The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-boxmediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock ''gated'' pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock D19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

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.

The thiazolidinedione (TZD) class of antidiabetic drugs, which are ligands for peroxisome proliferator-activated receptor (PPAR)-gamma, has been shown to possess potent anti-inflammatory and antineoplastic actions. Here, we show in mesangial cells that PPAR-gamma agonists inhibit fibronectin expression by transforming growth factor (TGF)-beta 1. TGF-beta 1 enhanced fibronectin mRNA expression, and this enhancement was abrogated by pretreatment with pioglitazone. Electrophoretic mobility shift assay identified that pioglitazone inhibited TGF-beta 1-induced DNA binding of activator protein-1 (AP-1). Pioglitazone inhibited AP-1 reporter activity but not Smad binding elements reporter activity without affecting TGF-beta 1-induced activation of mitogen-activated protein kinases (MAPKs) or Smad2. PPAR-gamma overexpression inhibited TGF-beta 1-induced fibronectin expression as well as the activation of AP-1. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural PPAR-gamma ligand, also inhibited TGF-beta1-induced fibronectin expression by suppressing AP-1 activation by TGF-beta 1. 15d-PGJ(2) inhibited the TGF-beta 1-induced MAPK activation. Dominant-negative PPAR-gamma (Delta PPAR-gamma) completely abrogated the inhibitory effect of pioglitazone and incompletely blocked its effect of 15d-PGJ(2) on TGF-beta 1-induced AP-1 reporter activity. Delta PPAR-gamma overexpression did not affect the inhibitory effect of 15d-PGJ(2) on TGF-beta 1-induced MAPK activation. In conclusion, pioglitazone inhibits TGF-beta 1-induced fibronectin expression by inhibiting AP-1 activation dependent on PPAR-gamma, while 15d-PGJ(2) acts through a dual mechanism independent of and dependent on PPAR-gamma activation in mouse mesangial cells.

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Whether baseline metabolic tumor volume (TMTV) and total lesion glycolysis (TLG) measured by FDG-PET/CT affected prognosis of patients with lymphoma was controversial. We searched PubMed, EMBASE and Cochrane to identify studies assessing the effect of baseline TMTV and TLG on the survival of lymphoma patients. Pooled hazard ratios (HR) for overall survival (OS) and progression-free survival (PFS) were calculated, along with 95% confidence intervals (CI). Twenty-seven eligible studies including 2,729 patients were analysed. Patients with high baseline TMTV showed a worse prognosis with an HR of 3.05 (95% CI 2.55–3.64, p<0.00001) for PFS and an HR of 3.07 (95% CI 2.47–3.82, p<0.00001) for OS. Patients with high baseline TLG also showed a worse prognosis with an HR of 3.44 (95% CI 2.37–5.01, p<0.00001) for PFS and an HR of 3.08 (95% CI 1.84–5.16, p<0.00001) for OS. A high baseline TMTV was significantly associated with worse survival in DLBCL patients treated with R-CHOP (OS, pooled HR = 3.52; PFS, pooled HR = 2.93). A high baseline TLG was significantly associated with worse survival in DLBCL patients treated with R-CHOP (OS, pooled HR = 3.06; PFS, pooled HR = 2.93). The negative effect of high baseline TMTV on PFS was demonstrated in HL (pooled HR = 3.89). A high baseline TMTV was significantly associated with worse survival in ENKL patients (OS, pooled HR = 2.24; PFS, pooled HR = 3.25). A high baseline TLG was significantly associated with worse survival in ENKL patients (OS, pooled HR = 2.58; PFS, pooled HR = 2.99). High baseline TMTV or TLG predict significantly worse PFS and OS in patients with lymphoma. Future studies are warranted to explore whether TMTV or TLG could be integrated into various prognostic models for clinical decision making.

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

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BackgroundThe prognostic significance of tumor-associated macrophages (TAM) in adult classical Hodgkin lymphoma (cHL) remains controversial. Here, we report a meta-analysis of the association of CD68 and CD163 infiltration on the clinical outcome of adult cHL.MethodsA comprehensive search to identify relevant articles was performed in PubMed, Embase, and Google Scholar on January 31, 2016. Using the fixed effect or random effects model of DerSimonian and Laird, hazard ratios (HR) or odds ratios (OR) with 95 % confidence intervals (CIs) were used as the effect size estimate.ResultsTwenty-two eligible studies with a total of 2959 patients were identified. Our analysis indicated that a high density of CD68+ TAMs in the tumor microenvironment of adult cHL predicted poor overall survival (OS) (HR: 2.41; 95 % CI, 1.92–3.03), shorter progression-free survival (PFS) (HR: 1.78; 95 % CI, 1.45–2.18), and poor disease-specific survival (HR: 2.71; 95 % CI, 1.38–5.29). High density of CD163+ TAMs in the tumor microenvironment of adult cHL also predicted poor OS (HR: 2.75; 95 % CI, 1.58–4.78) and poor PFS (HR: 1.66; 95 % CI, 1.22–2.27). In addition, we demonstrated that a high density of either CD68+ or CD163+ TAMs was associated with the presence of Epstein-Barr virus in neoplastic cells (ORCD68: 3.13; 95 % CI, 2.02–4.84; ORCD163: 2.88; 95 % CI, 1.55–5.34). A high density of either CD68+ or CD163+ TAMs tend to be associated with a more advanced clinical stage (ORCD68: 1.25; 95 % CI, 0.93–1.67; OR CD163: 1.19; 95 % CI, 0.86–1.63), B-symptoms (ORCD68: 1.35; 95 % CI, 0.90–2.01; ORCD163: 2.19; 95 % CI, 0.96–5.03), higher International Prognostic Factors Project Score (ORCD68: 1.20; 95 % CI, 0.67–2.15; ORCD163: 2.00; 95 % CI, 0.92–4.35), and bulky disease (ORCD68: 1.47; 95 % CI, 0.88–2.47; ORCD163: 1.19; 95 % CI, 0.72–1.96).ConclusionsOur analyses suggest that a high density of either CD68+ or CD163+ TAMs is a robust predictor of adverse outcomes in adult cHL. Increased TAMs should be taken into account to further improve prognostic stratification and the planning of appropriate therapeutic strategies.