Dysregulation of CLOCK Gene Expression in Hyperoxia-induced Lung Injury

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circadian rhythms, hyperoxia, inflammation, acute lung injury

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Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3−/− mice were exposed to room air or hyperoxia for 24, 48, or 72 h. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, or 100% O2) or room air for 72 h. The mRNA and protein levels of lung CLOCK genes, based on quantitative PCR and Western blot analysis, respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared with controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared with controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared with controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared with controls. Additionally, data suggest a dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3−/− was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3−/− mice compared with wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression.

acute lung injury (ali) affects a large number of patients worldwide, with mortality rates reported up to 40% (16). Many patients with ALI require oxygen supplementation to maintain adequate tissue oxygenation; unfortunately, it can exacerbate the condition as it may lead to hyperoxia-induced acute lung injury (HALI) (4). Exposure to hyperoxia can have pathological effects such as lung inflammation and edema accompanied by epithelial and endothelial cell death, suggesting that oxygen supplementation, although necessary, may potentially perpetuate or exacerbate ALI (2, 3). Inflammatory cells flood the lung tissue and proinflammatory cytokines like IL-1β are produced as a result of hyperoxia-induced ALI (11). Recent reports from our laboratory suggest the IL-1β processing machinery known as the “inflammasome” plays an important role in hyperoxic lung injury (14). Inflammasomes are master switches that are involved in caspase-mediated processing of proinflammatory cytokines (14).

Circadian rhythms play important roles in physiology and behavior; disruption of these rhythms can become a major cause in disease development (23). Recent evidence has implicated an important role for CLOCK genes in inflammation in relation to chronic diseases like diabetes and hypertension (17, 21). However, their role in ALI and how CLOCK gene expression is altered due to inflammation in ALI and other pulmonary associated diseases has been unexplored.

The mammalian circadian CLOCK is composed of at least 10 core circadian CLOCK proteins. Circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) transcription factors form a heterodimeric complex. This complex binds to E-boxes in the promoters of various target genes, including those encoding for negative [e.g., period homolog 1 (Per1), Per2, cryptochrome 1 (Cry1), and Cry2] or positive (e.g., Bmal1) loop components, as well as target genes, including D site of albumin promoter-binding protein (DBP), nuclear receptor subfamily 1, group D, member 1 (Rev-erb-α), and peroxisome proliferator-activated receptor-γ (PPARγ). It is well known that CLOCK genes are expressed rhythmically in the suprachiasmatic nucleus of the hypothalamus, the master circadian pacemaker in mammals. Recently, it has become clear that CLOCK genes also express and function in various peripheral tissues (24). In particular, CLOCK genes exhibit circadian expression patterns in organs that play critical roles in blood pressure homeostasis, including the vasculature, more specifically, the mouse aorta (18), heart (19, 25, 27), and kidney (19, 25). However, it is unknown whether CLOCK gene expression levels are altered in mice with HALI. In the present study, we hypothesize that chronic hyperoxia can induce inflammation in mouse lungs which may alter CLOCK gene expression and lead to the development of HALI.

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American Journal of Physiology-Cell Physiology, v. 306, issue 11, p. C999-C1007