Am J Physiol Lung Cell Mol Physiol 309: L1056–L1075, 2015.
First published September 11, 2015; doi:10.1152/ajplung.00152.2015.
Review
Circadian molecular clock in lung pathophysiology
Isaac K. Sundar,1 Hongwei Yao,1 Michael T. Sellix,2 and Irfan Rahman1
1
Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center,
Rochester, New York; and 2Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of
Rochester Medical Center, Rochester, New York
Submitted 12 May 2015; accepted in final form 8 September 2015
PER2; BMAL1; REV-ERB␣; ROR␣; biomarkers; asthma; COPD; fibrosis
disease (COPD) and asthma
are major health concerns worldwide with considerable and
well-documented influence on quality of life and mortality (60,
153). Patients with airway diseases including COPD and
asthma develop more frequent and severe exacerbation with an
increased rate of emergency room visits and hospitalization,
mostly at night and in the early morning hours when lung
function is lowest (119, 149, 182, 184, 186). Exacerbations
lead to deterioration of the disease state and are associated with
a rapid decline in lung function (119, 148, 149, 182, 186).
Environmental exposomes (measure of all exposures) or individual exposure, such as by air pollutants/particulates, allergens/pollens, tobacco/cigarette smoke (CS), as well as respiratory viral (influenza and rhinoviruses) and bacterial infections can lead to exacerbations of COPD and asthma (149,
184 –186). The circadian timing system drives daily changes of
airway caliber, airway resistance, respiratory symptoms, mucus hypersecretion and immune-inflammatory responses that
CHRONIC OBSTRUCTIVE PULMONARY
Address for reprint requests and other correspondence: I. Rahman, Dept. of Environmental Medicine, Univ. of Rochester Medical Center, Box 850, 601 Elmwood
Ave., Rochester, NY 14642 (e-mail: [email protected]).
L1056
underlie this daily variation in exacerbation frequency and
occurrence. The effect of virus-induced COPD/asthma exacerbations on clock function in the lung and/or the role of the
lung’s clock in the pathogenesis of COPD/asthma and its
associated exacerbations are largely unknown. Nonetheless,
there is a connection between the circadian timing system and
the decline in lung function/exacerbations common to both
COPD and asthma. Recently, we have defined the role of the
molecular clock in regulation of lung cellular and molecular
functions, particularly in response to environmental insults [CS
and influenza A virus (IAV)] (77, 177, 178, 210). However, the
basic role of the molecular clock and the impact of circadian
disruption in various lung pathophysiological conditions, the
use of clock molecules as biomarkers for pulmonary dysfunction, and the potential for novel chronopharmacological approaches for the treatment of lung disease have yet to be
described.
Despite numerous therapeutic advancements, the strength of
pharmacotherapy for COPD continues to be reliance on corticosteroids and/or bronchodilators (e.g., ␤-adrenoreceptor agonists) that do little to reduce mortality, inflammation, or the
daily decline in lung function in patients with COPD and in
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Sundar IK, Yao H, Sellix MT, Rahman I. Circadian molecular clock in lung
pathophysiology. Am J Physiol Lung Cell Mol Physiol 309: L1056 –L1075, 2015.
First published September 11, 2015; doi:10.1152/ajplung.00152.2015.—Disrupted
daily or circadian rhythms of lung function and inflammatory responses are
common features of chronic airway diseases. At the molecular level these circadian
rhythms depend on the activity of an autoregulatory feedback loop oscillator of
clock gene transcription factors, including the BMAL1:CLOCK activator complex
and the repressors PERIOD and CRYPTOCHROME. The key nuclear receptors
and transcription factors REV-ERB␣ and ROR␣ regulate Bmal1 expression and
provide stability to the oscillator. Circadian clock dysfunction is implicated in both
immune and inflammatory responses to environmental, inflammatory, and infectious agents. Molecular clock function is altered by exposomes, tobacco smoke,
lipopolysaccharide, hyperoxia, allergens, bleomycin, as well as bacterial and viral
infections. The deacetylase Sirtuin 1 (SIRT1) regulates the timing of the clock
through acetylation of BMAL1 and PER2 and controls the clock-dependent
functions, which can also be affected by environmental stressors. Environmental
agents and redox modulation may alter the levels of REV-ERB␣ and ROR␣ in lung
tissue in association with a heightened DNA damage response, cellular senescence,
and inflammation. A reciprocal relationship exists between the molecular clock and
immune/inflammatory responses in the lungs. Molecular clock function in lung
cells may be used as a biomarker of disease severity and exacerbations or for
assessing the efficacy of chronotherapy for disease management. Here, we provide
a comprehensive overview of clock-controlled cellular and molecular functions in
the lungs and highlight the repercussions of clock disruption on the pathophysiology of chronic airway diseases and their exacerbations. Furthermore, we highlight
the potential for the molecular clock as a novel chronopharmacological target for
the management of lung pathophysiology.
Review
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CIRCADIAN CLOCK IN THE LUNG
Table 1. Role of molecular clock in lung physiology
Circadian Clock Studies
Role of the timing system in rhythmic
respiratory function
Role of bronchiolar epithelial cells in
lung clock function
Experimental Design
Major Outcomes/Findings
⫺/⫺
C57BL/6 and Cry1,2
mice. Cervical vagotomy
and sympathectomy were performed. Bilateral
electrolytic lesion of the SCN (DD and LD
12:12).
C57BL/6J and PER2::LUC transgenic mice ex vivo.
Lung explants cultured from PER2::LUC mice.
Primary culture of mouse Clara/club cells. (LD
12:12).
Molecular link between the circadian
clock and xenobiotic detoxification
WT and clock mutant (Clk/Clk) mice injected with
benzo[␣]pyrene at 09:00 or 21:00. (LD 12:12).
Diurnal oscillations in the lung
transcriptome
Male Wistar rats (LD 12:12).
9
58
180
173
AhR, aryl hydrocarbon receptor; CRY, cryptochrome; DD, dark-dark; LD, light-dark; SCN, suprachiasmatic nucleus; WT, wild-type.
patients with severe asthma who smoke. A considerable number of recent studies support the fact that other environmental
factors and exposomes, including air pollutants/particulates
(151), xenobiotic detoxification (147, 180), CS (54, 77, 177,
192), shift work (107, 173, 198), jet lag (42, 65), hypoxia/
hyperoxia (100, 203), ventilator-induced lung injury (106), and
pathogens (bacteria/virus) (57, 174) can disturb molecular
clock function in the lungs and hasten the development of lung
pathophysiology (Tables 1 and 2). One can conclude from
these findings that perturbation of clock function is responsible
for changes in various cellular and molecular lung functions
during the pathogenesis of chronic airway disease. This concept was recently the focus of an NHLBI workshop (http://
www.nhlbi.nih.gov/research/reports/2014-circadian-clocklung-health), and our own brief translational perspective on
molecule clock function in airway diseases (178). Thus there is
a growing interest within the lung biology community regarding the mechanism whereby lung physiology and pathology are
influenced by circadian clock function.
The development of novel small molecule activators or
inhibitors of clock function has stimulated interest in the
potential clinical application of chronopharmacology. Chronopharmacology refers to the use of small molecules to target the
timing and amplitude of clock or clock-dependent gene expression for treating disease (41). In this review, we have amalgamated the findings in chronobiology and cell and molecular
biology of lung pathophysiology and merged them in light of
emerging details regarding the regulation of clock function by
environmental stressors and and/or the molecular clocks role in
the development of chronic airway disease. We propose that
the levels of molecular clock components in cells from systemic circulation and/or sputum may be useful as novel biomarkers of disease severity and exacerbations and for assessing
the viability and effectiveness of circadian-based therapy for
disease management. Finally, we elucidate the potential therapeutic targets and approaches of interest for the management
of chronic lung diseases using chronotherapy.
The Circadian Timing System and Pulmonary Physiology
Circadian rhythms are intrinsic biological oscillations with a
period near 24 h driven in mammals by the circadian timing
system (3). Circadian rhythms are generated at the cellular
level by an autoregulatory feedback loop of interlocked transcription factors referred to collectively as clock genes (124)
(Fig. 1). In mammals, the BMAL1:CLOCK activator complex
regulates expression of period (Per1-3) and cryptochrome
(Cry1-2) genes. PER and CRY form heterodimers, are phosphorylated, and translocate back to the nucleus, where they
repress their own transcription by blocking the activity of the
BMAL1:CLOCK complex (64). The counteracting key nuclear
receptors REV-ERB␣ (Nr1d1; nuclear receptor subfamily 1,
group D, member 1) and retinoic acid-related orphan receptor-␣ (ROR␣) provide stability to the oscillator by regulating
the timing and amplitude of Bmal1 expression. Molecular
clock proteins are heavily influenced by posttranslational modifications, including acetylation and phosphorylation, which
affect both their activity and stability (73, 129). The detailed
molecular mechanisms of each modification and their impacts
on clock-dependent cellular functions in peripheral organs,
including the lungs, are largely unknown.
The central pacemaker of the timing system is located within
the suprachiasmatic nucleus (SCN) of the basal hypothalamus,
though peripheral tissues including the lung are also comprised
of cell autonomous oscillators (3, 9). The circadian clock in the
lung plays a critical role in optimizing the organization of
cellular function and responses to environmental stimuli (103,
156). Any significant change in the timing or amplitude of
clock gene expression, commonly referred to as circadian
disruption, can affect downstream clock-controlled output
genes or physiological processes and has been implicated in
chronic disease (50, 112). In addition to clock disruption at the
tissue level, desynchrony between central and peripheral oscillators is known to increase the risk of metabolic, endocrine,
and cardiovascular disease. In healthy individuals, circadian
misalignment results from an acute change in the phase of
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Examined the timing system in the respiratory
tract and the role of acetylcholine
neurotransmission in rhythms of respiratory
function.
Clara/club cells are critical for maintaining
clock function in lung; coexpression of the
glucocorticoid receptor and clock proteins
establishes a likely interface between SCN
output and lung physiology.
CLOCK protein affects the expression of
detoxification enzymes through modulating
the activity of AhR.
Genes involved in the metabolism and
transport of endogenous compounds,
xenobiotics, and therapeutic drugs; other
biomarkers or potential therapeutic targets
genes for lung disease exhibit diurnal
oscillations, suggesting clock control of
lung physiology and pathophysiology.
Reference
Review
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CIRCADIAN CLOCK IN THE LUNG
Table 2. Role of molecular clock in lung pathology by environmental agents using animal models
Circadian Clock Disruption
Models/Agents
Cigarette smoke (CS)
Experimental Design
· Sprague-Dawley rats exposed for 2, 7, and 13 wk
(LD 12:12).
· C57BL/6J and AJ (male) mice (LD 12:12) exposed
to whole smoke for a period of 10 days.
· C57BL/6J and Bmal1⫺/⫺ mice were given a single
intraperitoneal LPS (12 mg/kg) (LD 12:12; LL
12:12 and DD 12:12).
Bleomycin
Chronic jet lag models (Chronic shiftlag/simulated jet lag/chronic phase
advance)
· C57BL/6J, Nrf2⫺/⫺, PER2⬋Luc and Clock⌬19
mice intratracheally instilled with bleomycin (1
mg/kg) or vehicle at either ZT0 or ZT12. Rescue
studies, sulforaphane (10 mg/kg ip) or vehicle
administered at ZT6 prior to a bleomycin
challenge at ZT12 and every other day for 7 days.
(LD 12:12 and DD 12:12).
· Male Fischer (F344) rats were exposed to LD 12:
12 or a chronic shift-lag (10 repeated 6-h photic
advances occurring every 2 days, followed by 5–7
days of constant darkness).
· C57BL/6J female and male mice were maintained
on a LD (12:12) control or exposed for 4 wk to a
shifting light regimen (SJL).
· Chronic phase advance (SJL) protocol involved 6-h
phase advances (earlier light onset) every 4 days
for 8 wk. (LD 12:12 and DD 12:12) in WT and
Bmal1⫺/⫺, Per2::Luc mice.
Hyperoxia
· WT and NALP3⫺/⫺ mice were exposed to room
air or hyperoxia for 24, 48, or 72 h. Different
concentrations of O2 were used for hyperoxia (50,
75, or 100%) or room air for 72 h.
· Neonatal (⬍12-h-old) or adult mice (2-month-old)
WT and p50⫺/⫺ mice exposed to either 21% O2
or room air or 95% O2 for 72 h.
Ventilator-induced lung injury (VILI)
· Sprague-Dawley rats endotracheally intubated and
placing on a mechanical ventilator (tidal volume
of 40 ml/kg or 10 ml/kg without positive endexpiratory pressure).
Cigarette smoke ⫹ Influenza A virus
(IAV)
· C57BL/6J exposed to chronic air or CS (6 mo),
followed by IAV infection (postinfection days
1–9) and WT littermates and Bmal1⫺/⫺ mice
infected with IAV (postinfection days 1–9). (LD
12:12).
Citation
54
· Genetic ablation of the clock gene Bmal1 (also called
Arntl or MOP3) in bronchiolar cells disrupts
rhythmic Cxcl5 expression demonstrating circadian
clock role in host defense to bacterial infection and
pulmonary inflammation.
· Genome-wide approach to investigate alterations in
clock gene expression during LPS-induced lung
inflammation produces a complex reorganization of
cellular and molecular circadian rhythms
(reprogramming) that are important in early events
that occur during lung injury.
· Circadian clock as an endogenous regulatory
mechanism controlling the rhythmic activity of the
redox-sensitive transcription factor Nrf2/glutathionemediated antioxidant defense pathway.
· Chronic shift-mediated circadian disruption promotes
tumorigenesis, alters gene expression of Per2 and
Bmal1 and cytolytic factors that correlate with
suppressed circadian expression of NK cytolytic
activity.
· Circadian disruption alters lung mechanics by shifting
light regimen (SJL) and clock gene expression in a
sexually dimorphic manner.
· Chronic phase advance alters physiological rhythms
and peripheral molecular clocks; SJL advanced the
phase of the rhythm immediately after a shift and
mice placed into free-running conditions (DD) for 2
wk after SJL also showed molecular clock shifted in
the lung.
· Alterations in clock genes are associated with
increased inflammatory markers in bronchoalveolar
lavage fluid of hyperoxic mice and a dose-dependent
increase in clock gene expression with increased O2
concentrations.
· Clock genes are regulated by oxidative stress and
inflammation in the neonatal lung hyperoxia model;
REV-ERB␣ play an essential role in lung cellular
function and injury
· Rev-erb␣ mRNA and protein is decreased in high tidal
volume mechanical ventilation group compared with
spontaneous group; treatment with REV-ERB␣
agonists greatly diminished VILI-induced lung
edema, inflammatory cell infiltration and
proinflammatory cytokine (TNF-␣) release.
· Chronic CS exposure combined with IAV infection
altered the timing of clock gene expression, reduced
locomotor activity along with increased lung
inflammation, emphysema, and disrupted rhythms of
lung function; BMAL1 KO mice infected with IAV
showed pronounced detriments in behavior, survival,
lung inflammatory and profibrotic responses in vivo.
192
77
177
57
70
147
107
65
198
100
203
106
174
CS, cigarette smoke; CT, circadian time; CPA, chronic phase advance; CRY, cryptochrome; DD, dark-dark; HEPA, high-efficiency particulate air; IAV,
influenza A virus; LD, light-dark; Nr1d1, nuclear receptor subfamily 1, group D; NK cells, natural killer cells; PER, period; SJL, simulated jet lag; TPM, total
particulate matter; VILI, ventilator-induced lung injury; ZT, zeitgeber time.
light-exposure (photoperiod), dissociating internal timing from
the external environment (e.g., “jet lag” or shift work) (198).
Circadian misalignment influences physiology through its impact on internal circadian organization, defined as the coordinated timing of clock-dependent cell and molecular functions.
Internal desynchrony occurs as a result of the differential
resetting or “shifting” rates (also called entrainment kinetics)
of central and peripheral oscillators (50). Although the malaise
of jet lag is generally considered a minor inconvenience, it has
been shown that a single 6-h advance repeated over 8 wk can
increase mortality in older mice (43). Mice exposed to non24-h days for short duration (20-h day; ⬃10 wk), a treatment
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Lipopolysaccharide (LPS)
· C57BL/6J, Bmal1 Cre-CC10 knockout, Sirt1⫹/⫺
and Sirt1 transgenic mice exposed to acute (3
days and 10 days) or chronic CS (6 mo) TPM:
100–300 mg/m3). Inverted LD 12:12 for acute 10
days and regular LD 12:12 for 3 days and 6 mo.
· C57BL/6J mice exposed to acute (3 days and 10
days) or chronic CS (6 mo) (TPM: 100–300 mg/
m3). Inverted LD 12:12 for acute 10 days and
regular LD 12:12 for 3 days and 6 mo.
· Conditional club cell Bmal1-knockout mice (CcspBmal1⫺/⫺) and LysM-Bmal1⫺/⫺ mice were
treated with intraperitoneal LPS (1 mg/kg) at
CT0. (LD 12:12 and DD 12:12).
Major Outcomes/Findings
· Transcriptomic changes in specific program of defense
(phase I and II detoxifying), innate and adaptive
immune, inflammation and circadian clock gene
expression.
· Nr1d1 gene expression is suppressed by CS, which
may affect clock function and play a complementary
role in CS-induced lung respiratory tract
pathobiology and/or lung tumorigenesis.
· CS exposure alters clock gene expression and reduced
locomotor activity by disrupting central and
peripheral clocks and augments lung inflammation,
leading to COPD/emphysema via the SIRT1-BMAL1
pathway.
· CS exposure affected both the timing and amplitude
of the daily rhythms of plasma corticosterone and
serotonin (stress hormones).
Review
CIRCADIAN CLOCK IN THE LUNG
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that mimics the effects of a daily 4 h advance, exhibit features
of metabolic, endocrine, and neurophysiological disease (82).
Moreover, human subjects exposed to similar non-24-h days
for short duration show a dramatic decline in metabolic,
endocrine, and autonomic nervous function (22). Notably,
internal desynchrony may also arise in response to other
environmental exposomes or physiological perturbations (e.g.,
CS exposure) independent of changes in the photoperiod and
may be a major factor in the etiology of clock-dependent
pathophysiology (50, 74, 158, 198). Although evidence has
accumulated for significant negative effects of circadian misalignment on metabolism, immune function, cognition, and
fertility, similar evidence for an impact on pulmonary physiology or lung pathophysiology has yet to be acquired.
Rhythms of clock gene expression have been reported in the
lungs (138, 139), including bronchial epithelial cells (58). Emerging studies show the role of clock dysfunction in pulmonary
physiology and pathology, particularly in response to proinflammatory mediators like CS. The timing and amplitude of clock
genes and putative clock-controlled gene (CCG) expression in
rodent lungs is altered by exposure to CS (54) and proinflammatory mediators like LPS and TNF-␣ (29). The magnitude of phasic
responses to proinflammatory factors can be quite significant.
Data have shown that neutrophil influx recorded during LPSmediated lung inflammation is three- to sixfold higher in Ccsp-
Bmal1⫺/⫺ mice (which specifically lack Bmal1 in the epithelial/
club cells) compared with Bmal1fl/fl littermate controls, resulting
in a complete loss of rhythmicity (57). This increased neutrophil
response was associated with a loss of barrier function as confirmed by elevated IgM levels in bronchoalveolar lavage (BAL)
fluid and increased CXCL5 expression in phase with the peak of
LPS-induced neutrophil influx. CXCL5 in BAL fluid of LPStreated Ccsp-Bmal1⫺/⫺ mice was also two- to threefold higher
compared with Bmal1fl/fl controls at both CT0 and CT12 (CT;
circadian time, subjective time base relative to the activity period
such that CT12⫽activity onset) (57). These findings are in agreement with early reports revealing that LPS-induced serum cytokines [IL-6, IL-12(p40), CXCL1, CCL5, and CCL2] measured at
either CT0 or CT12 showed significant time-dependent variation
in response magnitude (59). Similarly, CS-induced lung inflammation in lung epithelial cell-specific BMAL1 knockout (KO)
displayed a two- to threefold increase in proinflammatory cytokine gene expression (ccl1/mcp1 and cxcl1/kc) and cytokine
release (MCP-1 and KC) compared with air exposed controls
(ZT0 vs. ZT12; ZT, zeitgeber time, objective time base relative to
the 12:12 L:D cycle such that ZT12⫽lights off) (77). We recently
observed similar effects on proinflammatory MCP-1 release (twofold increase; ZT6/12 vs. ZT18/24) in chronic CS-exposed mice
infected with influenza virus compared with air-exposed mice
infected with influenza virus (174). We have also recently re-
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Fig. 1. Schematic representation of the core clock mechanism and rhythmic output of key target genes that regulate the molecular clock function in the lung.
The molecular clock in mammals is composed of a heterodimer of the transcription factors CLOCK and BMAL1, which regulates the transcription of Per (Period)
and Cry (Cryptochrome) via E-box sequences within their promoters. PER and CRY heterodimerize and block E-box-dependent transcriptional activity of the
CLOCK:BMAL1 complex. The timing of BMAL1 expression depends on the counteracting activity of REV-ERBs and ROR (retinoic acid receptor-related
orphan receptor) proteins. CHRONO negatively regulates circadian oscillations by directly interacting with BMAL1. Environmental agents influence lung cells
(e.g., epithelial cells) and macrophages through activation of kinases (MAP kinase, casein kinase2, CKII, glycogen synthase kinase GSK3 ␤, protein kinase C,
and PI-3 kinase) that lead to posttranslational modification (phosphorylation/acetylation) of molecular clock proteins (e.g., BMAL1 and PER2). Molecular clock
dysfunction negatively affects lung functions and may be a significant factor in the pathobiology of chronic airway diseases. CKIε/␦, casein kinase 1 ε/␦;
CHRONO, ChIP-derived repressor of network oscillator; CLOCK, circadian locomotor output cycles kaput; CCG, clock controlled genes; CRY 1,2,
cryptochrome 1,2; REV-ERB␣, nuclear receptor subfamily 1, group D, member 1; PER1,2, period 1,2; ROR␣, retinoic acid receptor-related orphan
receptor ␣.
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CIRCADIAN CLOCK IN THE LUNG
Chronobiology of Lung Pathophysiology and Inflammation
Though circadian rhythms of lung function in healthy individuals have been documented, the data remain somewhat
controversial and the majority of evidence actually stems from
the study of lung function in asthmatic patients (23). More
pronounced troughs at night in forced vital capacity, forced
expiratory volume in 1 s (FEV1), and peak expiratory flow are
found in smokers than in nonsmokers (16, 27, 152). This could
be due to oxidative/carbonyl stress imposed by CS stressdependent effects on rhythms of surfactant protein levels,
mucus retention/secretion, and lung inflammation (87). The
early morning deterioration in pulmonary function observed in
asthmatic patients is associated with decreased serum epinephrine levels, increased vagal nerve tone and cholinergic activity,
esophageal reflux, and increased circulating eosinophils due to
time-of-the-day response (23). It has also been reported that
airway resistance peaks in the morning and reaches its nadir at
noon, followed by an increase in the afternoon (75). This
pattern is very similar in healthy individuals and also in
patients with restrictive and stable obstructive disease (75).
In addition to altered patterns of lung function, patients with
COPD or asthma also have poor sleep quality, increased sleep
latency, decreased total sleep time, increased waking after
sleep onset, and decreased rapid eye movement and non-rapid
eye movement sleep episodes (104, 144). These individuals are
also commonly diagnosed with sleep disorders including obstructive sleep apnea (113). Psychological distress in those
with COPD/asthma is associated with a decline in lung function, increased exacerbation frequency, and worsening of cardiovascular disease, further disrupting sleep in these patients
(114). Insomnia, excessive daytime sleepiness, and nocturnal
oxygen desaturation are also common in patients with COPD/
asthma. Disrupted sleep in patients with COPD/asthma correlates with respiratory symptoms (cough, sputum production,
wheezing), nocturnal oxygen desaturation, hypercapnia, and
circadian changes in airway caliber and resistance (105). A
recent report revealed that disruption of circadian molecular
clock function following bacterial endotoxin challenge alters
the innate immune response without marked sleep loss, suggesting that the effects of circadian disruption on immune
function are independent of their impact on sleep/wake
rhythms (150). Similarly, changes in the timing and amplitude
of lung function also occur in nocturnal asthmatic patients.
Importantly, there is evidence that steroids and bronchodilators
may be less potent at night, when the circadian nadir in FEV1
renders these patients most likely to experience an exacerbation (18, 52, 115, 142). Furthermore, corticosteroids and/or
bronchodilators (e.g., ␤-adrenoreceptor agonists), the mainstays of pharmacotherapy for COPD, fail to reduce mortality or
inflammation or to improve the rhythmic decline of lung
function in patients with COPD (24, 25). These results highlight the need for development of novel strategies based on
circadian molecular clock for the treatment of these pulmonary
disorders that account for the rhythmic variation of lung
function across the 24-h day.
Circadian Disruption and Redox Regulation by Oxidants
The impact of molecular clock dysfunction on lung inflammatory responses via redox cellular changes has been investigated by using mice with loss-of-function clock gene mutations. Antioxidant redox-sensitive transcription factor Nrf2
activity in the lungs was altered in mice expressing a dominant
negative mutation of the CLOCK protein (Clock⌬19). This
effect of the clock⌬19 mutation on Nrf2 is complemented by
reduced glutathione levels, increased protein oxidation, and a
spontaneous fibrotic-like phenotype (147). In the same study,
rhythms of Nrf2 levels/activity in Nrf2/GSH pathway were
demonstrated in vivo in mouse lungs (147). Recently, it was
revealed that dioxin-mediated activation of the aryl-hydrocarbon receptor (Ahr) signaling pathway is influenced by
CLOCK, suggesting clock-dependent regulation of phase I
detoxifying genes (159, 180). Clock mutant mice, which ex-
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ported that lung function varies significantly during time of day
(day vs. night) among chronic air, CS, and their combination with
virus-infected mice (77, 174). Together, these results highlight the
involvement of a local/peripheral clock in lung cells of both
mouse and humans that can both be modified by inflammation
and regulate the timing of inflammatory responses. Accumulating
evidence also suggests that circadian disruption has a profound
influence on pulmonary function and lung pathophysiology, particularly in lung epithelium (50, 57, 58, 65, 70, 77, 79, 147, 174).
Clock function in lung tissue is clearly altered by CS stress both
in vivo and in vitro. Furthermore, using targeted deletion with
conditional KO mice, these studies have begun to define the
functional relationship between the molecular clock and pulmonary physiology, particularly as it relates to airway disease produced by environmental factors.
Normally, pulmonary function exhibits a robust circadian
rhythm, with a noon maximum (12:00 h) and an early morning
minimum (04:00 h) in healthy individuals. The early morning
surge in lung function coincides with exacerbations of COPD/
asthma in susceptible individuals (11). Other symptoms of
airway diseases also vary as a function of time (11). These
symptoms include increased hoarseness in the morning hours
and increased wheezing in the middle of the night. Though the
mechanisms behind these daily variations in symptomology are
complex, it has been speculated that autonomic input to airway
tissues via the vagal nerve plays an essential role (127). The
autonomic nervous system communicates timing signals from
the SCN to peripheral oscillators via two routes: 1) sympathetic
(e.g., intermediolateral cell column) and 2) parasympathetic
(e.g., dorsal motor nucleus of the vagus and the region nearby
the nucleus ambiguous) innervation (17). Bando et al. (9) for
the first time showed that vagal nerves convey rhythmic signals
to the respiratory clock in airway tissues (i.e., larynx, trachea,
bronchus, and lung). These oscillations were abolished in
arrhythmic Cry1⫺/⫺Cry2⫺/⫺ KO mice and also in wild-type
mice after electrolytic lesion of the SCN (9). Hemivagotomy
completely suppressed clock gene expression in the (operated)
ipsilateral submucosal glandular cells but only moderately
suppressed clock gene expression in epithelial cells. In contrast, clock gene expression was not affected after sympathectomy. Since SCN lesions abolish respiratory rhythms it is
presumed that the vagal nerve is the dominant route whereby
circadian timing signals are conveyed from SCN to the respiratory tract. These findings support the assertion that cells
involved in pulmonary system contain a functional clock that is
influenced by the SCN (9) but may regulate cell-specific
function semi-independently of central timing cues (Table 1).
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CIRCADIAN CLOCK IN THE LUNG
Circadian Disruption and Its Influence on Inflammatory and
Immune Responses in the Lungs
Immune-inflammatory parameters fluctuate across the 24-h
day, and disturbance of these rhythms has been associated with
infectious and inflammatory diseases (30, 38). Recent evidence
suggest that the molecular clock regulates fundamental aspects
of the immune-inflammatory response (38), including Toll-like
receptor 9 (TLR9) signaling and repressing chemokine (C-C
motif) ligand 2 (CCL2) expression (163, 166). The clock
protein and nuclear heme receptor REV-ERB␣ attenuates the
activation of IL-6 expression (59). REV-ERB␣ binds to nuclear factor-␬B (NF-␬B) RelA/p65 and can activate NF-␬Bdependent transcription (172). Transcription factors activator
protein 1 (AP-1) and NF-␬B share unique motifs that overlap
consensus sequences from the REV-ERB␣ promoter, suggesting a role for REV-ERB␣ in regulation of oxidative stress and
inflammation (203). REV-ERB␣ regulates clock-output genes
by binding to and recruiting NCoR and HDAC3 to the promoter region of target inflammatory genes. BMAL1 KO ani-
mals display upregulation of the prostaglandin synthase gene
(ptgs2), TNF-␣, and IL-6, suggesting that molecular clock
disruption can lead directly to inflammatory responses (126).
Targeted deletion of Bmal1 in myeloid cells enhances the
number of specific monocytes (Ly6Chi) in the spleen and
circulation during peritoneal inflammation in a time-dependent
manner (134). This study also revealed that BMAL1 binds to
E-boxes in the promoters of Ccl2, Ccl8, and S100a8 (encoding
S100 calcium binding protein A8) and recruits members of the
polycomb repressor complex (PRC2). This action stimulates
repressive histone marks and blocks transcription, thereby
suppressing Ly6Chi monocyte numbers and inflammation at
the site of damage (134). Together, these data confirm a role
for the clock in the control of the inflammatory response in the
lungs and suggest that diminished clock function can attenuate
normal responses to environmental inflammatory agents like
CS.
Recent data show that circadian clocks regulate immune
function (99) since molecular clock disruption can lead to both
pathogen-associated molecular patterns (PAMPs) and damageassociated molecular patterns (DAMPs) responses. PAMPs are
typically microbial or viral macromolecular components. Evidence suggests that several endogenous molecules are released
from damaged cells and tissues and these molecules were
collectively termed DAMPs (123). These DAMPs are also
capable of inducing inflammatory responses similar to that
produced by PAMPs (10, 123). Some of the signaling pathways central to PAMPs and DAMPs show a daily variation in
the response to their ligands. For example, TLR4 is shown to
oscillate and have differential inflammatory responses during a
specific period of its activation. TLR9, which is bound by
unmethylated repeats of the dinucleotide CpG, shows a daily
variation of lung function particularly in animal models of
sepsis. The molecular clock in immune cells influences the
release of cytokines and chemokines through pattern recognition receptors and cellular functions including phagocytosis
and migration of inflammatory/immune cells to the site of
infection. Support for circadian control of innate immunity
comes from recent work in macrophages (39). Myeloid cells
show temporal regulation of inflammatory responses and
BMAL1 inhibits activation of NF-␬B and miR-155 induction,
protecting mice against LPS-induced sepsis. Finally, circadian
disruption leads to altered sleep and immune responses in mice
(150). This suggests that circadian disruption-induced modulation of inflammatory and immune responses may underlie the
reduced quality of sleep reported in patients with chronic lung
disease.
Bacterial/Virus Infections and Molecular Clock Dysfunction
in the Lungs
Recent studies have also shown that bacterial infection alters
the timing and amplitude of clock gene expression in the lungs
(14, 59, 134), though the mechanism of this response is largely
unknown. The clock appears to play a role in the degree of
inflammation, the timing of cytokine gene expression, and
bacterial colonization (14, 59). This could be in part due to
increased leukocyte numbers and/or dependence on circadian
regulation of antimicrobial peptides (14). Similarly, mouse
macrophages showed enhanced ability to ingest particles near
the onset of activity, indicating significant daily variation in
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press a dominant negative CLOCK protein lacking histone
acetyltransferase (HAT) activity, also lack a rhythm of Ahr
gene expression (180). This highlights the role of circadian
disruption in abnormal metabolism of inhaled toxicants. As
such, the levels of Ahr mRNA in the lungs of Clock⌬19 mutant
mice remained lower compared with wild-type mice (180).
Arrhythmic bmal1⫺/⫺ mice also show signs of advanced aging
and underlying pathologies, correlated with increased levels of
reactive oxygen species and inflammation (92, 93).
It has been reported that circadian clock genes regulate
oxidative stress (e.g., markers of lipid peroxidation are increased in Bmal1 KO mice), scavengers of reactive aldehydes
during mitochondrial respiration (including ALDH2), and NADPH dehydrogenases [such as NQO1, which encodes quinone
1 (126)]. The cellular redox state alters circadian oscillation
through its influence on Bmal1 expression and several mitochondrial proteins are rhythmically expressed, suggesting a
potential reciprocal relationship between the core molecular
clock mechanism and the cellular redox state (72, 94, 137,
214). Circadian clock-dependent cycles of cellular NAD⫹
levels can drive mitochondrial oxidative metabolism via SIRT1
and regulate rhythm patterns of oxidative enzyme expression
and cellular respiration. This implicates the involvement of
NAD⫹ bioavailability in modulating both mitochondrial oxidative metabolism and circadian clock function (146). REVERB␣ modulates oxidative capacity by regulating mitochondrial biogenesis in skeletal muscle (197). Muscle dysfunction
is known to occur in COPD, further linking chronic inflammatory lung diseases, molecular clock function, and oxidative
stress. Additional data suggest that the timing of clock gene
expression in mammalian tissues is affected by redox modulation [e.g., regulation of cytochrome P-450 and regulation of
ROR elements via interaction with REV-ERB␣ (95)]. Similarly, alcohol consumption, which alters the redox status of the
cells and phase II genes, can affect the molecular clock. This
effect may be augmented in smokers due to the formation of
aldehyde adducts on molecular clock proteins. Overall, this
suggests that redox status of the cells or redox modulation of
molecular clock gene expression plays an integral role in
various cellular and molecular functions in the lung.
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Molecular Clock as Biomarkers of Lung Inflammatory and
Therapeutic Responses
Recent literature implies that systemic monitoring of the
molecular clock may be used as a biomarker or chronobiomarker (time-of-day sampling of genomic/transcriptomic, proteomic, and metabolomic biomarkers that was termed CircadiOmics) (118, 145, 187) of disease and or the response to
treatment. These emerging biomarkers may provide key information about the inflammatory response (e.g., recruitment of
inflammatory/immune cells) and the release of proinflammatory mediators at baseline or in response to treatments.
As previously noted, circadian misalignment during
chronic jet lag alters clock gene expression and pulmonary
function in mouse lungs (65). Circadian disruption may also
occur as a direct result of tissue and/or cell specific abnormalities in the timing and amplitude of clock gene expression, independent of changes in the external environment
(e.g., photic shifts). This form of circadian disruption can
perpetuate internal circadian misalignment due to dissociation between the effected tissue and remainder of the animal. Specifically, molecular clock dysfunction is known to
affect pulmonary physiology and injurious/damaging responses (57, 65, 77, 174, 177). Similarly, environmental CS
exposure or exposomes and viral infection result in circadian disruption, producing molecular clock dysfunction and
enhanced inflammatory responses in the lungs (55, 77, 174).
Recent data reveal that passive tobacco/cigarette smoking
can also affect molecular clock function (135). These data
suggest that different forms of circadian disruption can have
converging effects on the molecular clock in lung tissue,
leading to enhanced injurious and inflammatory responses.
The effects of CS on the molecular clock appear to be
Sirtuin 1 (SIRT1)-dependent as they lead to increased acetylation and degradation of BMAL1 in lung tissue. SIRT1 is
also known to affect the expression of PER2, and the
combined effects of SIRT1 on both BMAL1 and PER2
expression underlies the pathological responses to CS. We
have recently shown that molecular clock dysfunction along
with SIRT1 reduction contributes to abnormal inflammatory
responses in smokers and COPD patients (210). The levels
of clock proteins including BMAL1, PER2, and REV-ERB␣
are reduced in human peripheral blood mononuclear cells
(PBMCs), sputum cells, and lung tissues associated with
inflammatory responses in patients with COPD compared
with nonsmokers (210). This highlights the role of the
molecular clock in the pathogenesis of airway disease,
making it an excellent candidate as a biomarker of disease
severity [e.g., in terms of regulating local/systemic inflammatory responses (44)]. We and others have established that
environmental CS, shiftwork, and chronic jet lag can have
near parallel influences on the timing system and the inflammatory response (43, 65, 77, 107, 164, 177, 192). Other
reports indicate that inflammation due to septic shock also
leads to circadian disruption (28, 70, 140, 163, 198). Hence,
it is likely that circadian disruption-induced molecular clock
dysfunction augments lung inflammatory responses to environmental factors including CS and infectious agents. Altered clock function following exposure to various environmental factors may predispose the system to exaggerated
inflammatory responses and hasten the development of
pathophysiology in chronic airway disease. The cellular
metabolome, which is affected as a result of nutrient intake,
is regulated by the molecular clock and SIRT1 (141).
Recently, it has been shown that metabolites produced by
oxidation of fatty acids can regulate SIRT expression and
the clock (132, 133, 183). As an example, palmitate inhibits
the SIRT1-dependent Bmal1-Clock interaction and disrupts
clock gene oscillations. Furthermore, assessment of molecular clock function may be used as a biomarker of ongoing
inflammatory responses and for monitoring the severity of
lung pathophysiology (Table 2).
Recently, the role of the circadian clock in mouse physiology and behavior was assessed by use of RNA-seq and
transcriptome arrays. This analysis revealed that key drugs
used in respiratory medicine are linked to circadian regulatory genes (including Advair Diskus for asthma and COPD;
Serpina6, Pgr, Nr3c2, Adrb2, Pla2g4; Combivent for asthma
and COPD; Slc22a5, Slc22a4, Chrm2, Adrb1, Adrb2 and
ProAir HFA for asthma and COPD; Adrb1 and Adrb2)
(212). These targets are potentially clock dependent and
may show gene- and time-specific responses to treatment,
supporting the notion that clock-driven genes may be useful
as biomarkers for drug efficacy in respiratory diseases.
These authors have characterized the role of the circadian
clock in physiology and found that the expression of many
oscillating genes peaked during transcriptional “rush hours”
preceding dawn and dusk. Strikingly, heart and lung were
notable exceptions, with phase distributions that diverged
substantially from other organs. They also found oscillations in the expression of more than 1,000 known and novel
noncoding RNAs (ncRNAs). It was found that the majority
of best-selling drugs and World Health Organization essential medicines directly target rhythmic genes. Many of these
drugs have short half-lives and may benefit from timed
dosage or chronotherapeutic strategies (212).
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regulating host defense (71). The Toll-like receptor 4 (TLR4)
pathway in peritoneal macrophages is regulated by the clock
and may drive rhythmic variation in immune responses (86). In
lung tissue, the epithelial clock and glucocorticoid hormones
control diurnal variation of pulmonary inflammatory responses
to bacterial infection (57). Furthermore, endotoxemia-induced
lung inflammation reorganizes circadian rhythms of leukocytes
in a Bmal1-dependent manner (70). Likewise, rhythms of
pulmonary function define the time-dependent sensitivity to
steroids and ␤2-agonists in patients with nocturnal asthma and
asthmatic patients who smoke (18, 52). It is likely that the
mechanism that couples the molecular clock and bronchiolar
glucocorticoid receptors to pulmonary immune-inflammatory
responses plays an important role during COPD exacerbations
and respiratory infections (178). Viral respiratory infection
(influenza A) also causes molecular clock dysfunction in the
lungs and increases mortality in bmal1 KO mice, suggesting
that diminished clock function depresses the immune response
to respiratory infection (174). Although we have observed
direct effects of infection on clock function, inflammatory
responses, and mortality, it is unclear whether these effects are
core clock dependent or indirectly related to dysfunctionmediated changes in non-clock-dependent cellular programming in lung tissue.
Review
CIRCADIAN CLOCK IN THE LUNG
Circadian Disruption and Its Impacts on Stress-Induced
Lung Cellular Senescence
vances by decreasing SIRT1 activity in response to reduced
NAD⫹ levels. This competitive inhibition may operate through
protein acetylation and phosphorylation to alter the timing of
the clock during DNA damage (108, 109). The molecular clock
also plays a role in apoptosis and cell cycle arrest by altering
the expression of apoptosis-related genes. The protein levels of
c-Myc and Cyclin B1 are both downregulated and p53 levels
are upregulated during circadian disruption. Data suggest that
the clock may play an important role in carcinogenesis by
inhibiting apoptotic cell death via attenuating proapoptotic
signaling (194). Certain genes that function in cell proliferation
and tumor suppression are CCG. Several of these, including
c-myc and the tumor suppressor p53, are involved in regulating
the cell cycle and apoptosis. Other putative CCG involved in
cell cycle regulation include the caspases, c-myc, and cyclins.
The deregulation of c-myc is associated with various cancers
and with the hyperplastic growth of mammalian tissues. Previous studies have also indicated that c-myc overexpression
may enhance cell cycle progression in the presence of genomic
DNA damage as seen in lungs of patients with COPD and
idiopathic pulmonary fibrosis (IPF).
A model has been developed to connect the molecular clock
to DNA damage responses and metabolism via the regulation
of chromatin remodeling. This bioinformatics model predicts a
molecular signaling mechanism through which multiple forms
of perturbation, as a result of DNA damage, lead to alteration
in molecular circadian clock (109). Importantly, SIRT1 and
PARP1 appear to modulate the timing of the molecular clock
in response to genotoxic stress (109). This model further
revealed the integration of multiple forms of posttranslational
modifications by environmental stress, including acetylation of
NAMPT and other kinases including CHK2. It also predicts the
role of SIRT1 in regulating histone/nonhistone protein deacetylation in relation to core clock function. The SIRT1-PARP1
system regulates clock function in respiratory diseases via the
effects of posttranslational modifications, such as acetylation,
methylation, sumoylation, and ubiquitination.
The Molecular Clock and the Temporal Control of “Omics”
The CircadiOmics (http://circadiomics.igb.uci.edu/) provides a consolidated model that integrates metabolomic data
along with exposomics, genomics, transcriptomics, and proteomics, to better understand time-of-day coordination of
pathophysiology. It is possible to include additional -omics
approaches, such as lipidomics or breathomics, that could also
be developed in the future as valuable clinical tools for personalized medicine in respiratory disease (145, 196). Epigenomic alterations, DNA methylation, histone modifications,
proteomics, lipidomics, and recent data on the role of long
noncoding RNAs in rhythmic changes have recently been
shown (37). In this aspect, long noncoding RNAs (lncRNAs)
are recently identified genetic elements involved in gene expression. It has been shown that lncRNAs are involved in
circadian biology, e.g., differential night/day expression of
lncRNAs (0.3 to ⬎50 kb) occurs in the rat pineal gland.
Approximately one-half of these changes reflect nocturnal
increases. Organ culture studies indicate that expression of
these lncRNAs in the pineal is regulated by norepinephrine
acting through cAMP (37). These findings point to a dynamic
role of lncRNAs in the circadian system especially when
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Aging differentially affects central and peripheral clock
function (74, 164). Conversely, it is likely that cellular senescence due to molecular clock dysfunction is enhanced by
chronic CS exposure, persistent shiftwork, or respiratory infection in susceptible smokers. Tobacco smoking is a wellknown factor in premature lung aging reflected as a rapid
decline in lung function. Circadian disruption has established
adverse effects on the elderly population and susceptible smokers (60, 191). As mentioned above, clock dysfunction significantly increases mortality in aged mice. Thus circadian disruption may cause and/or enhance stress-induced cellular senescence via a molecular clock-dependent mechanism,
particularly in aged population/patients with chronic airway
diseases and susceptible smokers that suffer from frequent
exacerbations. In fact, data suggest that clock disruption in
Bmal1 KO mice leads to enhanced rates of cellular senescence
in multiple tissues including the lungs (92, 93). Though certainly related, a clear link between altered circadian organization, circadian clock function, and aging at the cellular, molecular, and physiological level has yet to be established.
Environmental tobacco smoke is shown to cause molecular
clock dysfunction in the lungs in connection with DNA damage and impaired double-strand break (DSB) repair, which is
aggravated in COPD (26, 45, 189, 190). Lungs of COPD
patients show persistent DNA damage associated with stressinduced premature senescence (SIPS) and a senescence-associated secretory phenotype (SASP) (160, 161). DNA damage
leads to an inflammatory phenotype in senescent cells (160,
199). Nonhomologous end joining (NHEJ) in response to stress
also occurs in lung cells (110). The molecular clock is shown
to regulate the strengths of cellular responses to DNA damage
(53, 162). Mice deficient in Bmal1 exhibit early signs of aging
(92, 93) via augmented DNA damage/impaired DNA repair
(NHEJ), cellular senescence, and inflammatory responses (reviewed in Ref. 178). Circadian clock proteins also interact with
other factors including Ku70, Ku80, and ataxia-telangiectasia
mutated (ATM) involved in DNA damage checkpoints following genotoxic stress, thereby mediating DNA damage/repair
responses (53, 56, 80, 90, 112, 162). Recently, it has been
demonstrated that PER1 interacts with ATM/Chk2 complex to
modulate DSB damage induced by ionizing radiation (56). It
has been shown that BMAL1 negatively regulates prosenescent
gene p21, which is required for cell cycle arrest upon DNA
damage, further supporting a role for BMAL1-dependent gene
expression in cellular senescence (63). Expression of XPA, the
rate-limiting factor in nucleotide excision repair, displays a
clear circadian rhythm (81). Hence, it is possible that CSmediated clock dysfunction in the lungs may enhance the
susceptibility to DNA damage, thus promoting lung cellular
senescence during the pathogenesis of COPD and other chronic
airway diseases by inhaled toxicants.
Further role of NAD utilization in the control of circadian
rhythms during DNA damage response via SIRT1/PARP1 axis
is recently shown (108, 109). It has been reported that genotoxic stress-mediated DNA damage response affects circadian
clocks directly/indirectly via the key DNA damage response
proteins, SIRT1 and PARP1, which utilize NAD (108, 109).
Increased PARP1 activity triggers SIRT1-induced phase ad-
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CIRCADIAN CLOCK IN THE LUNG
cAMP level is altered in conditions of chronic inflammation.
Varying levels of cAMP regulate apoptosis and efferocytosis in
exacerbations of chronic lung disease.
We have recently identified a role for mitochondrial alterations in the pathogenesis of COPD (1), and a circadian acetylome (which is affected by environmental stresses) appears to
regulate mitochondrial metabolic pathways. This suggests that
rhythmic acetylation plays a significant role in the timing of
oxidative metabolism, a cellular function that is dramatically
altered by environmental stressors like CS (116).
Stress Response and Regulation of Molecular Clock Proteins
by Posttranslational Modifications
Phosphorylation/dephosphorylation. Posttranslational modifications of molecular clock proteins can affect their stability,
activity, and subcellular localization (3) and are directly linked
to intracellular signaling pathways (Fig. 2). Phosphorylation
and acetylation of both BMAL1 and PER2 are critical and
fundamental activities in the basic operation of the molecular
clock (64, 73, 129). It is possible that signal transduction in
response to extracellular stimuli, such as oxidative/carbonyl
stress imposed by CS, results in the activation of a kinase
cascade and phosphorylation of molecular clock proteins. Extracellular regulated kinase (ERK), MAP kinase, casein kinase
I ε/␦, CK2, and GSK-3 are reported to produce changes in
phasing/rhythmicity of the clock and phosphorylate clock proteins in flies, plants, and mammals (2, 4, 66). In mammals,
phosphorylation of CLOCK and BMAL1 is regulated during
the circadian cycle by rhythmic kinase activity (2, 102). Phosphorylation leads to nuclear translocation and degradation of
CLOCK (91), but the identities and function of the kinases
responsible are unknown in lung cells. Phosphorylation of
PER2 and CRY2 regulates nucleocytoplasmic shuttling of the
repressor complex and largely defines the rate of protein
degradation, though specific evidence for this activity in lung
cells is lacking (3). It is possible that phosphorylation of
BMAL1 and PER2 by kinases occurs in response to CS in lung
cells, whereas dephosphorylation by ser/thr phosphatase leads
to ubiquitination and proteasomal degradation (Fig. 2). It is
reasonable to assert that drugs designed to target the activity of
enzymes like CK1ε either directly or indirectly may be able to
alter the timing of the clock (151) or correct perturbations to
the oscillator induced by proinflammatory mediators like CS.
Acetylation. Acetylation plays a central role in circadian
timing as CLOCK has intrinsic HAT activity (46). CLOCK
acetylates histone H3 at Lys14, histone H4 at Lys16, BMAL1
at Lys537, and Per2 (7, 13, 46, 73) (Fig. 2). Acetylation of
BMAL1 potentiates its binding with CRY1 which leads to
transcriptional inhibition (73). CLOCK interacts with p300/
CBP and regulates rhythmic acetylation of histone H3 and
specific gene expression (40, 68). Thus acetylation plays a role
in both activation and repression of clock gene expression (40).
CS exposure increases histone H3 (Lys9) and H4 (Lys12)
acetylation in vitro in human bronchial epithelial cells (H292)
and in mouse lung (36, 175, 176, 205). Our data suggest that
CS reduces BMAL1 levels in large part through reduced
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Fig. 2. Cigarette smoke-induced modification of molecular clock function in the lungs depends on extracellular control of posttranslational modifications.
Oxidative/carbonyl stress (environmental tobacco smoke)-induced activation of extracellular regulated kinase signaling stimulates posttranslational modification
of molecular clock proteins in lung epithelial cells and macrophages, altering their activity and degradation rates. As reported, posttranslational modifications
as phosphorylation (P), ubiquitination (Ub), acetylation (Ac), SUMOylation (SUMO), S-nitrosylation (S-Ni), and carbonylation (4-HNE) in clock protein play
an essential role in its regulation. For example, phosphorylation of BMAL1 and PER2 leads to ubiquitination and eventually degradation, which influences both
the timing of the clock and downstream clock-dependent gene expression. CLOCK (circadian locomotor output cycles protein kaput), HAT (histone
acetyltransferases), HDAC (histone deacetylases), and SIRT1 (Sirtuin 1) are involved in the acetylation and deacetylation processes respectively. MAPK
(mitogen-activated protein kinase), CK1␦/ε (casein kinase 1␦/ε), CKII (casein kinase II), GSK3␤ (glycogen synthase kinase 3␤), PKC (protein kinase C), and
PP1 (protein phosphatase I) are involved in phosphorylation of clock proteins.
Review
CIRCADIAN CLOCK IN THE LUNG
Role of Sirtuins in Regulating Clock Protein Deacetylation
During Cellular Senescence and Inflammatory Responses
Evidence suggests that the molecular clock is involved in the
DNA damage response, cellular senescence, and cellular/organismal aging. It is well established that the timing system, as
a unit, declines in amplitude and robustness as a function of
age. SCN-dependent rhythms decline with age in parallel with,
and most likely as a function of, a decline in the robustness of
the molecular clock. How aging at each level (SCN network,
molecular clock) influences the rate and amount of cellular
senescence is a matter of conjecture. In Bmal1 KO mice, it was
reported that cellular aging is attributed to molecular clock
disruption. However, in the case of environmental stresses via
DNA damage, this mechanism cannot be reconciled especially
when considered in light of chronic lung disease. For example,
CS alters the molecular clock through its influence on SIRT1
activity but it also directly affects DNA damage and cellular
senescence. It is not known which comes first, whether the
effects are parallel and additive, and how much overlap there is
between the pathways (i.e., via kinase signaling). The role of
mitochondria and reactive oxygen species in the regulation of
cellular senescence occurs via the cytochrome oxidase system
in cell cycle survival pathways. This system involves metabolism via NAD⫹-dependent pathways, which also influence the
level and activity of SIRT1 (12).
The NAD⫹-dependent deacetylase-SIRT1 has been reported
to counteract the HAT activity of CLOCK (13, 128, 129). The
NAD⫹ substrate dependency of SIRT1 suggests that it constitutes a functional link between metabolism and clock function
(129, 130). SIRT1 mediates deacetylation of BMAL1 at
Fig. 3. Molecular mechanisms of SIRT1-mediated regulation of BMAL1 and
PER2 during inflammation. Oxidative/carbonyl stress (cigarette smoke)-induced reduction in SIRT1 levels/activity in the lungs leads to hyperacetylation
of clock proteins including BMAL1 and PER2 and transcription factor NF-␬B
RelA/p65. Acetylation of BMAL1 and PER2 results in increased ubiquitination and degradation and reduced E-box-mediated transcriptional activity.
Circadian clock disruption occurs due to enhanced clock gene acetylation leads
to reduced efficacy of anti-inflammatory drugs including steroids and ␤-agonists. Activation of SIRT1 in the presence of NAD⫹ by using specific SIRT1
activators (e.g., SRT1720) can enhance its deacetylase activity and facilitate
normal molecular clock function. Moreover, deacetylation of RelA/p65 (NF␬B) by SIRT1 activation can modulate inflammatory response indirectly
through its influence on the molecular clock. Hence, modulation of SIRT1
levels in the lung plays a protective role against inflammation and circadian
disruption in the pathogenesis of chronic airway diseases.
Lys537, PER2, and CRY1, thereby regulating the transcription
of clock genes (7, 13). SIRT1 level/activity shows daily variation in the lungs, correlated with rhythmic acetylation of
BMAL1, PER2, and histone H3 (13). SIRT1 activity is decreased in human lung epithelial cells, macrophages, and lungs
of mice exposed to CS and in the lungs of patients with COPD
(154, 206). SIRT1 activation reduces CS-induced acetylation
and degradation of BMAL1 in mouse lungs and monocytes.
Thus targeting circadian-dependent pharmacological activation
of SIRT1 may have potential as a chronopharmacological
intervention for the management of chronic airway disease
(210).
CS causes SIRT1 reduction and chromatin modifications due
to histone acetylation on proinflammatory gene promoters (8,
154, 204, 206). There is emerging evidence that circadian
rhythms govern proinflammatory cytokine gene expression
(193, 211). It is also known that inflammation and alteration in
clock gene expression are intimately associated with fatigue
and reduced activity (29, 32, 140). Hence, it is possible that
SIRT1 regulates acetylation/activation of RelA/p65 (NF-␬B)
and the inflammatory response indirectly through its influence
on the molecular clock (32, 154, 204, 206) (Fig. 3). SIRT1deficient mice develop exaggerated lung inflammation and
cellular senescence, whereas SIRT1-overexpressing transgenic
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deacetylase activity of SIRT1 (77). This appears to affect the
timing of the clock in both the lungs and the brain. Recently,
independent reports have clearly demonstrated a direct
BMAL1:CLOCK target gene, gene model 129 (Gm129), later
renamed CHRONO (computationally highlighted repressor of
the network oscillator), as a novel regulator of the feedback
loop (6). CHRONO interacts with BMAL1 and represses the
activity of the BMAL1:CLOCK transcriptional complex (5)
(Fig. 1). Bimolecular fluorescence complementation and coimmunoprecipitation analysis revealed that CHRONO acts by
abrogating the binding of BMAL1 to its transcriptional coactivator CBP (5). CHRONO KO mice showed prolonged locomotor activity rhythms (5, 61), and endogenous CHRONO is
rhythmically expressed antiphase with BMAL1 (61). Overexpression of CHRONO leads to suppression of BMAL1:
CLOCK activity in a histone deacetylase-dependent manner
(61). CHRONO KO mice also show alterations in the rhythmic
expression of core clock genes and an impaired response to
stress (61). CHRONO forms a complex with the glucocorticoid
receptor and modulates the response to circulating glucocorticoids (61). CHRONO is a part of the negative repressor
complex and is a potential link between the clock, environmental circadian disruption, and chronic airway disease,
though this relationship remains unexplored. Although these
data represent a significant advance, there remains a great deal
we do not yet understand regarding the influence of environmental stress (oxidative/carbonyl stress by CS) on intrinsic
CLOCK-HAT activity, direct clock protein acetylation, and/or
histone acetylation at CCG promoters in the lung.
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genes in the liver (117). SIRT6 interacts directly with BMAL1:
CLOCK and SREBP-1, forming a cis-regulatory complex that
regulates cyclic expression of genes involved in fatty acid and
cholesterol metabolism (117). The role of SIRT6 in the timing
of gene expression, cellular senescence, and DNA damage/
repair in the lungs remains to be determined.
Molecular Clock Dysfunction in Specific Lung Diseases
Recent studies reveal that molecular clock dysfunction can
occur in lung cells from patients or in experimental animal
models of lung disease. Various environmental agents that are
involved in the pathogenesis of airway diseases can alter
molecular clock function, influencing both the timing system
and inflammatory responses. Study of the regulation or alterations of molecular clock function in various airway diseases is
rapidly developing with many recent and exciting discoveries.
Chronic Obstructive Pulmonary Disease
COPD is a common and devastating chronic airway disease
marked by a decline in lung function and marked daily variation in exacerbation frequency and intensity. Using a mouse
model of COPD we have shown that tobacco smoke/CS exposure reduces SIRT1 activity and produces clock dysfunction in
the lungs (77). A recent study from our group has also determined that a reduction in SIRT1 activity is associated with
telomere attrition and inflammosenescence in patients with
COPD (209). Recently, a rat model of passive CS exposure
was used to demonstrate CS-mediated changes in molecular
clock function within intervertebral discs (IVD) (135). Passive
smoking for 8 wk in rats altered the timing of clock gene
expression in IVD, producing marked phase shifts of peak
clock gene expression (135). It was also revealed that CS
exposure (both acute and chronic) produced marked changes in
the sleep/wake rhythm and timing of clock gene expression in
the SCN (77). Sleep disorders, not limited to hypopneas, are
strongly comorbid with COPD (113, 114). These data suggest
that the altered quality and duration of sleep in those with
COPD results from direct impacts of CS on the sleep/wake
centers in the brain (77). Thus the bulk of evidence supports
the notion that molecular clock function in the lungs is altered
during the etiology of COPD, in large part owing to extended
periods of exposure to CS. Of note is the finding in rat IVDs
indicating that even secondhand smoke exposure can lead to
changes in molecular clock function. It remains to be seen
whether molecular clock dysfunction is a causative factor in
the timing and amplitude of exacerbations in those with COPD.
There is clearly significant potential for molecular clock gene
expression as a biomarker of disease severity and/or the effectiveness of treatment strategies for COPD.
Asthma
Abnormal rhythms of molecular clock gene expression have
been detected in the surface epithelium and isolated BAL cells
from patients with asthma (51). It was found that bronchial
airway epithelial cells from severe asthmatic patients have
increased gene expression of ARNTL2 (BMAL2) compared
with mild asthmatic patients and healthy subjects (51). In
contrast, Per2 gene expression was higher in healthy subjects
compared with mild and severe asthmatic patients, which could
impact both the incidence and severity of asthma symptoms
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mice are protected against CS-induced lung premature aging
(i.e., cellular senescence, inflammation, lung function decline,
and air space enlargement/emphysema) (207). It is likely that
SIRT1 can affect core clock function via its regulation of clock
protein levels, particularly in the lungs. Acetylation of BMAL1
potentiates its binding with the repressor CRY1, which leads to
suppression of CLOCK:BMAL1 mediated transcription (73).
Hence, it is possible that CS-mediated reduction of SIRT1
level/activity leads to hyperacetylation of clock gene-encoded
proteins, culminating in abnormal circadian periodicity and
acetylation of histones on proinflammatory and prosenescent
genes.
SIRT1 regulates cellular senescence (SIPS and SASP) in
response to CS and toxicant stress by protecting genomic DNA
against double-strand break in lung cells (136, 207, 208).
SIRT1 KO mice show increased lung cell senescence possibly
via DNA damage and impaired DNA damage repair (DDR),
associated with augmented inflammatory responses (207).
SIRT1 functions in DDR to promote DNA repair and downregulates inflammation by deacetylating specific histones. The
role of SIRT1 in molecular clock function as it relates to DNA
damage/repair, as well as to SIPS and SASP in the lungs, is
currently unknown. Furthermore, it is not known whether there
is a relationship between the decline of SCN function in aging
and SIRT1 activity in SCN pacemaker neurons. Furthermore, it
is unclear whether this role for SIRT1 is a factor in the
increased rate of COPD/IPF among the aged (31).
As in the lungs, SIRT1 also regulates BMAL1:CLOCK
activity in the pacemaker neurons of the SCN. In aged mice,
SIRT1 levels in the SCN are decreased (similar to its reduction
by CS and in patients with COPD), giving rise to a longer
intrinsic period, a more disrupted activity pattern, and an
inability to readily adapt to acute perturbation in the environment (i.e., phase shifts). Hence, SIRT1 is a significant part of
the molecular oscillator regulating the activity of SCN pacemaker neurons. SIRT1 is critical for the maintenance of robust
circadian rhythms in young animals (which may be the case in
the lung), and decay in this activity by smoking/environmental
stresses may play an important role in aging, particularly in the
pathogenesis of chronic inflammatory lung diseases. Resveratrol and other dietary proanthocyanidins are known to modulate the molecular clock via a SIRT1/NAD/NAMPT pathway
and may be the mechanism whereby these compounds attenuate cellular senescence (157).
SIRT6 is the only sirtuin constitutively localized to chromatin and is prominently present at the transcription start sites of
active genomic loci (155). SIRT6 has been reported to be
dynamic in its chromatin binding in response to TNF-␣,
thereby altering the transcriptional landscape of aging- and
stress-related genes (85). SIRT6 is known for its deacetylase
activity at histones, acting primarily at H3K9 (84, 121) and
H3K56 (122, 202) to modulate gene expression, telomere
maintenance, and genomic instability (111, 181). SIRT6 activity has been linked to metabolic homeostasis with studies
showing that Sirt6⫺/⫺ mice die at 2– 4 wk of age owing to
accelerated aging and persistent hypoglycemia. This increase
in mortality is largely due to altered rates of glycolysis, glucose
uptake, and mitochondrial respiration (125, 200). Analysis of
the hepatic circadian transcriptome revealed that SIRT1 along
with SIRT6 influences distinct cohorts of oscillating transcripts, suggesting a SIRT-specific clustering of rhythmic
Review
CIRCADIAN CLOCK IN THE LUNG
Idiopathic Pulmonary Fibrosis
A recent study reports the cell-type dependent upregulation
of ROR␣ in fibrotic lung tissues from IPF and cryptogenic
organizing pneumonia patients. Though expression of ROR␣
was enhanced in fibroblasts present in fibrotic areas, fibroblasts
away from these foci did not show any ROR␣ expression
(188). The localization of ROR␣ expression was limited to the
nucleus in normal Type II epithelial cells of controls or
unaffected areas of the lungs from fibrotic patients. Nevertheless, ROR␣ expression changes dramatically in the lung epithelial cells lining the fibroblastic foci (188). Future studies
should examine the role of ROR␣ in pulmonary fibrosis,
through its interaction with other canonical signaling pathways
such as WNT/␤-catenin and the AKT/PI3K pathways previously linked to cell proliferation and the epithelial to mesenchymal transition.
Acute Lung Injury
Acute lung injury (ALI)-associated inflammation due to
septic shock can lead to circadian disruption. For example,
studies indicate that the core clock protein PER2 regulates
the expression of the hypoxia-inducible factor-1 (HIF-1)
and may play a role in the activation of mucosal HIF-1 in
ALI (47, 49). Furthermore, it has been shown that PER1
interacts with the ␣-subunit of HIF-1 (34). Prior studies
have clearly shown that PER2 levels were induced similar to
HIF-1 during mechanical ventilation. Induction of Per2
expression was reported in a murine model of ALI by
mechanical ventilation (ventilator-induced lung injury) (48).
Additionally, to test the hypothesis that constant light can
induce Per2 expression in the lungs, mice were maintained
under constant light (LL) for a period of 1 wk. Results
revealed an enhanced Per2 rhythm in the lungs over a 48 h
time period in LL (48). A recent review highlights the
importance of “Chronomics”: timing and rhythm in intensive care unit (ICU), circadian physiology during acute
stress, and the effects of ICU milieu upon circadian rhythms
to stress the value of considering circadian-immune disturbance as a vital tool for personalized treatment (143).
Overall, these observations suggest that activation of PER2dependent gene expression during ALI represents a novel
pathway for targeting the treatment and management of ALI
(48).
Infections
To examine the impact of circadian disruption on the
severity of responses to pulmonary infection, investigators
exposed wild-type and BMAL1 KO mice to viral pneumonia
(Sendai virus) (69). Bmal1-null mice exhibited 100% mortality to viral pneumonia by postinfection day 8 compared
with 0% mortality for wild-type littermates (69). Increased
morbidity was apparent as early as postinfection day 3 and
was characterized by a drop in absolute body weight that
continued until day 21 postinfection. These data strongly
support the notion that circadian disruption exerts a significant effect on acute responses to respiratory viral infection
in vivo (69). This observation concurs with a recent study
from our group showing that the influenza A virus alters
clock function via a Bmal1-dependent pathway (174). The
impact of circadian disruption on the immune response to
infection is well known (28). It is clear that persistent
exposure to external or internal chronodisruption, as experienced during chronic jet lag or shift work or in animal
models after genetic manipulation of the clock, dramatically
reduces the effectiveness of the immune response to infection. Pharmacological manipulations of molecular clock
function in chronic airway diseases or during respiratory
infections represent novel and potentially effective therapies
for chronic lung diseases and associated exacerbations.
Sleep Apnea
The role of respiratory physiology and its alterations in
the progression of sleep apnea and its comorbidity are well
known. Recent advances in our understanding of molecular
clock function and its role in sleep biology may shed new
light on the etiology of sleep disturbances in patients with
hypoventilation syndromes, including sleep apnea. It was
reported that the timing system contributes to the timing and
duration of apneic periods during obstructive sleep apnea.
This may be due to a direct influence of the molecular clock
and could be in some way regulated by the activity of the
SIRT1-BMAL1 pathway (21). This area is emerging and
more research is needed to address the role of the molecular
clock in the pathophysiological sleep disruption that occurs
during sleep apnea.
Chronopharmacology for the Treatment of Lung
Pathophysiological Conditions
Recently, a circadian gene expression atlas in mammals and
its implications in biology and medicine suggest that more than
50% of all (top-selling) drugs target circadian clock genes or
their protein products. Many of these drugs have direct influence in pulmonary physiology. However, there is a fundamental gap between the limited, scattered research on circadian
genes and their importance in regulation of lung pathophysiological responses. A detailed study characterizing the role of
the circadian clock noted key pharma drugs used in asthma and
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(51). ARNTL2/PER2 ratio was increased in severe asthmatic
patients compared with healthy subjects and an increase in
daytime ARNTL2 was associated with nocturnal symptoms
(51). The impact of asthma on Arntl2 and Per2 gene expression
rhythms in bronchial airway epithelial cells was further supported by experiments in vitro using cultured BAL cells. It is
possible that restoring the balance between repressor (Per2)
and activator (Bmal2) expression in lung cells could reduce the
frequency and severity of nocturnal exacerbations in asthmatic
patients (51).
ROR␣, a member of the nuclear receptor superfamily of
transcription factors and a component of the core clock, was
reported to influence the Th17 and Th2 responses in asthma
(78), enhance fibrosis through the innate immune lymphoid
type 2 cells (ILC2) (67), and play a role in DNA damagemediated apoptosis and emphysema in patients with COPD
(165). Finally, murine studies suggest molecular clock disruption may drive circadian rhythms in IgE/mast cell-mediated
allergic reactions (131). Thus the regulation of molecular clock
function in various immune-inflammatory cells may both predict the severity/exacerbations of asthma and provide a novel
pathway toward new and effective treatments.
L1067
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CIRCADIAN CLOCK IN THE LUNG
COPD (Advair Diskus regulating circadian-influenced genes
Serpina6, Pgr, Nr3c2, Adrb2, Pla2g4; Combivent regulating
Slc22a5, Slc22a4, Chrm2, Adrb1, Adrb2; and ProAir HFA for
Adrb1 and Adrb2) have been shown (212). Furthermore, circadian-dependent targets based on molecular clock function in
the lung and during pathological conditions have been discussed. Understanding various genes and targets may aid in
developing the chronotherapeutics for the treatment of pulmonary conditions (83).
Role of REV-ERB␣
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Several novel compounds that regulate the timing and amplitude of clock gene expression either directly or indirectly via
SIRT1 have been studied (Table 3). We have shown that
SIRT1 and REV-ERB␣ activators/agonists can have an important role in regulating certain abnormalities seen in COPD and
may be protective in a mouse model of emphysema (77, 210).
Important advancements have been made for the role of molecular clock output nuclear receptor REV-ERB␣ in coupling
molecular clock, metabolism, and inflammation (20, 213).
Recent evidence highlighted the role of REV-ERB␣ in adipogenesis possibly in lung cell regeneration upon noxious
stresses. This highlights that molecular clock protein may be
intimately linked to repair of lung cellular dysfunction (132,
133) (Table 3).
Interestingly, REV-ERB␣ functions as a core repressor in
the molecular clock, a critical regulator of metabolic/inflammatory gene expression, and a metabolic sensor. Zhang et al.
(213) report that multitasking transcription factor REV-ERB␣
plays an essential role in the regulation of both clock and
metabolic genes through distinct mechanisms. REV-ERB␣
binds directly to specific DNA sequences of clock genes by
displacing its competing transcription factor ROR. REV-ERB␣
does not interact with DNA sequences in metabolic genes but
instead interacts with other transcription factors that are involved in the regulation of metabolic gene expression in a
tissue-specific manner (20, 213). Clock control requires REVERB␣ to bind directly to the genome at its cognate sites, where
it competes with the activator ROR␣ to regulate the timing and
amplitude of Bmal1 expression. REV-ERB␣ is a receptor for
heme, an indicator of energy status that is depleted during
periods of negative energy balance. REV-ERB␣ regulates
metabolic/inflammatory genes primarily by recruiting the
HDAC3 corepressor to sites to which it is tethered by cell
type-specific transcription factors. These two mechanisms interplay with the availability of REV-ERB␣ in the nucleus. It is
possible that SIRT1 regulates the acetylation and decacetylation of REV-ERB␣ and its binding to respective promoters.
The direct competition between REV-ERB␣ and ROR␣ provides a mechanism for self-sustained control of the universal
molecular clock, whereas REV-ERB␣ uses epigenomic factors
to regulate metabolic/inflammatory genes that may be required
during the inflammatory responses.
Recently, there has been a new focus on small molecules
that target the REV-ERB and ROR nuclear receptors with an
emphasis on clinical applications in pulmonary and cardiac
disease (89, 167). Since REV-ERB␣ also plays a key role in
regulating mitochondrial content and the oxidative capacity of
skeletal muscle, it may be prudent that pharmacological activation of REV-ERB␣ will be useful in skeletal muscle related
diseases (197). Dysfunction of skeletal muscle is the hallmark
in the pathogenesis of COPD. Dual function can be achieved
since REV-ERB␣ agonists can regulate sleep architecture and
emotion in mice, and thus these compounds may be useful in
the treatment of sleep disorders and anxiety as seen as a
comorbid conditions in various lung diseases (10) (Table 3).
Among the other most effective treatments for alleviating
inflammation in chronic airway diseases are drugs that modulate glucocorticoid (GC) signaling and sympathomodulatory
drugs including the ␤2-adrenergic agonists. Not surprisingly,
activated glucocorticoid receptors (GR) modulate transcription
of molecular clock genes through glucocorticoid response
elements (8, 101). Clock gene expression is induced by adrenal
steroids in human bronchial epithelial cells, PBMCs, lymphocytes and rat fibroblasts (8, 19). Bronchodilators, including
␤2-adrenoreceptor agonists also induce Per gene expression in
bronchial epithelial cells (179). Hence, the induction of clockdependent output genes may be mechanistically linked to the
anti-inflammatory effects of GCs and ␤2-adrenoreceptor agonists. These data further highlight the potential for the molecular clock, in particular the role of REV-ERB␣ as a therapeutic
target for devising novel therapeutic strategies for minimizing
inflammatory responses in chronic airway disease.
It is worth noting that daily rhythms of GC secretion and
adrenergic signaling via the autonomic nervous output are
widely considered pathways whereby the SCN coordinates
and synchronizes central and peripheral oscillators (120).
Thus although these drugs may acutely alleviate inflammation they could have previously undefined long-term effects
on the timing system. A recent report revealed the inefficacy
of GC/␤2-adrenoreceptor agonists in treatment of COPD
exacerbations or asthmatic patients that smoke. Because GR
expression in lung tissue does not vary across the day, it is
most likely a downstream response, or lack thereof, to GR
agonists that is responsible for this lack of efficacy (58). It
is reasonable to implicate phasic variation in clock-dependent responses to GR and ␤2-adrenoreceptor signaling in
this diminished response, particularly in light of our own
data showing that the expression and timing of CCG expression are altered in an animal model of COPD. Thus chronopharmacological agents that increase the amplitude and
normalize the phase of clock and clock-dependent gene
expression in the lungs may be particular effective at regulating inflammatory responses and hold considerable promise for the management of chronic airway diseases and their
exacerbations.
Small molecular effectors of oscillator function have been
shown to target various components of the core clock,
leading to altered timing and amplitude of clock and CCG
expression (Table 3). These molecules may be given in
conjunction with steroids or ␤2-adrenoreceptor agonists as
part of a novel chronopharmacological treatment regimen
(33). It was recently reported that selenium, a trace metal
found in most commercial rodent diets and many foods,
increases BMAL1 levels and activity (76). Rodents provided
a diet high in selenium displayed considerably higher levels
of BMAL1 in the liver and showed resistance to mortality
following exposure to chemotherapeutic drugs (76). These
effects are specific to peripheral oscillators, since selenium
did not affect the timing or amplitude of BMAL1 expression
in the SCN. Natural ligands for REV-ERB␣, e.g., heme
Review
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CIRCADIAN CLOCK IN THE LUNG
Table 3. Chronotherapeutic drugs that can modulate molecular clock proteins
Small Molecules/Compounds/Drugs
SIRT1 activators
SRT 1720/SRT2183
Molecular Targets
CLOCK:BMAL1
REV-ERB␣ and BMAL1
BMAL1
REV-ERB agonists SR9009/SR9011
REV-ERB␣ and REV-ERB␤
REV-ERB␣ and REV-ERB␤
REV-ERB␣
REV-ERB␣
REV-ERB antagonist SR8278
REV-ERB␣
ROR␣/␥ agonist SR1078
ROR␣ and ROR␥
ROR␣/␥ inverse agonist SR1001
ROR␣ and ROR␥
ROR␣ and ROR␥
ROR␣/␥ inverse agonist T0901317
ROR␣ and ROR␥ (other receptor preference
LXR␣, LXR␤, PXR, FXR)
ROR␣ selective inverse agonist
SR3335
ROR␣
ROR␥-specific synthetic ligand SR1555
ROR␥
ROR␥-specific synthetic ligand SR2211
ROR␥
Citation
Altered CLOCK:BMAL1-mediated transcription of clockcontrolled genes. Activation of SIRT1 represses the
circadian gene expression and decreases H3 K9/K14
acetylation.
Attenuates REV-ERB␣ and BMAL1 protein levels and
inhibition of LPS-induced proinflammatory cytokine
release in PBMCs of nonsmokers compared to smokers
and patients with COPD
Unable to attenuate CS-induced lung inflammatory and
injurious responses in Bmal1 epithelial cell specific
KO mice
REV-ERBs are crucial for the coordination of genes
involved in metabolism either directly or indirectly,
through effects on the circadian clock.
REV-ERB agonist alters the expression of metabolic
genes in liver, skeletal-muscle and adipose tissue, and
increases energy expenditure. Synthetic REV-ERB
ligands are promising candidates for the treatment of
metabolic disease.
Inhibits expression of the circadian target gene Bmal1.
Also represses the expression of gluconeogenic genes
in liver cells and reduced glucose output in primary
hepatocytes.
Reduces LPS induced IL-6 but not IL-8 release in
primary human monocyte-derived macrophages
(MDMs) and primary human alveolar macrophages. In
primary human MDMs, combined application of LPS
and GSK4112 attenuated transcription of cytokine
genes such as il-6, ccl2, Cxcl11, Cxcl6, and il19.
Stimulates the expression of REV-ERB␣ target genes
involved in gluconeogenesis whereas GSK4112
(agonist) suppresses their expression. REV-ERB
antagonist, SR8278 represents a unique chemical tool
for probing REV-ERB function to explore the role of
circadian clock and metabolic pathways in vitro and in
vivo.
Functions as ROR␣/ROR␥ agonist by expressing two
important ROR target genes, glucose-6-phosphatase
(G6Pase) and fibroblast growth factor 21 (FGF21) in
the liver. Synthetic ROR␣/␥ agonist can be used as a
chemical tool to probe the function of RORs
A high-affinity synthetic ligand that is specific to both
ROR␣/␥, which inhibits TH17 cell differentiation and
function. SR1001 treatment in nonobese diabetic mice
reduces diabetes incidence and insulitis by targeting
TH17 cells.
Inhibits the development of murine TH17 cells and
reduces proinflammatory cytokine expression,
particularly TH17-mediated cytokines, autoantibody
production, and increases the frequency of
CD4(⫹)Foxp3(⫹) T regulatory cells
Represses ROR␣/␥ -dependent transactivation of RORresponsive reporter genes and reduces recruitment of
steroid receptor coactivator-2 by ROR␣ at an
endogenous ROR target gene (G6Pase).
SR3335 directly binds to ROR␣, suppresses the
expression of endogenous ROR␣ target genes involved
in hepatic gluconeogenesis including glucose-6phosphatase and phosphoenolpyruvate carboxykinase in
HepG2. SR3335-treated mice displayed lower plasma
glucose levels following the pyruvate challenge.
Inhibits TH17 cell development and function but also
increases the frequency of T regulatory cells.
SR2211, a selective ROR␥ modulator that potently
inhibits both the gene expression and production of
IL-17 in EL-4 cells. SR2211 is identified as a potent
modulator of ROR␥.
15
210
77
171
35
62
59
88
195
170
168
98
96
169
97
BMAL1, brain and muscle ARNT-like 1; CLOCK, circadian locomotor output cycles kaput; COPD, chronic obstructive pulmonary disease; IL-6; interleukin
6; LPS, lipopolysaccharide; MDMs, monocyte-derived macrophages; PBMCs, peripheral blood mononuclear cells; ROR␣/␥, retinoic acid receptor-related orphan
receptor ␣/␥; TH17 cells, T helper cells.
precursor 5-aminolevulinic acid, have also been described to
have similar effects (201). Finally, synthetic ligands for
REV-ERB␣ (e.g., GSK4112, SR9011, SR9009) may also
regulate inflammation, improve respiratory function, and
normalize sleep/wake rhythm in patients with asthma and
COPD (88, 89, 171).
Conclusions and Future Directions
In summary, abnormal rhythms of lung function in patients
with COPD and asthma are associated with molecular clock
dysfunction and enhanced airway inflammation in response to
environmental agents (Fig. 1). Moreover, exposomes such as
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REV-ERB agonist GSK4112
Major Outcomes/Findings
Review
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CIRCADIAN CLOCK IN THE LUNG
ACKNOWLEDGMENTS
We apologize to those authors whose excellent contribution in the field
could not be cited here because of space limitations.
GRANTS
This study was supported by the NIH 1R01HL097751 (to I. Rahman),
1R01HL092842 (to I. Rahman), American Lung Association RG-266456-N
(to H. Yao), American Lung Association RG-305393 (to I. K. Sundar),
University of Rochester Drug Discovery Pilot Award, and National Institute of
Environmental Health Sciences Environmental Health Science Center Pilot
Award (under NIEHS grant P30-ES01247 to M. T. Sellix).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
I.K.S. prepared figures; I.K.S., H.Y., M.T.S., and I.R. drafted manuscript;
I.K.S., H.Y., M.T.S., and I.R. edited and revised manuscript; I.K.S., H.Y.,
M.T.S., and I.R. approved final version of manuscript.
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environmental tobacco smoke, airborne particulates, and bacterial/viral infections trigger oxidative stress, premature cellular senescence, and DNA damage response that can also cause
molecular clock dysfunction in the lungs. CS stress-induced
oxidative/carbonyl stress reduces SIRT1 expression in the
lungs, leading to changes in the acetylation and degradation of
the clock protein BMAL1 and PER2. These posttranslational
modifications of clock protein levels can further enhance lung
inflammation and cellular senescence. Thus the timing system
may act under normal circumstances as a rheostatic modulator
of lung inflammoimmune responses that when challenged (as
in response to CS-associated stress) can also act to increase the
amplitude and duration of proinflammatory impact on lung
function. This bidirectional relationship reveals that the clock
is poised to act as a target for novel therapeutic interventions.
Assessment of molecular clock function itself can be used as a
biomarker of chronotherapeutics in lung pathophysiological
conditions. Further work is needed to understand the “omics”
of the pulmonary clock as it relates to the progression of airway
diseases. This approach to understand the influence of the
timing system on cellular and molecular lung function is likely
to expose novel targets for chronopharmacotherapy. Evidence
links the molecular clock to NF-␬B, TLR4, and Nrf2 pathways
in the lungs, particularly in the context of pulmonary responses
to exposomes, environmental agents, virus/bacteria, and “double hits” (combination of environmental agents/toxins, e.g.,
smoke, and infectious agents, e.g., viral/bacterial). We are
already on the cusp of exciting discoveries in this area. Novel
clock-targeting compounds like the SIRT1 activators, upcoming small molecular clock-enhancing molecules, and REVERB ligands may soon be used to target the molecular clock in
lung cells, thus normalizing the lung inflammatory and prosenescence responses and improving the efficacy of glucocorticoids and ␤2-agonists. Recent evidence also suggests that
REV-ERB␣ ligands may be used for the treatment of sleep and
anxiety disorders (10). Clinical evidence suggests that SIRT1
also affects the clock-dependent metabolome and mitochondrial functions/biogenesis, which are altered during the pathogenesis of airway diseases like COPD (123). Thus clocktargeting drugs may provide multimodal global benefit by
targeting pulmonary physiology and inflammation, sleep quality, and glucose metabolism. Understanding the mechanism
whereby the dysfunctional molecular clock regulates lung
pathophysiological functions could lead to new and effective
avenues for treatment based on chronopharmacological agents.
Furthermore, assessment of molecular clock components in
systemic monocytes and/or sputum cells may be useful biomarkers of therapeutic efficacy and outcomes in chronic airway
disease.
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