The intestinal exporter MRP2 plays an important role in disposition and elimination of a wide range of drugs. Here, we aimed to clarify the impact of circadian clock on intestinal MRP2, and to determine the molecular mechanisms for generation of diurnal MRP2 expression. Methods : The regulatory effects of Bmal1 on intestinal MRP2 expression were assessed using intestine-specific Bmal1 knockout ( Bmal1 iKO ) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Everted gut sac, cell viability and in situ intestinal perfusion experiments were performed to evaluate intestinal efflux of the MRP2 substrate methotrexate (MTX). Toxicity and pharmacokinetic experiments were performed with Bmal1 iKO mice and control littermates ( Bmal1 fl/fl mice) after oral gavage of MTX. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays. Results : Bmal1 iKO mice were generated by inter-crossing the mice carrying a Bmal1 exon 8 floxed allele ( Bmal1 fl/fl ) with Villin-Cre mice. Intestinal MRP2 expression exhibited a diurnal oscillation in Bmal1 fl/fl mice with a zenith value at ZT6. Bmal1 ablation caused reductions in Mrp2 mRNA and protein levels [as well as in transport activity (measured by MTX)], and blunted their diurnal rhythms. Intestinal ablation of Bmal1 abrogated circadian time-dependency of MTX pharmacokinetics and toxicity. Bmal1/BMAL1 regulation of rhythmic Mrp2/MRP2 expression was also confirmed in the colon cancer CT26 and Caco-2 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that Dbp activated and E4bp4 repressed Mrp2 transcription via specific binding to a same D-box (-100/-89 bp) element in promoter region. Further, Bmal1 directly activated the transcription of Dbp and Rev-erbα through the E-boxes, whereas it negatively regulated E4bp4 via the transcriptional repressor Rev-erbα. Positive regulation of Mrp2 by Rev-erbα was also observed, and attained through modulation of E4bp4. Conclusion : Bmal1 coordinates temporal expressions of DBP (a MRP2 activator), REV-ERBα (an E4BP4 repressor) and E4BP4 (a MRP2 repressor), generating diurnal MRP2 expression.

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To design potentially more effective therapies, we need to further understand the mechanisms underlying epilepsy. Here, we uncover the role of Rev-erbα in circadian regulation of epileptic seizures. We first show up-regulation of REV-ERBα/Rev-erbα in brain tissues from patients with epilepsy and a mouse model. Ablation or pharmacological modulation of Rev-erbα in mice decreases the susceptibility to acute and chronic seizures, and abolishes diurnal rhythmicity in seizure severity, whereas activation of Rev-erbα increases the animal susceptibility. Rev-erbα ablation or antagonism also leads to prolonged spontaneous inhibitory postsynaptic currents and elevated frequency in the mouse hippocampus, indicating enhanced GABAergic signaling. We also identify the transporters Slc6a1 and Slc6a11 as regulators of Rev-erbα-mediated clearance of GABA. Mechanistically, Rev-erbα promotes the expressions of Slc6a1 and Slc6a11 through transcriptional repression of E4bp4. Our findings propose Rev-erbα as a regulator of synaptic function at the crosstalk between pathways regulating the circadian clock and epilepsy.

Background and Purpose: Acute kidney injury (AKI) is a common and critical illness, resulting in severe morbidity and a high mortality. There is a considerable interest in identifying novel molecular targets for management of AKI. We investigated the potential role of the circadian clock components Rev-erb-α/β in regulation of ferroptosis and AKI. Experimental Approach: AKI model was established by treating mice with folic acid. Regulatory effects of Rev-erb-α/β on AKI and ferroptosis were determined using singlegene knockout (Rev-erb-α −/− and Rev-erb-β −/−) mice, incomplete double-knockout (icDKO, Rev-erb-α +/− Rev-erb-β −/−) mice and cells with erastin-induced ferroptosis. Targeted antagonism of Rev-erb-α/β to alleviate AKI and ferroptosis was assessed using the small-molecule antagonist SR8278. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation assays. Key Results: Loss of Rev-erb-α or Rev-erb-β reduced the sensitivity of mice to folic acid-induced AKI and eliminated the circadian time dependency in disease severity. This coincided with less extensive ferroptosis, a main cause of folic acid-induced AKI. Moreover, icDKO mice were more resistant to folic acid-induced AKI and ferroptosis as compared with single-gene knockout mice. Supporting this, targeting Rev-erb-α/β by SR8278 attenuated ferroptosis to ameliorate folic acid-induced AKI in mice. Rev-erb-α/β promoted ferroptosis by repressing the transcription of Slc7a11 and HO1 (two ferroptosis-inhibitory genes) via direct binding to a RORE cis-element. Conclusion and Implications: Targeted inhibition of Rev-erb-α/β limits ferroptosis to ameliorate folic acid-induced AKI in mice. The findings may have implications for improved understanding of circadian clock-controlled ferroptosis and for formulating new strategies to treat AKI.

Metabolic homeostasis of amino acids is essential for human health. Here, we aimed to investigate a potential role for the clock component reverse erythroblastosis virus α (Rev‐erbα) in circadian regulation of amino acid metabolism. RNA‐seq with Rev‐erbα‐/‐ mice showed expression changes in genes involved in amino acid metabolism, particularly, the urea cycle and methionine metabolism. Rev‐erbα ablation increased hepatic mRNA, protein, and enzymatic activity of betaine homocysteine methyltransferase (Bhmt), cystathionine β‐synthase (Cbs), and cystathionine γ‐lyase (Cth) and decreased the levels of plasma and liver homocysteine in mice. Cell‐based assays confirmed negative regulation of these three genes by Rev‐erbα. Combined luciferase reporter, mobility‐shift, and chromatin immunoprecipitation assays identified Rev‐erbα as a transcriptional repressor of Bhmt, Cbs, and Cth. Rev‐erbα ablation or antagonism alleviated chemical‐induced hyperhomocysteinemia in mice. This was accompanied by elevated expressions of Bhmt, Cbs, and Cth. Moreover, Rev‐erbα ablation or antagonism promoted urea production and ammonia clearance. Of urea cycle–related genes, arginase 1 (Arg1), ornithine transcarbamylase (Otc), and carbamoyl‐phosphate synthase 1 (Cps1) expressions were up‐regulated in Rev‐erbα‐/‐ mice. Negative regulation of these urea cycle genes by Rev‐erbα was validated using cell‐based experiments. Mechanistic studies revealed that Rev‐erbα inhibited CCAAT‐enhancer‐binding protein α transactivation to repress the transcription of Arg1, Cps1, and Otc. Conclusion: Rev‐erbα antagonism alleviates hyperhomocysteinemia and promotes ammonia clearance. Targeting Rev‐erbα represents an approach for the management of homocysteine‐ and ammonia‐related diseases.