The Circadian Clock Component BMAL1 Is a Critical Regulator of p21 Expression and Hepatocyte Proliferation

Abstract

Most living organisms show circadian (ϳ24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21 WAF1/CIP1 is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21 WAF1/CIP1 circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1 ؊/؊ knock-out mice. Biochemical and genetic data show that oscillation of p21 WAF1/CIP1 gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERB␣/␤ and ROR␣4/␥, which are core clock regulators. Importantly, p21 WAF1/CIP1 overexpressing Bmal1 ؊/؊ primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21 WAF1/CIP1 . These data establish a novel molecular link between clock and cell cycle genes and suggest that the G 1 progression phase is a target of the circadian clock during liver cell proliferation.Many physiological and behavioral processes display daynight oscillations in most organisms including mammals. These biological rhythms are controlled by endogenous selfsustained circadian (ϳ24 h) oscillators that operate not only in the central clock located in the suprachiasmatic nuclei of the hypothalamus but also in virtually all peripheral cells. Light is the main synchronizer of the central clock, which in turn coordinates the phases of peripheral oscillators regulating specific physiological outputs. Forward genetics and biochemical approaches have established the molecular basis underlying circadian oscillations in mammalian tissues (1-4). This mechanism involves complex interlocked positive and negative transcriptional/posttranslational feedback loops between the clock genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2 and their protein products. The robustness of the oscillations is ensured by additional regulators such as the REV-ERB␣ (NR1D1) and ROR␣ (NR1F1) orphan nuclear receptors (5, 6). Furthermore, extensive multilevel posttranslational regulation of various clock components has also been shown to play an important role in the molecular clock mechanism (7).There are substantial evidences that progression through the cell cycle occurs at specific times of the day/night cycle, suggesting that a function of the circadian clock system is to control this fundamental process. Notably, after the initial observation almost 40 years ago that cell division of the unicellular algae Euglena was controlled by an endogenous clock (8), numerous studies have shown a circadian variation of the proliferative activity in mammalian tissues such as the epithelia ...

Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration

“…43 Increased ARNTL levels in HET livers correlated with a significant increase in mRNA levels of its other target gene Wee1 (Supplementary Figure 5), a cyclic kinase that inactivates cdc2 to preclude mitosis. 44 Because ARNTL/ BMAL1 KO mice experience defective hepatocyte proliferation due to increased p21 transcription, 45 we anticipated that higher ARNTL/BMAL1 levels in HET livers might cause the opposite, that is, decrease p21 transcription. However, p21 mRNA levels remained unchanged with p21 protein levels being significantly higher in HET versus WT livers.…”

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle

“…Of the many processes being regulated by the circadian clock, some of the most profound are those related to specific cell-cycle events, DNA repair, and apoptosis (2,4,5,(21)(22)(23)(24)(25). The dynamics of the coupling we describe here are likely to be controlled through multiple and interacting pathways.…”

“…For instance, in mice the circadian clock regulates the cyclin-dependent kinase inhibitors p16 and p21, the G2/M kinase Wee1, as well as the checkpoint proteins CHK1 and 2, and genetic disruption of any of these links compromises cellular proliferation (2)(3)(4)(5). Although these molecular links provide a partial mechanistic basis for the coupling between these oscillators, the consequences for the joint dynamics are far from clear, as is the extent to which the cell cycle is coordinated by the clock, and vice versa.…”

14-3-3γ Inhibition of MDMX-mediated p21 Turnover Independent of p53

“…The protein level of p21 is oscillated during the cell cycle (11)(12)(13). Both ubiquitin-dependent and independent mechanisms have been shown to mediate p21 proteasomal turnover during the cell cycle or in response to specific signals (14 -17).…”