PubMed · 16501555
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Joel C Eissenberg, Sarah C R Elgin. 2006. Marking time.. https://doi.org/10.1038/ng0306-276
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Corneal transparency is critical for optimal visual function, and corneal neovascularization represents the primary cause of visual impairment globally. Recent studies have identified the transcription factor BMAL1 as a significant regulator of angiogenesis. However, its specific role and underlying mechanisms in endothelial cell apoptosis remain inadequately understood. This study seeks to elucidate the role and underlying mechanisms of BMAL1 in endothelial cell apoptosis by employing genetic modification, alkali-burned mouse corneal neovascularization models, lentiviral transfection, proteomic analysis, and other complementary methodologies. Our results showed that BMAL1 expression is significantly elevated in corneal neovascularization induced by alkali burn and removal of Bmal1 in endothelial cells resulted in the suppression of corneal neovascularization in alkali burn mouse models. In vivo experiments have demonstrated that the knockout of Bmal1 in endothelial cells leads to an increase in endothelial cell apoptosis. Complementary in vitro studies revealed that overexpression of BMAL1 in endothelial cells inhibits apoptosis, while knockdown of BMAL1 promotes apoptosis. Proteomic analysis identified STAT6 as a downstream target of BMAL1 involved in the regulation of endothelial cell apoptosis. Further cell salvage experiments confirmed that BMAL1 modulates endothelial cell apoptosis through the regulation of STAT6 expression. Finally, the results of dual-luciferase reporter assay demonstrated that BMAL1 exerts transcriptional repressive effects on the promoter bound by STAT6. This study elucidates the novel role and mechanism of BMAL1 in the regulation of angiogenesis and endothelial cell apoptosis, thereby identifying a potential therapeutic target for the treatment of vascular diseases such as corneal neovascularization.
CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix-loop-helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK-BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1-2 (cryptochromes 1-2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)-BMAL1(BMAL2) circadian transcriptional activation by CRY1-2.
Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.