Background: Circadian clock is an evolutionarily-conserved mechanism that exerts pervasive temporal control in stem cell behavior. This time-keeping machinery is required for orchestrating myogenic progenitor properties in regenerative myogenesis that ameliorates muscular dystrophy. Here we report a screening platform to discover circadian clock modulators that promote myogenesis, with the identification of chlorhexidine (CHX) as a clock-activating molecule with pro-myogenic activities. Methods: A high-throughput molecular docking pipeline was applied to identify candidate compounds with a structural fit for a hydrophobic pocket within the key circadian transcription factor protein, Circadian Locomotor Output Cycles Kaput (CLOCK). Secondary biochemical screen for clock-modulatory activities of these molecules were preformed, together with functional validations of myogenic regulations to identify modulators with pro-myogenic properties. Results: CHX was identified as a clock activator that promotes distinct aspects of myogenesis. CHX activated circadian clock that reduced cycling period length and augmented amplitude. This action was mediated by the targeted CLOCK structure via augmented interaction with heterodimer partner Bmal1, leading to enhanced CLOCK/Bmal1-controlled transcription with up-regulation of core clock genes. Consistent with its clock-activating function, CHX displayed robust effects on stimulating myogenic differentiation in a clock-dependent manner. In addition, CHX augmented the proliferative and migratory activities of myoblasts. Conclusion: Our findings demonstrate the feasibility of a screening platform to discover clock modulators with myogenic regulatory activities. Discovery of CHX as a pro-myogenic molecule could be applicable to promote regenerative capacities in ameliorating dystrophic or degenerative muscle diseases.
The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here we report that Nobiletin, a clock amplitude-enhancing molecule, displays anti-adipogenic properties via activating a clock-controlled Wnt signaling pathway that suppresses adipocyte differentiation. Nobiletin augmented clock oscillation with period length shortening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and core clock components. Consistent with its circadian clock-modulatory activity, Nobiletin inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the re-activation of Wnt signaling during adipogenic differentiation via transcriptional up-regulation of key components of this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and body weight reduction. Lastly, Nobiletin inhibited the maturation of primary preadipocytes and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development, implicating its potential therapeutic application in countering obesity and its associated metabolic consequences.
The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here we report that Nobiletin, a circadian clock amplitude-enhancing molecule, displays anti-adipogenic properties via activation of Wnt signaling pathway that is dependent on its clock modulation. Nobiletin augmented clock oscillatory amplitude with period lengthening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and clock components within the negative feedback arm. Consistent with its clock-modulatory activity, Nobiletin strongly inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the re-activation of Wnt signaling during adipogenesis via transcriptional up-regulation of key components within this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and reduction of body weight. Lastly, Nobiletin inhibited the differentiation of primary preadipocytes and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development in a clock-dependent manner, implicating its potential application in countering obesity and associated metabolic consequences.
The circadian clock is driven by a transcriptional-translational feedback loop, and Cryptochrome 2 (Cry2) represses CLOCK/Bmal1-induced transcription activation. Despite the established role of clock in adipogenic regulation, whether the Cry2 repressor activity functions in adipocyte biology remains unclear. Here we identify a critical cysteine residue of Cry2 that mediates interaction with Per2, and demonstrate that this mechanism is required for clock transcriptional repression that inhibits Wnt signaling to promote adipogenesis. Cry2 protein is enriched in white adipose depots and was robustly induced by adipocyte differentiation. Via site-directed mutagenesis, we identified that a conserved Cry2 Cysteine at 432 within the loop interfacing with Per2 mediates heterodimer complex formation that confers transcription repression. C432 mutation disrupted Per2 association without affecting Bmal1 binding, leading to loss of repression of clock transcription activation. In preadipocytes, whereas Cry2 enhanced adipogenic differentiation, the repression-defective C432 mutant suppressed this process. Furthermore, silencing of Cry2 attenuated, while stabilization of Cry2 by KL001 markedly augmented adipocyte maturation. Mechanistically, we show that transcriptional repression of Wnt pathway components underlies Cry2 modulation of adipogenesis. Collectively, our findings elucidate a Cry2-mediated repression mechanism that promotes adipocyte development, and implicate its potential as a clock intervention target for obesity.
The circadian clock is driven by a transcriptional-translational feedback loop that elicits ~24 hour rhythms in behavior and physiology. The cell-autonomous clock exerts temporal regulation in adipocyte development and clock disruption leads to the development of obesity. There is current intense effort to identify clock-targeting interventions for metabolic disease therapy. Cryptochrome 2 (Cry2) is a circadian clock repressor, and it binds with Period proteins as a heterodimer complex to inhibit CLOCK/Bmal1-activated clock transcription that constitutes the negative feedback arm of the clock circuit. Our previous studies demonstrated that clock activator Bmal1 inhibits adipogenesis via the Wnt signaling pathway. To date, the role of Cry2 in adipogenic regulation remains unknown. Here we show that Cry2 inhibits adipogenesis and identify a critical residue that mediates interaction with Per2 required for this regulation. Cry2 transcript and protein are markedly induced during adipogenic differentiation. Via site-directed mutagenesis, we identified that Cysteine 432 within the lid domain of Cry2 protein is required for interaction with Per2, and a C432A mutant abolished this interaction. As a result, Cry2 repression activity of CLOCK/Bmal1-mediated transcription was abolished by the C432A mutant, as shown by Per2-luc reporter luciferase assay. Cry2 C432 mutation also led to elevated ubiquitination that accelerated protein degradation, an additional mechanism contributing to its loss of function. In 3T3L1 preadipocyte, we found that ectopic expression of Cry2 enhanced adipogenesis, whereas its loss-of-function suppressed adipocyte differentiation. In line with these findings, a Cry-stabilizing compound KL001 markedly augmented adipogenic differentiation. As compared to wild-type Cry2 effect on promoting adipogenesis, Cry2 C432A mutant largely abolished differentiation consistent with loss of repressive function in clock modulation. Mechanistically, we show that Cry2 induces adipogenesis via negative regulation of Wnt signaling pathway leading to inhibition of CCAAT/enhancer binding protein α (C/EBPα) expression. Together, our findings reveal a novel function of Cry2 in promoting adipocyte development via a critical residue involved in interaction with Per2. Our study thus provides a mechanistic basis for clock-targeting interventions to inhibit Cry2 activity for the treatment of obesity and its associated complications This work is supported by NIDDK grants 1R01DK112794, 1R01DK130499 and AR-DMRI 2023 T2D Innovation Grant. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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