Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.

Angiotensin II (Ang II) induces reactive oxygen species (ROS) production by human vascular smooth muscle cells (hVSMCs). ROS have been implicated in the development of both acute stress-induced premature senescence (SIPS) and chronic replicative senescence. Global oxidative DNA damage triggers SIPS and telomere DNA damage accelerates replicative senescence, both mediated via p53. This study tests the hypothesis that DNA is an important target for Ang II–induced ROS leading to senescence via telomere-dependent and independent pathways. DNA damage was quantified using the Comet assay, telomere DNA length by Southern blotting and hVSMC senescence by senescence-associated β-galactosidase staining. Exposure to Ang II increased DNA damage in hVSMCs within 4 hours. Inhibition by an AT ₁ receptor antagonist (losartan metabolite: E3174) or catalase, confirmed that Ang II–induced DNA damage was AT ₁ receptor-mediated, via the induction of ROS. Acute exposure to Ang II resulted in SIPS within 24 hours that was prevented by coincubation with E3174 or catalase. SIPS was associated with increased p53 expression but was not dependent on telomere attrition because overexpression of human telomerase did not prevent Ang II–induced SIPS. Exposure to Ang II over several population doublings accelerated the rate of telomere attrition (by >2-fold) and induced premature replicative senescence of hVSMCs—an effect that was also attenuated by E3174 or catalase. These data demonstrate that Ang II–induced ROS-mediated DNA damage results in accelerated biological aging of hVSMCs via 2 mechanisms: (1) Acute SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with accelerated telomere attrition.

The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1 ^−/− macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1 ^−/− macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function.

The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis — all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism.

The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα^−/− mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity. Significance The circadian clock protein REVERBα is proposed to be a key regulator of liver metabolism. We now show that REVERBα action is critically dependent on metabolic state. Using transgenic mouse models, we show that the true role of REVERBα is to buffer against aberrant responses to metabolic perturbation, rather than confer rhythmic regulation to programs of lipid synthesis and storage, as has been thought previously. Thus, in the case of liver metabolism, the clock does not so much drive rhythmic processes, as provide protection against mistimed feeding cues. Understanding how the clock is coupled to metabolism is critical for understanding metabolic disease and the impacts of circadian disruptors such as shift work and 24-h lifestyles.