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
display abnormal circadian rhythms in lung function.
We determined the mechanism whereby environmental
tobacco/cigarette smoke (CS) modulates expression of
the core clock gene BMAL1, through Sirtuin1 (SIRT1)
deacetylase during lung inflammatory and injurious
responses. Adult C57BL6/J and various mice mutant
for SIRT1 and BMAL1 were exposed to both chronic (6
mo) and acute (3 and 10 d) CS, and we measured the
rhythmic expression of clock genes, circadian rhythms of
locomotor activity, lung function, and inflammatory and
emphysematous responses in the lungs. CS exposure
(100 –300 mg/m3 particulates) altered clock gene expres-
sion and reduced locomotor activity by disrupting the
central and peripheral clocks and increased lung inflam-
mation, causing emphysema in mice. BMAL1 was acety-
lated and degraded in the lungs of mice exposed to CS
and in patients with chronic obstructive pulmonary
disease (COPD), compared with lungs of the nonsmok-
ing controls, linking it mechanistically to CS-induced
reduction of SIRT1. Targeted deletion of Bmal1 in lung
epithelium augmented inflammation in response to CS,
which was not attenuated by the selective SIRT1 activa-
tor SRT1720 (EC50ⴝ0.16 ␮M) in these mice. Thus, the
circadian clock, specifically the enhancer BMAL1 in
epithelium, plays a pivotal role, mediated by SIRT1-
dependent BMAL1, in the regulation of CS-induced
lung inflammatory and injurious responses.— Hwang,
J.-W., Sundar, I. K., Yao, H., Sellix, M. T., Rahman, I.
Circadian clock function is disrupted by environmental
tobacco/cigarette smoke, leading to lung inflammation
Abbreviations: ANOVA, analysis of variance; BAL, bron-
choalveolar lavage; BW, body weight; COG, center of gravity;
COPD, chronic obstructive pulmonary disease; CRY, crypto-
chrome; CS, cigarette smoke; CSE, cigarette smoke extract;
E, tissue elastance; H&E, hematoxylin and eosin; IP-10,
CXCL10/interferon-␥ inducible protein 10; KC, CXCL1/
keratinocyte-derived chemokine; Lm, mean linear intercept;
MCP-1, CCL2/monocyte chemotactic protein; PER, Period;
qPCR, quantitative real-time PCR; QsC, quasi-static compli-
ance; R, lung resistance; SCN, suprachiasmatic nucleus;
SIRT1, Sirtuin1; Tg, transgenic; TPM, total particulate matter;
ZT, zeitgeber time
0892-6638/14/0028-0001 © FASEB
and injury via a SIRT1-BMAL1 pathway. FASEB J. 28,
000 – 000 (2014). www.fasebj.org
Key Words: oxidative stress 䡠 cytokines 䡠 chronic obstructive
pulmonary disease 䡠 locomotor activity 䡠 emphysema 䡠 mouse
Circadian rhythms are biological oscillations that
occur with a near-24-h period and are synchronized or
entrained to environmental cues, such as the day–night
transition (1). In mammals, the central circadian pace-
maker is localized in the suprachiasmatic nucleus
(SCN) of the hypothalamus (1). In addition to the
central pacemaker, peripheral tissues, including liver,
heart, and lung, are autonomous circadian oscillators
(2). The clock in each of these tissues plays a critical
role in optimizing cellular function and responses to
environmental stimuli (1, 2). The coordinated activity
of these oscillators can be referred to as the circadian
timing system (2). Disruption of the circadian timing
system has been shown to cause cellular dysfunction
and chronic diseases (3). Circadian rhythms of respira-
tory function have been described in both experimen-
tal animals (4, 5) and healthy human subjects (6, 7).
However, the effect of environmental stress on clock
function in the lung and its role in lung pathophysiol-
ogy are unknown.
The molecular clock drives intrinsic daily rhythms of
physiology and behavior. At the cellular level, the clock
is defined as a transcriptional and translational feed-
back loop (TTFL) oscillator (1). The core loop consists
of the CLOCK:BMAL1 heterodimer, which activates
the transcription of Period (PER) and Cryptochrome
(CRY). PER and CRY proteins form heterodimeric
complexes that inhibit their own transcription by sup-
1
These authors contributed equally to this work.
2
Correspondence: Department of Environmental Medi-
cine, University of Rochester Medical Center, Box 850, 601
Elmwood Avenue, Rochester 14642, NY, USA. E-mail:
irfan_rahman@urmc.rochester.edu
doi: 10.1096/fj.13-232629
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.
1
pressing the activity of CLOCK:BMAL1 (1). Emerging
evidence suggests that the molecular clock is intimately
associated with the response to environmental stress in
pathophysiology (8 –11). However, the role of the circa-
dian clock gene BMAL1 in oxidative stress–induced in-
flammation remains elusive.
It is well known that tobacco and cigarette smoke
(CS) induces oxidative stress and consequently leads to
excessive pulmonary inflammation in the pathogenesis
of chronic obstructive pulmonary disease (COPD) and
emphysema (12). Daily rhythms of lung function, bron-
chodilator responses, surfactant protein levels, steroid
efficacy, mucus secretion, and chronic cough are hall-
marks of obstructive lung diseases known to be clock
dependent (5, 7, 12–17). Patients with COPD display
rhythmic variation in respiratory symptoms, including
nocturnal breathlessness, insomnia, and an early-morn-
ing decline in lung function associated with increased
cough and mucus hypersecretion (18 –20). These symp-
toms may stem from altered clock function in lung
tissue, and it is reasonable to suspect that disrupted
clock function can have a profound influence on lung
function and lung pathology. However, it is not known
whether CS directly alters clock function during the
pathogenesis of COPD.
Sirtuin1 (SIRT1), an NAD⫹-dependent deacetylase,
affects clock function by binding with CLOCK:BMAL1
complexes and deacetylating BMAL1 and PER2 pro-
teins (21–25). CS reduces the levels and activity of
SIRT1, and patients with COPD have reduced lung
levels of SIRT1 (26 –28). Hence, it is likely that CS-
mediated reduction of SIRT1 leads to increased acety-
lation of circadian clock proteins, culminating in ab-
normal clock gene expression and proinflammatory
responses in the lung. We hypothesize that environ-
mental CS affects molecular clock function in lung
tissue via the reduction of SIRT1, which in turn leads to
inflammatory and injurious responses in the pathogen-
esis of COPD and emphysema. To address this hypoth-
esis, we determined whether BMAL1, as a substrate of
SIRT1, has a role in the regulation of CS-induced lung
inflammatory and injurious responses.
MATERIALS AND METHODS
Animals
Male C57BL/6J (C57) and Bmal1-floxed mutant mice were
purchased from the Jackson Laboratory (Bar Harbor, ME,
USA). To generate conditional deletion of Bmal1 in lung
epithelial cells (CC10-Cre/Bmal1floxed/floxed, hereafter re-
ferred as BMAL1-CC10 cre), Bmal1-floxed mutant mice were
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-
genic (Tg) mice overexpressing Sirt1 (SIRT1 Tg mice) were
obtained from Dr. Leonard Guarente (Massachusetts Insti-
tutes of Technology, Cambridge, MA, USA) and Dr.Wei Gu
(Columbia University, New York, NY, USA), and SIRT1
knockout mice (SIRT1-deficient mice) were from Dr. Michael
McBurney (University of Ottawa, Ottawa, ON, Canada; refs.
2 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org
30, 31). SIRT1 heterozygous knockout mice were used, be-
cause SIRT1 homozygous knockout mice have a low perinatal
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
regular diet and water ad libitum, unless otherwise indicated.
For acute (10 d) CS exposure, the mice were entrained for a
minimum of 2 wk to an inverted L:D cycle (lights on from 6
PM to 6 AM), except for wheel-running experiments, before
CS exposure during their active phase. For 3 d and chronic (6
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
experiment. All of the procedures described in this study
were approved by the University Committee on Animal
Research at the University of Rochester.
Tobacco/CS exposure
Eight-week-old mice were used for tobacco/CS exposure (27,
32, 33). We used both acute (3 and 10 d exposure, which
induces lung inflammatory responses) and chronic (6 mo
exposure, which causes pulmonary emphysema) mouse mod-
els of CS exposure to determine the effect and mechanism of
both acute and chronic CS exposure on circadian clock
function and lung inflammation. The mice were exposed to CS
from a mainstream-delivering Baumgartner-JaegerCSM2082i ciga-
rette-smoking machine (CH Technologies, Westwood, NJ,
USA) for 3 d or an environmental sidestream-delivering
Teague TE-10 smoking machine (Teague Enterprises, Davis,
CA, USA) for 10 d and 6 mo of CS exposure in the Inhalation
Facility at the University of Rochester Medical Center. For 3 d
CS exposure, the mice were placed in individual compart-
ments of a wire cage, which was then placed inside a closed
plastic box connected to the smoke source. The smoke was
generated from 3R4F research cigarettes containing 11.0 mg
total particulate matter (TPM), 9.4 mg tar, and 0.73 mg
nicotine/cigarette (University of Kentucky, Lexington, KY,
USA). The TPM per cubic meter of air in the exposure
chamber was monitored in real time with a MicroDust
Proaerosol monitor (Casella CEL, Bedford, UK) and verified
daily by gravimetric sampling (33). The smoke concentration
was set at ⬃300 mg/m3 TPM by adjusting the flow rate of the
diluted medical air, and the level of carbon monoxide in the
chamber was 350 ppm (33). The mice received two 1-h
exposures per day (1 h apart), daily for 3 consecutive days
according to the Federal Trade Commission protocol (1
puff/min of 2 s duration and 35 ml volume) and were
euthanized 24 h after the last exposure (33, 34). The control
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
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
long-term CS exposure (32). Each smoldering cigarette was
puffed for 2 s, once every minute for a total of 5 puffs, at a
flow rate of 1.05 L/min, to provide a standard puff of 35 cm3.
Mice received 5 h exposure per day, 5 d/wk for the duration
of exposure and were euthanized at 6-h intervals 24 h after
the last CS exposure. The 6 h sampling interval was based on
prior studies on circadian gene expression in rodents (35).
Locomotor activity recording
The mice were individually housed and allowed free access to
food and water. Locomotor activity was measured with the
Photobeam Activity System (San Diego Instruments, San
Diego, CA, USA), a computerized system that measures the
frequency of photobeam breaks along the side of the cage.
HWANG ET AL.
For wheel-running behavior, the mice were housed singly in
standard mouse cages fitted with a stainless-steel running
wheel (Coulbourn Instruments, Flushing, NY, USA) placed
within a light-tight chamber (Phenome Technologies, Lin-
colnshire, IL, USA). Total cage activity (photobeam break)
and wheel-running activity were recorded in 1 min intervals
and analyzed with ClockLab software (Actimetrics, Evanston,
IL, USA). For wheel-running analyses, the mice were exposed
to CS during their inactive phase for 10 d and then trans-
ferred to recording chambers. They remained under a 12:12
L:D cycle for 2 d followed by release into constant darkness.
The free-running period of wheel-running activity was deter-
mined during 2 wk of constant darkness with a ␹2 periodo-
gram (P⬍0.001), and data were analyzed with an unpaired t
test (GraphPad Prism, San Diego, CA, USA).
Real-time luminescence recording
Adult male Period2::luciferase knock-in (PER2::LUC) mice
(36) were euthanized by excess CO2 exposure 3 h before
lights off [zeitgeber time (ZT) 9 –12; lights off⫽ZT12]. Por-
tions of the lung and the brain were removed and collected in
cold, sterile Hanks’ balanced salt solution (HBSS). After the
tissue was blocked, the brain was sliced in the coronal plane
with a vibrating microtome at a thickness of 300 ␮m. A section
containing the bilateral SCN near the midpoint of the
rostrocaudal extent was recovered, and a minimum of tissue,
including the paired SCNs was removed with fine scalpels.
The paired SCNs were then separated by a precise midline
cut, producing 2 hemi-SCN tissue explants. Small (5 mm3)
fragments of lung tissue were isolated. The tissue specimens
were placed in 35-mm culture dishes with 1.2 ml of culture
medium [DMEM supplemented with B27 (Life Technologies,
Inc., Grand Island, NY, USA), 10 mM HEPES, 352.5 ␮g/ml
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-
scribed above (control) or with the same medium containing
cigarette smoke extract (CSE) and sealed with sterile vacuum
grease and a glass coverslip. Sealed cultures were maintained
at 35°C in a light-tight incubator, and luminescence was
continuously recorded (counts/second) with an automated
luminometer (LumiCycle; Actimetrics). Raw luminescence
data were detrended (24 h moving average) and smoothed (2
h moving average; Origin Pro 8.5, OriginLabs, Northampton,
MA, USA).
Lung morphometry
Mouse lungs (which had not been lavaged) were inflated by
1% low-melting-point agarose at a pressure of 25 cmH2O, and
then fixed with 4% neutral buffered formalin (27, 32). Fixed
lung tissues were dehydrated, embedded in paraffin, and
sectioned (4 ␮m) with a rotary microtome (Microm Interna-
tional GmbH, Walldorf, Germany). The lung sections were
deparaffinized, rehydrated by passing them through a series
of xylene and graded alcohol, and stained with hematoxylin
and eosin (H&E). Alveolar size was estimated from the mean
linear intercept (Lm) of the airspace, which is a measure of
airspace enlargement or emphysema performed with Meta-
Morph software (Molecular Devices, Sunnyvale, CA, USA;
refs. 27, 32). Lm was calculated for each sample on the basis
of 10 random fields/slide, observed at ⫻200. The airway and
vascular structures were eliminated from the analysis.
Measurements of lung mechanical properties
The mechanical properties of the mouse lungs were deter-
mined with the FlexiVent apparatus (Scireq, Montreal, QC,
TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM
Canada; refs. 27, 32). Quasi-static compliance (QsC), lung
resistance (R), and tissue elastance (E) were measured in the
mice, anesthetized by sodium pentobarbital [50 mg/kg body
weight (BW), intraperitoneally]]. A tracheotomy was per-
formed, and an 18-gauge cannula was inserted 3 mm into an
anterior nick in the exposed trachea and connected to a
computer-controlled rodent ventilator (FlexiVent; Scireq).
Initially, the mice were ventilated with room air (150 breaths/
min) at a volume of 10 ml/kg body mass. After 3 min of
ventilation, measurement of lung mechanical properties was
initiated by a computer-generated program to measure QsC,
R, and E at 3 cmH2O positive end expiratory pressure
obtained by fitting a model to each impedance spectrum. The
calibration procedure removed the impedance of the equip-
ment and tracheal tube within this system (37, 38). These
measurements were repeated 3 times for each animal.
Bronchoalveolar lavage (BAL)
The mice were anesthetized at 24 h after the last exposure by
an intraperitoneal injection of 100 mg/kg BW pentobarbital
sodium (Abbott Laboratories, Abbott Park, IL, USA) and
killed by exsanguination. The heart and lungs were removed
en bloc, and the lungs were lavaged 3 times with 0.6 ml of
saline (0.9% sodium chloride) via a cannula inserted into the
trachea (4). The lavaged fluid was centrifuged, and the
cell-free supernatants were frozen at ⫺80°C for later analysis.
The BAL inflammatory cell pellet was resuspended in 1 ml
saline, and the total number of cells was counted with a
hemocytometer. Cytospin slides (Thermo Shandon, Pitts-
burgh, PA, USA) were prepared at 50,000 cells/slide, and
differential cell counts (⬃500 cells/slide) were performed on
cytospin-prepared slides stained with Diff-Quik (Dade Beh-
ring, Newark, DE, USA).
RNA isolation and quantitative PCR (qPCR)
Total RNA was isolated from brain and nonlavaged lung
tissue specimens (stored in RNAlater; Ambion, Austin, TX,
USA) with an RNeasy kit (Qiagen, Valencia, CA, USA). RNA
yields were determined by UV absorbance with a NanoDrop
instrument (ND-1000 Spectrophotometer; NanoDrop Tech-
nologies, Wilmington, DE, USA). cDNA was synthesized from
0.5 ␮g of total RNA by using the RT2 First Strand Kit
(SABioscience, Frederick, MD, USA). To validate the expres-
sion of diverse genes in brain and lung tissues, qPCR was
performed with a Bio-Rad iCycler real-time system and SYBR
Green qPCR Master mix from SABioscience. In chronic CS
exposure, qPCR data were gathered from circadian gene and
proinflammatory cytokine gene expression at the ZT24 time
point (n⫽2/air group) in all datasets. All the specific primers
were purchased from SABioscience. Gene expression was
normalized to 18s rRNA levels. Relative RNA abundance was
quantified by the comparative 2⫺⌬⌬Ct method. Significant
rhythms of gene expression were verified with CircWave
software (version 1.4). In addition, the center of gravity
(COG), or peak phase, was determined for each rhythm by
using CircWave (39).
Proinflammatory mediator analysis
The levels of proinflammatory mediators, such as CCL2/
monocyte chemotactic protein-1 (MCP-1), CXCL1/keratino-
cyte derived chemokine (KC), and CXCL10/interferon-␥
inducible protein 10 (IP-10) in lung homogenates were
measured by enzyme-linked immunosorbent assay (ELISA)
performed with the respective duo-antibody kits (R&D Sys-
tems, Minneapolis, MN, USA), according to the manufacturer’s
3
instructions. The results were expressed in the samples as
picograms per milligram protein.
Immunoblot and immunoprecipitation
Protein samples from lung homogenates were separated by
6 –10% SDS-PAGE. The separated proteins were electroblot-
ted onto nitrocellulose membranes (Amersham Biosciences,
Piscataway, NJ, USA). The membranes were blocked for 1 h at
room temperature with 5% BSA and then probed with
anti-SIRT1, anti-Ac Lys (1:1000; Cell Signaling Technology,
Danvers, MA, USA), anti-BMAL1 and anti-CLOCK, (1:1000-
1:2000; Abcam, Cambridge, MA, USA), and anti-acetyl
BMAL1 (1:1000; Millipore, Billerica, MA, USA) antibodies, to
determine the respective proteins. After 3 washing steps (10
min each), the levels of protein were detected with secondary
antibody (1:5000 dilution) in 2.5% BSA in PBS containing
0.1% Tween 20 (v/v) for 1 h linked to horseradish peroxidase
(Dako, Carpinteria, CA, USA), and bound complexes were
detected by ECL (Perkin Elmer, Wellesley, MA, USA). Equal
loading of the samples was determined by quantitation of
proteins and by stripping and reprobing the membranes
for ␤-actin (Oncogene, Cambridge, MA, USA). Immuno-
precipitation was performed to determine the acetylation
of BMAL1 in human whole-lung homogenates from non-
smokers, smokers, and patients with COPD, as described
previously (26, 27, 34). ImageJ 1.41densitometry software
(U.S. National Institutes of Health, Bethesda, MD, USA)
was used for gel band quantitative densitometry.
Administration of SRT1720
SRT1720 (100 mg/kg, ⬎95% pure by C-13 NMR and LCMS,
synthesized by Life Chemicals, Niagara-on-the-Lake, ON, Can-
ada) was administered to the acute air- and CS-exposed mice
daily for 3 d through oral gavage 1 h before CS exposure (40).
Human samples
Lung tissue specimens from nonsmokers, smokers, and pa-
tients with COPD were obtained as described previously (26,
27, 34).
Statistical analysis
The period of PER2::LUC expression in each tissue explant
was determined with a ␹2 periodogram analysis (LumiCycle
Analysis Software; Actimetrics). A minimum of 3 d of data
were used to calculate the period of PER2::LUC expression in
each explant of lung and SCN tissue. Period data from the
lung tissue were analyzed with 1-factor analysis of variance
(ANOVA) and the Newman-Keuls post hoc test. Period data
from the SCN tissue (2 groups) were analyzed with an
unpaired t test. Data are presented as the mean ⫾ se. For
statistical analysis of qPCR data, CircWave 1.4 software was
used. In addition to CircWave analysis, statistical significance
between the air- and CS-exposed groups was calculated by
Fisher’s multiple comparison, with 2-way ANOVA. To empha-
size the waveform of the data, representative protein expres-
sion was subjected to nonlinear regression analysis with a
sixth- order polynomial (Prism; Graphpad). Statistical analysis
of significance was calculated by StatView software in a 1-way
ANOVA followed by Tukey’s post hoc test for multigroup
comparisons. Values of P ⬍ 0.05 represented a significant
difference.
4 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org
RESULTS
CS exposure differentially affected the amplitude
and phase of circadian clock gene expression in the
lungs and brain
We used qPCR to investigate the effect of environmen-
tal CS exposure on rhythmic expression of core clock
genes in mouse lungs. Both chronic (6 mo exposure,
which causes pulmonary emphysema) and acute (3 and
10 d exposure, which induces a lung inflammatory
response) environmental CS exposure models were
used to determine the effects of CS on rhythmic
expression of core clock genes in lung tissue and the
functional relationship between altered clock function
and lung inflammation. Most of the core clock genes
(bmal1, clock, per1, per2, cry1, cry2, rev-erb␣, and rev-erb␤)
were rhythmically expressed in lung tissue from mice
exposed to air or CS, either acutely (10 d; Fig. 1A, B) or
chronically (6 mo; Supplemental Fig. S1A, B). CircWave
analysis confirmed statistically significant (P⬍0.001)
rhythms of bmal1, clock, per1, per2, rev-erb␣, and rev-erb␤
gene expression (Fig. 1A, B, Table 1, Supplemental Fig.
S1A, and Table 2). Of the known core clock genes, only
ror␣ mRNA was not rhythmic in the lungs of the mice
exposed to air or CS for 6 mo (Supplemental Fig. S1A).
In both the air- and CS-exposed mice, the expression of
bmal1, clock, and cry1 displayed nocturnal acrophases,
peaking in the mid to late portions of the dark phase
(ZT18 –24; ZT0⫽lights on; ZT12⫽lights off; Fig. 1A, B
and Supplemental Fig. S1A, B). As expected, in both
groups, the peaks of per1, per2, cry2, rev-erb␣, and rev-erb␤
were nearly antiphase to bmal1, which peaked between
ZT6 and ZT12 (Fig. 1A, B and Supplemental Fig. S1A,
B). Chronic CS exposure caused a modest reduction in
the amplitude of bmal1 and rev-erb␣ expression and
substantially reduced the amplitude of per1 expression
in the lungs (Supplemental Fig. S1A, B). Further,
CircWave analyses indicated that chronic CS exposure
altered the phase of per1 and per2 expression in the
lungs (Supplemental Fig. S1B).
As with the chronic-exposure model, acute CS expo-
sure also reduced the amplitude of bmal1, clock, and per2
mRNA expression, but only modestly affected the am-
plitude of per1 expression in the lungs (Fig. 1A).
Surprisingly, acute CS exposure appeared to have time-
dependent suppressive effects on cry1, cry2, and rev-erb␤
expression (Fig. 1A). Finally, CircWave analyses indi-
cated that acute CS slightly delayed the phase of rev-erb␣
expression in the lungs (Fig. 1B). Together, data from
both the acute and chronic models reveal that the
amplitude and phase of clock gene expression in the
lungs were differentially affected by CS exposure, al-
though the effects of acute exposure were more robust.
Parallel effects of CS exposure on the phase and
amplitude of clock gene expression were observed in
brain tissue of the acutely CS-exposed mice (Supple-
mental Fig. S3), suggesting that the effects of CS on
clock function are not limited to lung tissue. The effect
of acute CS exposure on clock gene expression in the lung
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
lung tissue explants (Fig. 2A). Of note, treatment with
0.1% CSE did not affect the amplitude of PER2::LUC
expression in the SCN explants, compared to that in
the lung tissue (Fig. 2B). However, the same treatment
significantly shortened the period of PER2::LUC ex-
pression in the CS-exposed SCN explants when com-
pared with that in the untreated control (Fig. 2B).
Overall, CSE treatment reduced the amplitude and
differentially affected the period of PER2::LUC expres-
sion in brain and lung tissue explants ex vivo.
CS exposure reduced total locomotor activity and
shortened the free-running period of wheel-running
behavior in mice
Seeing that CS exposure altered the amplitude and
period of clock gene expression in lung and brain
tissue, we investigated its effect on the circadian
rhythms of behavior in mice. The total cage activity of
individual C57BL/6J mice was recorded by infrared-
beam breaks during the 6 mo of CS exposure (17 d
before starting and continuing until the end of chronic
CS exposure). Mice exposed to chronic CS showed a
significant reduction in total cage locomotor activity
(Supplemental Fig. S2A). Analysis of total ambulatory
counts across the 24 h day revealed that chronic CS
exposure significantly reduced activity in the WT mice
within 5 d of the first exposure, and the decrease
persisted until the beginning of the 6th mo of CS
exposure (Supplemental Fig. S2B). When daytime and
nighttime activity were analyzed separately, the ampli-
tude of the response increased, indicating that the
primary effect of CS exposure was a reduction in
nighttime activity (Supplemental Fig. S2B).
The effect of acute CS exposure on total cage loco-
motor activity and wheel-running behavior were mea-
sured to determine whether the reduction in activity
that we observed is initiated at an early stage of smok-
ing. We used our beam break monitor to record total
cage activity for 2 wk before CS exposure, during the
active phase for 10 d of CS exposure and during a 2 wk
period after CS exposure (clean air). The mice were
exposed to CS during their active phase for 5 h/d over
10 consecutive days. Locomotor activity was almost
immediately attenuated in the CS-exposed mice, and
was persistently lower after CS exposure ended (Fig. 3A).
Although activity increased during recovery, the mice
managed to approach only ⬃70% of their original
activity level after 2 wk of breathing clean air (Fig. 3B).
Activity onset time or phase angle of entrainment was
estimated with Clocklab software (Actimetrics) and did
not appear to be affected by CS (Fig. 3C). Analysis of
wheel-running behavior revealed a small, but signifi-
cant, reduction in the free-running period of activity in
mice after acute exposure to CS (␶⫽23.61⫾0.06) dur-
ing their inactive phase when compared with the air-
exposed mice (␶⫽23.80⫾0.03; P⬍0.05) (Fig. 4). Short-
ening of the free-running period of wheel-running
activity suggests that CS alters clock gene expression in
the central clocks that regulate behavioral rhythms, a
finding supported by our analysis of PER2::LUC expres-
sion in SCN tissue explants (Fig. 2B) and clock gene
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
resulted in increased inflammatory responses
To investigate whether CS alters circadian rhythmicity
of proinflammatory cytokine responses, we determined
the expression of proinflammatory cytokine genes after
chronic and acute CS exposure. In air-exposed mice
from the acute exposure experiments, lung mRNA
expression of both monocyte chemotactic protein–1
(ccl1/mcp-1) and keratinocyte chemoattractant (cxcl1/kc)
peaked during the dark phase (Fig. 5A–C and Supple-
mental Fig. S4A–C). CircWave analysis confirmed signifi-
cant circadian rhythms of both genes in air-exposed mice
(P⬍0.001). In contrast, the expression of interferon-␥-
induced protein 10 (cxcl10/IP10) peaked during the light
phase, but did not have a significant circadian rhythm of
expression in air-exposed mice (Fig. 5C and Supplemen-
tal Fig. S4C). Acute and chronic CS exposure disrupted
rhythms of ccl1/mcp-1 and cxcl1/kc expression in the lungs
(Fig. 5A, B and Supplemental Fig. S4A, B). These results
suggest that proinflammatory cytokine gene expression is
rhythmic in the lungs, and is disrupted by both acute and
chronic CS exposure.
To understand the functional link between CS-medi-
ated alterations in circadian rhythmicity and proinflam-
matory cytokine responses, we determined the abun-
dance of proinflammatory cytokines (ccl1/mcp-1, cxcl1/kc,
and MIP-2) in mouse lungs after CS exposure. Acute CS
exposure appeared to cause a phase shift in the rhythms
of MCP-1 and KC release, such that MCP-1 peaked in the
middle of the day (ZT6 vs. ZT18 in air-exposed group),
and KC levels peaked closer to lights off (ZT12; Fig. 5D,
E). Acute CS also appeared to increase the amplitude of
KC release in the mouse lung tissue (Fig. 5E). Chronic CS
exposure had differential and cytokine-specific effects on
MCP-1, KC, and MIP-2 levels in the tissue (Supplemental
Fig. S4D–F). The levels of MCP-1 and KC were increased
in the latter portion of the dark phase (ZT18-24) after
chronic CS exposure.
The increased level of cytokines was associated with a
rhythmic influx of inflammatory cells in the lung in
response to acute (Fig. 6A) and chronic (Fig. 6B) CS

TOBACCO/CIGARETTE SMOKE DISRUPTS CIRCADIAN RHYTHM 7


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
significantly during the late night (ZT24), whereas
there was no significant difference in the number of
macrophages and total cells in BAL fluid (Fig. 6B).
Thus, our data show that the disruption of circadian
clock function in the lung was associated with aug-
mented proinflammatory responses during both acute
and chronic CS exposure.
Disruption of circadian clock function was associated
with airspace enlargement and emphysema along with
alteration in lung mechanical properties and function
To investigate the potential effect of circadian clock
dysfunction due to CS-induced airspace enlargement and
emphysema, we subjected mice to chronic CS exposure
and determined their susceptibility to CS-induced air-
space enlargement and emphysema by lung histopatho-
logic and functional measurements. There was a sig-
nificant difference in lung histopathology (H&E)
between air- and CS-exposed mice after 6 mo, which
was assessed by determining the Lm (air 49.56⫾3.25
vs. CS 61.40⫾1.60; P⬍0.01, n⫽4 – 6 mice/group). Sim-
ilarly, the overall lung E was significantly decreased and
R was decreased, but not significantly, in the mice
exposed to chronic CS. However, lung QsC was in-
creased significantly in chronic CS-exposed mice as
compared to that in air-exposed mice (Table 3). We
also found a significant decrease in lung E and R in the
chronic CS-exposed mice at ZT18, but not at other
times of the day (ZT0, ZT6, ZT12, and ZT24), when
compared to those parameters in air-exposed mice
(Tables 3 and 4). However, when we combined the data
8 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org
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-
from day (ZT0 and ZT6) and night (ZT12 and ZT18),
E was significantly reduced, whereas lung compliance
increased in the chronic CS-exposed mice (Table 3).
There was no statistically significant difference in BW
or mortality between the chronic CS- and air-exposed
mice.
Rhythmic expression of SIRT1 was disrupted by CS
exposure, resulting in increased acetylation of
BMAL1 in lungs
To investigate the mechanisms of CS-induced disruption
of the lung inflammatory response, we determined the
levels of BMAL1 acetylation and the abundance of SIRT1
in mouse lung after an acute (10 d) CS exposure. In the
lungs of the control air-exposed mice, the protein abun-
dance of SIRT1 and BMAL1 showed clear rhythmic
oscillations that peaked in the night (Fig. 7A, B). How-
ever, CS reduced the abundance of SIRT1 and BMAL1
across the entire day, which was consistent with gene
expression data (Fig. 1A and Supplemental Fig. S1A). We
observed a similar effect of CS on the rhythm of sirt1 gene
expression in the lung and brain tissues (Fig. 1C and
Supplemental Fig. S3C). Furthermore, the mice with CS
exposure showed a reduction in BMAL1 levels (Fig. 7A).
Similarly, the BMAL1 levels were modestly reduced in the
smokers and significantly reduced in lungs of the patients
with COPD, compared with levels in lungs of the non-
smokers (Fig. 8A, B). CS caused increased BMAL1 acety-
lation in the mouse lungs (Fig. 7A, B), and a similar
increase in BMAL1 acetylation was observed in the lungs
of the patients with COPD and the smokers, compared to
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
acetylation in the CS-exposed mice was associated with
reduced locomotor activity (Figs. 9 and 10). Given that
SIRT1 modulates the circadian clock mechanism by con-
trolling BMAL1 acetylation, we hypothesize that acetyla-
tion of BMAL1 is increased by the reduction of SIRT1
levels after CS exposure.
To determine whether SIRT1 reduction is responsi-
ble for increased BMAL1 acetylation, SIRT1-deficient
(SIRT1⫹/⫺) and SIRT1-overexpressing (Tg) mice were
exposed to acute CS for 3 d, and the level of BMAL1
acetylation was measured in lung tissue. Acute CS exposure
significantly increased the acetylation of BMAL1 in the
SIRT1-deficient mice relative to both the air-exposed
SIRT1-deficient mice and the CS-exposed WT mice
(Fig. 7C). This response was attenuated in the SIRT1 Tg
mice, most likely because of the persistently elevated
level of SIRT1 deacetylase activity (Fig. 7C). Moreover,
locomotor activity levels were significantly reduced
during acute (3 d) CS exposure in SIRT1-deficient but
not in SIRT1-overexpressing mice (Figs. 9 and 10).
These results suggest that SIRT1 reduction in response
to CS exposure leads to an increase in BMAL1 acetyla-
tion and reduced levels of BMAL1, causing altered
molecular clock function and increased proinflamma-
tory responses in the lungs.
Lung epithelial cell–specific BMAL1 deletion
increased lung inflammation, which was not
attenuated by treatment with a selective
pharmacological SIRT1 activator
To determine the role of the SIRT1-BMAL1 pathway in
CS-induced circadian disruption and inflammatory re-
sponses, we established lung epithelial cell–specific
BMAL1⫺/⫺ (BMAL1-CC10 cre) mice and exposed
them to an acute period (3 d) of CS. The BMAL1-CC10
cre mice exhibited normal rhythms of locomotor activ-
ity (ambulatory count and onset time; Fig. 11) and, as
in the WT mice, the activity in these mice was signifi-
cantly reduced by CS exposure (Fig. 11A, B). As in the
WT mice, we detected rhythmic expression of proin-
flammatory cytokine genes, including ccl1/mcp-1 and
cxcl1/kc, in the BMAL1-CC10 cre mice that were af-
fected by CS, such that the expression of ccl1/mcp-1 was
increased at ZT0 but remained unchanged at ZT12
(Fig. 12A, B). Cxcl1/kc gene expression was elevated at
both times in the CS-exposed animals. We found simi-
lar effects of lung-specific BMAL1 deletion on rhythms
of proinflammatory cytokine release (Fig. 12B). Over-
all, lung epithelial cell–specific clock disruption altered
the inflammatory response to CS. To further examine
the involvement of SIRT1 and BMAL1 in these proin-
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
selective pharmacologic SIRT1 activator SRT1720. The
BMAL1-CC10 cre, global SIRT1-deficient, and SIRT1
Tg mice exhibited normal diurnal patterns of locomo-
tor activity (Figs. 9 –11). As expected, locomotor activity
during the night was reduced in these mice during the
acute CS exposure and returned to normal levels after
4 d of clean-air recovery (Figs. 9 –11). Treatment with
SRT1720 attenuated proinflammatory cytokine release
in the CS-exposed WT mice, but not in the CS-exposed
BMAL1-CC10 cre mice (Fig. 12C). These data strongly
suggest that epithelial BMAL1 plays a critical role in the
regulation of CS-induced inflammatory responses and
that the effect of BMAL1 is modulated by SIRT1.
DISCUSSION
Patients with COPD experience circadian disruption
(14, 17–20). Hence, CS exposure may affect circadian
clock function in the lung, leading to inflammatory and
injurious responses. We examined the putative mecha-
nisms of clock disruption in response to chronic envi-
ronmental CS exposure in lung and brain tissues in a
mouse model of COPD and emphysema. We deter-
mined the effect of COPD and emphysema on molec-
ular clock function in the lung. We show for the first
time that CS exposure alters circadian clock gene
expression in both the lungs and brain. Our results
show that both acute and chronic CS exposure signifi-
cantly reduced the amplitude of locomotor activity in
mice. This effect persisted even after CS exposure
ended, with locomotor activity levels not fully recover-
ing until 14–30 d after smoking cessation in the chronic-
exposure model. Despite this effect on locomotor
activity levels, the phase angle of entrainment and
consolidation of activity in the dark phase were not
disrupted following CS exposure. Further, acute CS
exposure had a small but significant effect on the
free-running period of wheel-running activity, suggest-
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-
ioral rhythms. This interpretation is strengthened by
our observation of altered clock gene expression in
whole brain following acute CS in vivo and the period
of PER2::LUC expression in SCN explants ex vivo.
Further, it is worth noting that the shortened period of
wheel-running after acute CS paralleled the effects of
CSE on SCN tissue explants. Together, these data
suggest that CS exposure affects both lung and brain
clock function. Although the effects of CS as an agent
of circadian disruption may not be as severe as the
effects of jet lag or shift work, it is reasonable to
speculate that even subtle alteration of clock function
can have a substantial long-term effect on clock-
dependent physiology. The differential and opposing
effects of CSE on the period and phase of clock gene
expression in the lung and SCN suggest that internal
circadian disruption due to phase dissociation between
these central and peripheral clocks is a factor in the
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
immune functions are governed, in part, by the circa-
dian timing system (41– 43). However, it is not known
to what extent circadian rhythms in the immune in-
flammatory system are modulated by timing cues deliv-
ered to this system from sources such as the SCN or are
regulated locally by peripheral clocks. Circadian desyn-
chrony or environmental circadian disruption during
experimental jet lag increases the susceptibility to in-
flammation through dysregulation of innate immune
responses (44). Consistent with other peripheral tis-
sues, circadian timing in the lung, although able to
oscillate independently (14 –16), is synchronized with
other tissues and the external environment by the
central clock in the SCN (1). We have shown that CS
exposure disrupts circadian expression of clock and
clock-controlled genes in the lung, resulting in altered
patterns of inflammatory cytokine gene expression and
secretion. It is possible that the effects of acute CS
exposure (10 d) in mice were amplified by the fact that
CS exposure was conducted during the active phase in
mice housed in an inverted L:D cycle (lights on from 6
PM to 6 AM). Because of the limitations of our facility,
it was not feasible to conduct long-term (chronic CS)
exposure studies in an inverted L:D cycle to confirm
this possibility. Hence, we do not see a strong correla-
tion in the effects of CS on clock gene expression
between the acute and chronic treatment groups. Al-
though certainly adequate, a 6-h time resolution is a
less than ideal sampling frequency for detection of
subtle variation in gene expression and protein levels
across the 24-h day. That said, we were able to detect
significant changes in the phase of several clock genes
after both acute and chronic CS exposure with that
sampling frequency. Studies designed to interrogate
subtle changes in clock gene expression following in
vivo CS exposure at more frequent sampling rates are
warranted. Similar effects of CS on circadian rhythms of
cardiac function and lung tumorigenesis have been
reported (45– 47). Given the parallel nature of the
responses, it is reasonable to define CS exposure as a
form of environmental circadian disruption, capable of
altering the molecular clock and clock-dependent pro-
cesses in a manner similar to repeated jet lag or
rotating shift work. These effects may be more adverse,
given that the disruption occurs in the presence of a
regular 12:12 L:D cycle and apparently normal entrain-
ment. Furthermore, it is possible that CS-induced pro-
inflammatory cytokines contribute to circadian disrup-
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-
tory responses (12, 51). In acute CS exposure, neutro-
phil influx was observed at ZT24, but not at ZT0, which
is in fact the same time point. However, neutrophil
influx in the lung usually increases 16 h after the last CS
exposure. Thus, we expected to see a delayed increase
in neutrophil influx. Earlier reports from our labora-
tory have clearly demonstrated an increase in proin-
flammatory cytokine release and neutrophil influx
after acute CS exposure at a concentration of 300
mg/m3 TPM, which was achieved with a mainstream CS
exposure system (Baumgartner-Jaeger CSM2072i, CH
Technologies; ref. 33). This response is not attainable
with the Teague smoke exposure system [a mixture of
sidestream (89%) and mainstream (11%) smoke] used
in the current study (⬃100 mg/m3 TPM). Further-
more, in our protocol, there was no influx of neutro-
phils into the BAL fluid with the use of the Teague
machine for CS exposure.
The role of clock proteins in increased susceptibility
to oxidative stress–mediated inflammatory and injuri-
ous responses is implicated. For example, PER2 mutant
mice are known to be sensitive to oxidative stress and to
be susceptible or prone to the development of cancer
(9). Recently, it was reported that BMAL1-deficient
mice have an increased sensitivity to oxidative stress
that correlates with cellular senescence (11). Although
the nuclear receptor REV-ERB␣, which is known as a
negative regulator of BMAL1, has been reported to
have a role in the regulation of inflammatory mediators
(41, 44, 52), evidence supporting the role of BMAL1 in
oxidative stress-induced inflammation remains elusive.
Cigarette smoking not only induces oxidative stress,
but is also associated with clinical depression (53, 54)
and sleep disorders (55). Patients with COPD have an
early morning surge in respiratory symptoms (cough, spu-
tum production, and wheezing) and sleep abnormalities
(13, 14, 17, 19, 20). Disrupted sleep in patients with
COPD correlates with respiratory symptoms (cough,
sputum production, and wheezing), nocturnal oxygen
desaturation, hypercapnia, and circadian changes in
airway caliber and resistance (20, 56). Each of these
effects of chronic tobacco/CS is closely related to a
significant deterioration in quality of life in smokers
and contributes to the development of COPD. In these
patients depression often lasts even after smoking ces-
sation (57) and is a frequent comorbidity of COPD
(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-
ties are not fully understood. In this study, both acute
and chronic CS exposure reduced nighttime activity in
mice. Surprisingly, chronically exposed mice were less
active only through 3 mo of CS exposure. Contrary to
our expectations, locomotor activity was similar in the
air- and CS-exposure groups after 6 mo. When we
compared the overall activity levels in mice from the
beginning (1st mo) of the experiment until completion
(6th mo), there was a clear age-dependent decline in
total activity levels. Hence, the overall reduction in
activity in aged mice may have masked the effects of
chronic CS exposure. Furthermore, it is possible that
the effects of chronic CS exposure on the brain are less
pronounced because of the habituation of neural re-
sponses to CS. Regardless, our data suggest that re-
duced sleep quality in patients with COPD is a direct
consequence of altered clock function in sleep-
regulatory centers of the brain, including the central
pacemaker in the SCN.
SIRT1 is an NAD⫹-dependent deacetylase involved in
regulation of various biological processes. The levels of
SIRT1 are reduced through protein carbonylation in
response to CS exposure in the lung (26, 62). A
reduction in the levels and activity of SIRT1 in response
to CS leads to lung inflammation, emphysema, and
senescence (27). Recently, it has been reported that
SIRT1 has an important role in the regulation of
circadian clock mechanisms by affecting circadian
clock gene transcription in an NAD⫹-dependent man-
ner (24, 25, 63). Another interesting report showed
that SIRT1 in the brain governs central circadian
control and activates transcription of the BMAL1:
CLOCK transactivator complex by amplifying the
SIRT1-PGC-1␣-Nampt axis (64). Altered SIRT1 levels in
the brain also exert moderate changes in the intrinsic
circadian periods of young and old mice (64). SIRT1
plays an essential role in the age-dependent decline in
central clock function, and targeting these circadian
regulated loops in the brain (SIRT1, PGC-1␣, and
Nampt) may provide novel strategies for ameliorating
the negative effects of aging on circadian clock func-
tion (64). SIRT1 affects the molecular clock by binding
with CLOCK:BMAL1 complexes and deacetylating the
BMAL1 and PER2 proteins (21–24). Thus, CS could
affect clock function by reducing SIRT1, leading to
increased inflammatory responses in the lung. Our
preliminary observations showed that PER2 levels,
which are CLOCK:BMAL1 dependent, are reduced in
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
by SIRT1 in lungs of mice exposed to CS. Our results
show that SIRT1 expression is rhythmic in the lungs of
air-exposed mice, which is consistent with a previous
report (24). CS significantly reduced SIRT1 protein
abundance, concomitant with a reduction in BMAL1
levels, and increased acetylation on lysine residue K537
of the remaining BMAL1 protein. These results were
confirmed in the lungs of SIRT1-deficient and SIRT1-
overexpressing mice that were exposed to CS and
paralleled a report showing that acetylation of BMAL1
on lysine residue K537 is regulated by SIRT1 (25). We
found a modest decrease in BMAL1 levels in lung tissue
from patients with COPD, although it was still signifi-
cant when compared with that in lung tissue of non-
smokers. We also observed a considerable variation in
the expression levels of BMAL1 in the lungs of patients
with COPD, compared to those of nonsmokers, which is
not unexpected, given a large variation in the intensi-
ties and grades of COPD severity (GOLD stages I–IV),
timing of sample collections, the duration and amount
of cigarette inhalation and exposure, and the possible
influence of prescribed drugs, which varies greatly
between patients. To our knowledge, this is the first
study showing that SIRT1 is expressed with a circadian
rhythm under clock control in lung tissue. Further, we
report for the first time that BMAL1 acetylation in the
lung in vivo is due to a CS-dependent reduction in
SIRT1 levels. There is emerging evidence that circadian
rhythms govern proinflammatory cytokine gene expres-
sion (41, 44, 50). It is also known that inflammation
and alteration in clock gene expression (BMAL1 and
PER2) are intimately associated with fatigue and dimin-
ished locomotor activity (65– 67). Hence, it is possible
that SIRT1 acts as a critical link between core clock
function and inflammatory response in the lung.
BMAL1, as part of the primary enhancer complex with
CLOCK, has a pivotal role in regulation of circadian clock
function through transactivation of clock components
and downstream clock-controlled genes. Acetylation is
linked to BMAL1 phosphorylation (25, 67, 68), possibly
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
CS exposure induced increased acetylation of BMAL1
and its loss of stability. Intriguingly, as shown in a previous
study (25), we observed that the level of BMAL1 was
greater in the lungs of SIRT1-deficient mice, suggesting
the SIRT1 is involved in BMAL1 stability. Further study is
needed to investigate SIRT1-regulated mechanisms in-
volved in BMAL1 stability, shuttling, phosphorylation, and
acetylation in response to CS exposure and in the devel-
opment of COPD.
BMAL1-deficient mice show signs of advanced aging
and an age-related phenotype, correlating with increased
levels of ROS and cellular senescence (69). CS exposure
reduced the levels of BMAL1 mRNA and protein, and
simultaneously evoked lung inflammatory responses, sug-
gesting that the loss of BMAL1 in response to CS exposure
increases inflammatory responses through deregulation
of CS-induced oxidative stress. It has been shown that
BMAL1 interacts with the CCL1/MCP1 promoter, en-
hancing CCL1/MCP1 gene expression. We found a sharp
surge of CCL1/MCP1 in the lungs of CS-exposed WT
mice, which was augmented in the lungs of CS-exposed
mice harboring lung epithelium-specific deletion of
bmal1. Although the specific mechanisms remain un-
known, it is likely that BMAL1 has a role in the regulation
of oxidative stress and could be a target of SIRT1 in
oxidative stress–induced inflammatory responses. A re-
cent study showed a similar pharmacologic activation of
SIRT1 in modulation of circadian rhythms in liver via a
reduction in H3K9/K14 acetylation (70). Furthermore,
augmented inflammatory responses observed in CS-ex-
posed mice harboring a lung epithelium–specific bmal1
deletion compared to WT mice suggest the involvement
of the epithelial molecular clock in regulation of CS-
induced lung inflammation.
In conclusion, our data show that environmental CS
exposure in mice caused alterations in circadian clock
gene expression in brain and lung tissue, altered rhythms
of locomotor activity, and increased lung inflammation
and emphysema. BMAL1 levels were reduced in lung
tissue from patients with COPD, most likely owing to
increased turnover mediated by enhanced acetylation of
BMAL1 by SIRT1. These data clearly indicate that circa-
dian rhythms of lung function are dampened in patients
with COPD, which is associated with abnormal airway
inflammation. We also report that BMAL1 regulated
CS-induced lung inflammatory responses through SIRT1-
controlled acetylation and stability of BMAL1 in lung
epithelium (Fig. 13). Thus, our results highlight the
importance of the molecular clock in the regulation of
lung inflammation and injurious responses caused by CS.
Overall, CS-mediated disruption of circadian clock func-
tion has implications in the pathogenesis of COPD. Un-
derstanding molecular clock function in the lung and its
association with daily lung physiological function, partic-
ularly as it relates to the response to tobacco/CS, may
strengthen the rationale for chronotherapy in COPD
management.
The authors thank Dr. Thomas J. Mariani (University of
Rochester, Rochester, NY) for providing the Cre recombinase
transgenic mice with CC10 promoter, Dr. Michael McBurney
(University of Ottawa, Ottawa, ON, Canada) for providing
SIRT1-knockout mice (SIRT1-deficient mice), Dr. Leonard
Guarente (Massachusetts Institutes of Technology, Cam-
bridge, MA, USA) and Dr. Wei Gu (Columbia University, New
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
Bachmann, Lindsay Marchetti, Zachary Murphy, Drew Phil-
lips, Suzanne Bellanca, and Stephanie Uhrinek (University of
Rochester, Rochester, NY, USA) for technical assistance. This
study was supported by grants from the U.S. National Insti-
tutes of Health (1R01HL097751, 1R01HL092842) to I.R. and
a grant from the National Institute of Environmental Health
Sciences (NIEHS) Environmental Health Science Center
(P30-ES01247). The authors declare no conflicts of interest.
Author contributions: J.H. performed mouse studies, cell
counts, immunoblotting, real-time PCR analysis, ELISA, and
manuscript writing; I.S.K. performed cell counts, real-time
PCR analysis, ELISA, mean linear intercept (Lm) analysis,
immunoblotting, immunoprecipitation, and overall data col-
lection and analysis; J.H. and I.S.K. performed: data analysis
for circadian experiments and cell counts; H.Y., I.S.K., M.T.S.,
and I.R. performed: interpretation of results and manuscript
editing; M.T.S. performed wheel running activity assay and,
PER2::luciferase assays; I.R. performed study design and
manuscript writing.
Dedication: The authors dedicate this work to the loving
memory of Dr. Vuokko L. Kinnula (Pulmonary Division,
Department of Medicine and Pathology, University of Hel-
sinki and Helsinki University Hospital, Helsinki, Finland),
who provided the human tissue samples and left us suddenly
during the preparation of this article.
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Received for publication April 18, 2013.
Accepted for publication August 26, 2013.
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