time that CS exposure alters circadian clock gene
4 d of clean-air recovery (Figs. 9 –11). Treatment with expression in both the lungs and brain. Our results
SRT1720 attenuated proinflammatory cytokine release show that both acute and chronic CS exposure signifi-
in the CS-exposed WT mice, but not in the CS-exposed cantly reduced the amplitude of locomotor activity in
BMAL1-CC10 cre mice (Fig. 12C). These data strongly mice. This effect persisted even after CS exposure
suggest that epithelial BMAL1 plays a critical role in the
ended, with locomotor activity levels not fully recover-
regulation of CS-induced inflammatory responses and
ing until 14–30 d after smoking cessation in the chronic-
that the effect of BMAL1 is modulated by SIRT1.
exposure model. Despite this effect on locomotor
activity levels, the phase angle of entrainment and
DISCUSSION consolidation of activity in the dark phase were not
disrupted following CS exposure. Further, acute CS
Patients with COPD experience circadian disruption exposure had a small but significant effect on the
(14, 17–20). Hence, CS exposure may affect circadian free-running period of wheel-running activity, suggest-
clock function in the lung, leading to inflammatory and ing that CS may alter the timing of clock gene expres-

TABLE 3. Lung mechanical properties measured at different day and night ZTs in WT mice exposed to chronic CS

QsC (ml/cmH2O) R (cmH2O/s/ml) E (cmH2O/ml)

Day/night (ZT) Air CS Air CS Air CS

ZT0 ⫹ ZT6 0.051 ⫾ 0.002 0.051 ⫾ 0.002 0.747 ⫾ 0.045 0.726 ⫾ 0.036 19.680 ⫾ 0.792 19.654 ⫾ 0.633
ZT12 ⫹ ZT18 0.046 ⫾ 0.001 0.052 ⫾ 0.002* 0.747 ⫾ 0.045 0.708 ⫾ 0.038 21.905 ⫾ 0.359 19.502 ⫾ 0.565**

Data are shown as as means ⫾ se (n⫽4/group). *P ⬍ 0.05, **P ⬍ 0.01 vs. air-exposed mice.

10 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


TABLE 4. Lung mechanical properties measured at different ZTs in WT mice exposed to chronic CS

Compliance (ml/cmH2O) R (cmH2O/s/ml) E (cmH2O/ml)

ZT Air CS Air CS Air CS

ZT0 0.048 ⫾ 0.001 0.051 ⫾ 0.002 0.766 ⫾ 0.105 0.741 ⫾ 0.052 20.968 ⫾ 0.647 19.759 ⫾ 0.718
ZT6 0.054 ⫾ 0.001 0.052 ⫾ 0.003 0.729 ⫾ 0.015 0.711 ⫾ 0.061 18.392 ⫾ 0.169 19.548 ⫾ 1.217
ZT12 0.045 ⫾ 0.002 0.050 ⫾ 0.002 0.730 ⫾ 0.043 0.725 ⫾ 0.008 22.027 ⫾ 0.840 19.963 ⫾ 0.917
ZT18 0.046 ⫾ 0.0003 0.053 ⫾ 0.002 0.861 ⫾ 0.038 0.691 ⫾ 0.083* 21.783 ⫾ 0.189 19.041 ⫾ 0.736*
ZT24 0.048 ⫾ 0.001 0.053 ⫾ 0.002 0.736 ⫾ 0.016 0.682 ⫾ 0.021 20.776 ⫾ 0.525 18.853 ⫾ 0.751

Data are shown as means ⫾ se (n⫽4/group). *P ⬍ 0.05 vs. air-exposed mice.

sion in brain regions, like the SCN, that drive behav- of circadian disruption may not be as severe as the
ioral rhythms. This interpretation is strengthened by effects of jet lag or shift work, it is reasonable to
our observation of altered clock gene expression in speculate that even subtle alteration of clock function
whole brain following acute CS in vivo and the period can have a substantial long-term effect on clock-
of PER2::LUC expression in SCN explants ex vivo. dependent physiology. The differential and opposing
Further, it is worth noting that the shortened period of effects of CSE on the period and phase of clock gene
wheel-running after acute CS paralleled the effects of expression in the lung and SCN suggest that internal
CSE on SCN tissue explants. Together, these data circadian disruption due to phase dissociation between
suggest that CS exposure affects both lung and brain these central and peripheral clocks is a factor in the
clock function. Although the effects of CS as an agent development of diseases such as COPD.

Figure 7. Circadian rhythm of SIRT1 expression in lung tissue was disrupted by CS exposure, resulting in elevated BMAL1
acetylation. A) Immunoblot analysis of SIRT1, total BMAL1, acetylated BMAL1 (Ac BMAL1), and CLOCK in lung tissue
homogenates from mice after 10 d of air or CS exposure. Images are representative of ⱖ2 separate experiments. B) Oscillation
patterns of SIRT1, BMAL1, and Ac BMAL1 protein as shown by immunoblot analysis. After densitometric analysis, levels of
SIRT1 and BMAL1 were both normalized against ␤-actin, and Ac BMAL1 was normalized against total BMAL1. Data from air-
and CS-exposed samples are representative of the immunoblot data, and were analyzed with nonlinear regression, as described
in Materials and Methods (R2⫽1 for all data). C) Genetic manipulation of SIRT1 regulated BMAL1 acetylation in response to
CS in the lungs. Immunoblot analysis of SIRT1, BMAL1, and acetylated BMAL1 performed in lung tissue homogenates from
SIRT1 heterozygous knockout (SIRT1⫹/⫺) and SIRT1-overexpressing (SIRT1 Tg) mice following 3 d of air or CS exposure.
Images are representative of ⱖ2 separate experiments. Reassembly of noncontiguous gel lanes is demarcated by white spaces
and boxes, aligned so as to reflect the overall representative data. Data from air- and CS-exposed tissue are shown as means ⫾
se (n⫽2–3/group) for each time point.

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 11


Figure 8. BMAL1 was down-regulated in lungs of smokers and patients with COPD. A) Immunoblot analysis of BMAL1 and
CLOCK in whole-tissue lysates extracted from lung tissue of nonsmokers, smokers, and patients with COPD. B) After
densitometric analysis, the levels of BMAL1 and CLOCK in lung tissue were normalized against ␤-actin (loading control).
Relative level of BMAL1 was significantly decreased in lung tissue from smokers and patients with COPD when compared with
that of nonsmokers. C) Whole-tissue lysates extracted from lung tissues (collected at midmorning and midday) of nonsmokers,
smokers, and patients with COPD (26, 27, 34) were immunoprecipitated with anti-BMAL1 antibody and probed with
anti-acetylated lysine antibody. d) Relative intensity of acetylated lysine/BMAL1 represents the increased acetylation of BMAL1
in lungs of smokers and patients with COPD as compared to nonsmokers. Images and data represent means ⫾ se
(n⫽3– 4/group). *P ⬍ 0.05, **P ⬍ 0.01 vs. nonsmokers.

Emerging evidence indicates that inflammation and this possibility. Hence, we do not see a strong correla-
immune functions are governed, in part, by the circa- tion in the effects of CS on clock gene expression
dian timing system (41– 43). However, it is not known between the acute and chronic treatment groups. Al-
to what extent circadian rhythms in the immune in- though certainly adequate, a 6-h time resolution is a
flammatory system are modulated by timing cues deliv- less than ideal sampling frequency for detection of
ered to this system from sources such as the SCN or are subtle variation in gene expression and protein levels
regulated locally by peripheral clocks. Circadian desyn- across the 24-h day. That said, we were able to detect
chrony or environmental circadian disruption during significant changes in the phase of several clock genes
experimental jet lag increases the susceptibility to in- after both acute and chronic CS exposure with that
flammation through dysregulation of innate immune sampling frequency. Studies designed to interrogate
responses (44). Consistent with other peripheral tis- subtle changes in clock gene expression following in
sues, circadian timing in the lung, although able to vivo CS exposure at more frequent sampling rates are
oscillate independently (14 –16), is synchronized with warranted. Similar effects of CS on circadian rhythms of
other tissues and the external environment by the cardiac function and lung tumorigenesis have been
central clock in the SCN (1). We have shown that CS reported (45– 47). Given the parallel nature of the
exposure disrupts circadian expression of clock and responses, it is reasonable to define CS exposure as a
clock-controlled genes in the lung, resulting in altered form of environmental circadian disruption, capable of
patterns of inflammatory cytokine gene expression and altering the molecular clock and clock-dependent pro-
secretion. It is possible that the effects of acute CS cesses in a manner similar to repeated jet lag or
exposure (10 d) in mice were amplified by the fact that rotating shift work. These effects may be more adverse,
CS exposure was conducted during the active phase in given that the disruption occurs in the presence of a
mice housed in an inverted L:D cycle (lights on from 6 regular 12:12 L:D cycle and apparently normal entrain-
PM to 6 AM). Because of the limitations of our facility, ment. Furthermore, it is possible that CS-induced pro-
it was not feasible to conduct long-term (chronic CS) inflammatory cytokines contribute to circadian disrup-
exposure studies in an inverted L:D cycle to confirm tion via TLRs/NF-␬B (48 –50), as CS components may

12 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


Figure 9. Locomotor activity of WT, heterozygous SIRT1-knockout (SIRT⫹/⫺), and SIRT1-overexpressing (SIRT1 Tg) mice
during acute CS exposure. Wild-type (WT), SIRT1⫹/⫺, and SIRT1 Tg mice were kept in a 12:12 L:D cycle throughout the
experiment. Mice were not exposed to CS for the first 3 d (preexposure); were exposed to CS during the light phase (ZT4-8)
for 3 consecutive days (CS exposure); and then were kept in room air without CS exposure for 4 d (postexposure).
Representative actograms of locomotor activity from mice exposed to air or CS for 3 d. During the period of CS exposure, activity
was not recorded (light-gray or dark-gray shaded area during the 12-h light phase). Locomotor activity during the dark phase
was plotted as ambulatory counts. Shading indicates the days of CS exposure.

recognize TLRs and activate NF-␬B for proinflamma- (9). Recently, it was reported that BMAL1-deficient
tory responses (12, 51). In acute CS exposure, neutro- mice have an increased sensitivity to oxidative stress
phil influx was observed at ZT24, but not at ZT0, which that correlates with cellular senescence (11). Although
is in fact the same time point. However, neutrophil the nuclear receptor REV-ERB␣, which is known as a
influx in the lung usually increases 16 h after the last CS negative regulator of BMAL1, has been reported to
exposure. Thus, we expected to see a delayed increase have a role in the regulation of inflammatory mediators
in neutrophil influx. Earlier reports from our labora- (41, 44, 52), evidence supporting the role of BMAL1 in
tory have clearly demonstrated an increase in proin- oxidative stress-induced inflammation remains elusive.
flammatory cytokine release and neutrophil influx Cigarette smoking not only induces oxidative stress,
after acute CS exposure at a concentration of 300 but is also associated with clinical depression (53, 54)
mg/m3 TPM, which was achieved with a mainstream CS and sleep disorders (55). Patients with COPD have an
exposure system (Baumgartner-Jaeger CSM2072i, CH early morning surge in respiratory symptoms (cough, spu-
Technologies; ref. 33). This response is not attainable tum production, and wheezing) and sleep abnormalities
with the Teague smoke exposure system [a mixture of (13, 14, 17, 19, 20). Disrupted sleep in patients with
sidestream (89%) and mainstream (11%) smoke] used COPD correlates with respiratory symptoms (cough,
in the current study (⬃100 mg/m3 TPM). Further- sputum production, and wheezing), nocturnal oxygen
more, in our protocol, there was no influx of neutro- desaturation, hypercapnia, and circadian changes in
phils into the BAL fluid with the use of the Teague airway caliber and resistance (20, 56). Each of these
machine for CS exposure. effects of chronic tobacco/CS is closely related to a
The role of clock proteins in increased susceptibility significant deterioration in quality of life in smokers
to oxidative stress–mediated inflammatory and injuri- and contributes to the development of COPD. In these
ous responses is implicated. For example, PER2 mutant patients depression often lasts even after smoking ces-
mice are known to be sensitive to oxidative stress and to sation (57) and is a frequent comorbidity of COPD
be susceptible or prone to the development of cancer (58 – 61). However, the underlying molecular and cel-

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 13


Figure 10. Ambulatory counts (A) and activity onset time (ZT) by acute CS exposure (B) in WT, heterozygous SIRT1-knockout
(SIRT ⫹/⫺), and SIRT1-overexpressing (SIRT1 Tg) mice. Activity onset relative to lights off (ZT12; phase angle of entrainment)
during the period of preexposure (d 1–3), CS exposure (d 4 – 6), and postexposure (d 7–10). Gray shading indicates the dark
phase (ZT12-24). Data from air- and CS-exposed mice represent means ⫾ se (n⫽3– 4 mice/group) for each time point.

lular mechanisms for CS-induced circadian abnormali- to CS leads to lung inflammation, emphysema, and
ties are not fully understood. In this study, both acute senescence (27). Recently, it has been reported that
and chronic CS exposure reduced nighttime activity in SIRT1 has an important role in the regulation of
mice. Surprisingly, chronically exposed mice were less circadian clock mechanisms by affecting circadian
active only through 3 mo of CS exposure. Contrary to clock gene transcription in an NAD⫹-dependent man-
our expectations, locomotor activity was similar in the ner (24, 25, 63). Another interesting report showed
air- and CS-exposure groups after 6 mo. When we that SIRT1 in the brain governs central circadian
compared the overall activity levels in mice from the control and activates transcription of the BMAL1:
beginning (1st mo) of the experiment until completion CLOCK transactivator complex by amplifying the
(6th mo), there was a clear age-dependent decline in SIRT1-PGC-1␣-Nampt axis (64). Altered SIRT1 levels in
total activity levels. Hence, the overall reduction in the brain also exert moderate changes in the intrinsic
activity in aged mice may have masked the effects of circadian periods of young and old mice (64). SIRT1
chronic CS exposure. Furthermore, it is possible that plays an essential role in the age-dependent decline in
the effects of chronic CS exposure on the brain are less central clock function, and targeting these circadian
pronounced because of the habituation of neural re- regulated loops in the brain (SIRT1, PGC-1␣, and
sponses to CS. Regardless, our data suggest that re- Nampt) may provide novel strategies for ameliorating
duced sleep quality in patients with COPD is a direct the negative effects of aging on circadian clock func-
consequence of altered clock function in sleep- tion (64). SIRT1 affects the molecular clock by binding
regulatory centers of the brain, including the central with CLOCK:BMAL1 complexes and deacetylating the
pacemaker in the SCN. BMAL1 and PER2 proteins (21–24). Thus, CS could
SIRT1 is an NAD⫹-dependent deacetylase involved in affect clock function by reducing SIRT1, leading to
regulation of various biological processes. The levels of increased inflammatory responses in the lung. Our
SIRT1 are reduced through protein carbonylation in preliminary observations showed that PER2 levels,
response to CS exposure in the lung (26, 62). A which are CLOCK:BMAL1 dependent, are reduced in
reduction in the levels and activity of SIRT1 in response lungs of patients with COPD. CS also decreased abun-

14 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


Figure 11. Locomotor activity of BMAL1-CC10 cre mice in response to acute CS exposure. BMAL1 CC10 cre (epithelial BMAL1
knockout) mice were kept in a 12:12 L:D cycle throughout the experiment. Mice were not exposed to CS for the first 3 d
(preexposure), were exposed to CS during the light phase (ZT4-8) for 3 consecutive days (CS exposure), and then were kept
in room air without CS exposure for 4 d (postexposure). A) Representative actograms of locomotor activity from WT or
BMAL1-CC10 cre mice exposed to air/CS for 3 d. During the period of CS exposure, activity was not recorded (light-gray or
dark-gray shaded area during the 12-h light phase). B) Locomotor activity during the dark phase was plotted as ambulatory
counts. Shading indicates the days of CS exposure. C) Activity onset relative to lights off (ZT12; phase angle of entrainment)
during the period of preexposure (d 1–3), CS exposure (d 4 – 6) and postexposure (d 7–10). Gray shading indicates the dark
phase (ZT12-24). Data from air- and CS-exposed mice represent means ⫾ se (n⫽3– 4 mice/group) for each time point.

dance of PER2, which may be acetylated and regulated of cigarette inhalation and exposure, and the possible
by SIRT1 in lungs of mice exposed to CS. Our results influence of prescribed drugs, which varies greatly
show that SIRT1 expression is rhythmic in the lungs of between patients. To our knowledge, this is the first
air-exposed mice, which is consistent with a previous study showing that SIRT1 is expressed with a circadian
report (24). CS significantly reduced SIRT1 protein rhythm under clock control in lung tissue. Further, we
abundance, concomitant with a reduction in BMAL1 report for the first time that BMAL1 acetylation in the
levels, and increased acetylation on lysine residue K537 lung in vivo is due to a CS-dependent reduction in
of the remaining BMAL1 protein. These results were SIRT1 levels. There is emerging evidence that circadian
confirmed in the lungs of SIRT1-deficient and SIRT1- rhythms govern proinflammatory cytokine gene expres-
overexpressing mice that were exposed to CS and sion (41, 44, 50). It is also known that inflammation
paralleled a report showing that acetylation of BMAL1 and alteration in clock gene expression (BMAL1 and
on lysine residue K537 is regulated by SIRT1 (25). We PER2) are intimately associated with fatigue and dimin-
found a modest decrease in BMAL1 levels in lung tissue ished locomotor activity (65– 67). Hence, it is possible
from patients with COPD, although it was still signifi- that SIRT1 acts as a critical link between core clock
cant when compared with that in lung tissue of non- function and inflammatory response in the lung.
smokers. We also observed a considerable variation in BMAL1, as part of the primary enhancer complex with
the expression levels of BMAL1 in the lungs of patients CLOCK, has a pivotal role in regulation of circadian clock
with COPD, compared to those of nonsmokers, which is function through transactivation of clock components
not unexpected, given a large variation in the intensi- and downstream clock-controlled genes. Acetylation is
ties and grades of COPD severity (GOLD stages I–IV), linked to BMAL1 phosphorylation (25, 67, 68), possibly
timing of sample collections, the duration and amount leading to its nuclear accumulation (68) and subsequent

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 15


Figure 12. Lung epithelial cell-specific BMAL1
knockout enhanced CS-induced lung inflam-
mation. A) Expression of proinflammatory cy-
tokine genes (ccl1/mcp-1, and cxcl1/kc) was per-
formed by qPCR. CircWave analysis confirmed
circadian rhythms of each proinflammatory cy-
tokine in air-exposed mice. *P ⬍ 0.05, **P ⬍ 0.01 vs.
corresponding air-exposed mice. B) Levels of pro-
inflammatory mediators, including CCL1/MCP-1
and CXCL1/KC, were measured in lung homoge-
nates obtained from air- or CS-exposed WT and
BMAL1 CC10 cre mice. Data are shown as means ⫾
se (n⫽3/group) for each time point. *P ⬍ 0.05,
**P ⬍ 0.01, ***P ⬍ 0.001 vs. corresponding
air-exposed mice. C) WT and BMAL1-CC10 cre
mice were treated with pharmacologic SIRT1
activator SRT1720 (SRT) or vehicle (Veh) dur-
ing CS exposure for 3 d. Levels of proinflam-
matory mediators, such as CCL1/MCP-1 and
CXCL1/KC, were measured in lung homoge-
nates obtained from air- or CS-exposed WT and
BMAL1-CC10 cre mice. Data are shown as
means ⫾ se (n⫽3 mice/group) for each time
point. *P ⬍ 0.05, **P ⬍ 0.01 vs. corresponding
air-exposed mice.

degradation (67). The results presented here reveal that of the epithelial molecular clock in regulation of CS-
CS exposure induced increased acetylation of BMAL1 induced lung inflammation.
and its loss of stability. Intriguingly, as shown in a previous In conclusion, our data show that environmental CS
study (25), we observed that the level of BMAL1 was exposure in mice caused alterations in circadian clock
greater in the lungs of SIRT1-deficient mice, suggesting gene expression in brain and lung tissue, altered rhythms
the SIRT1 is involved in BMAL1 stability. Further study is of locomotor activity, and increased lung inflammation
needed to investigate SIRT1-regulated mechanisms in- and emphysema. BMAL1 levels were reduced in lung
volved in BMAL1 stability, shuttling, phosphorylation, and tissue from patients with COPD, most likely owing to
acetylation in response to CS exposure and in the devel- increased turnover mediated by enhanced acetylation of
opment of COPD. BMAL1 by SIRT1. These data clearly indicate that circa-
BMAL1-deficient mice show signs of advanced aging dian rhythms of lung function are dampened in patients
and an age-related phenotype, correlating with increased with COPD, which is associated with abnormal airway
levels of ROS and cellular senescence (69). CS exposure inflammation. We also report that BMAL1 regulated
reduced the levels of BMAL1 mRNA and protein, and CS-induced lung inflammatory responses through SIRT1-
simultaneously evoked lung inflammatory responses, sug- controlled acetylation and stability of BMAL1 in lung
gesting that the loss of BMAL1 in response to CS exposure epithelium (Fig. 13). Thus, our results highlight the
increases inflammatory responses through deregulation importance of the molecular clock in the regulation of
of CS-induced oxidative stress. It has been shown that lung inflammation and injurious responses caused by CS.
BMAL1 interacts with the CCL1/MCP1 promoter, en- Overall, CS-mediated disruption of circadian clock func-
hancing CCL1/MCP1 gene expression. We found a sharp tion has implications in the pathogenesis of COPD. Un-
surge of CCL1/MCP1 in the lungs of CS-exposed WT derstanding molecular clock function in the lung and its
mice, which was augmented in the lungs of CS-exposed association with daily lung physiological function, partic-
mice harboring lung epithelium-specific deletion of ularly as it relates to the response to tobacco/CS, may
bmal1. Although the specific mechanisms remain un- strengthen the rationale for chronotherapy in COPD
known, it is likely that BMAL1 has a role in the regulation management.
of oxidative stress and could be a target of SIRT1 in
oxidative stress–induced inflammatory responses. A re- The authors thank Dr. Thomas J. Mariani (University of
cent study showed a similar pharmacologic activation of Rochester, Rochester, NY) for providing the Cre recombinase
SIRT1 in modulation of circadian rhythms in liver via a transgenic mice with CC10 promoter, Dr. Michael McBurney
(University of Ottawa, Ottawa, ON, Canada) for providing
reduction in H3K9/K14 acetylation (70). Furthermore, SIRT1-knockout mice (SIRT1-deficient mice), Dr. Leonard
augmented inflammatory responses observed in CS-ex- Guarente (Massachusetts Institutes of Technology, Cam-
posed mice harboring a lung epithelium–specific bmal1 bridge, MA, USA) and Dr. Wei Gu (Columbia University, New
deletion compared to WT mice suggest the involvement York, NY, USA) for providing Tg mice overexpressing Sirt1

16 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org HWANG ET AL.


Figure 13. Environmental circadian disruption after CS exposure is SIRT1-BMAL1 dependent, and is associated with increased
inflammation and reduced locomotor activity. CS exposure affects SIRT1 levels in the lung, which leads to BMAL1
acetylation/degradation, culminating in increased inflammation, circadian disruption (altered gene expression of clock and
clock-controlled genes), and reduced locomotor activity. Overexpression or pharmacologic activation of SIRT1 in cell-specific
BMAL1-knockout mice does not attenuate lung inflammation, suggesting that the effects of CS on circadian clock function and
inflammatory responses are mediated almost entirely by SIRT1-BMAL1-dependent mechanism.

(SIRT1 Tg mice), and Dr. Sangwoon Chung, Katherine 2. Dibner, C., Schibler, U., and Albrecht, U. (2010) The mamma-
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tutes of Health (1R01HL097751, 1R01HL092842) to I.R. and of breathing. Respir. Physiol. Neurobiol. 131, 91–100
a grant from the National Institute of Environmental Health 5. Hadden, H., Soldin, S. J., and Massaro, D. (2012) Circadian
Sciences (NIEHS) Environmental Health Science Center disruption alters mouse lung clock gene expression and
(P30-ES01247). The authors declare no conflicts of interest. lung mechanics. J. Appl. Physiol. 113, 385–392
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memory of Dr. Vuokko L. Kinnula (Pulmonary Division, clock to oxidative stress. Proc. Natl. Acad. Sci. U. S. A. 104,
Department of Medicine and Pathology, University of Hel- 15899 –15904
sinki and Helsinki University Hospital, Helsinki, Finland), 11. Khapre, R. V., Kondratova, A. A., Susova, O., and Kondratov,
who provided the human tissue samples and left us suddenly R. V. (2011) Circadian clock protein BMAL1 regulates cellular
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