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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.
(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.
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).
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
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
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
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
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.
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
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.
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
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-
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-
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
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
(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
Author contributions: J.H. performed mouse studies, cell 6. Boysen, P. G., Block, A. J., Wynne, J. W., Hunt, L. A., and Flick,
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for circadian experiments and cell counts; H.Y., I.S.K., M.T.S., (2011) Light-dark oscillations in the lung transcriptome: impli-
and I.R. performed: interpretation of results and manuscript cations for lung homeostasis, repair, metabolism, disease, and
editing; M.T.S. performed wheel running activity assay and, drug action. J. Appl. Physiol. 110, 1732–1747
PER2::luciferase assays; I.R. performed study design and 9. Fu, L., Pelicano, H., Liu, J., Huang, P., and Lee, C. (2002) The
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10. Zheng, X., Yang, Z., Yue, Z., Alvarez, J. D., and Sehgal, A. (2007)
Dedication: The authors dedicate this work to the loving FOXO and insulin signaling regulate sensitivity of the circadian
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
senescence in vivo. Cell Cycle 10, 4162–4169
during the preparation of this article. 12. Yao, H., and Rahman, I. (2011) Current concepts on oxidative/
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