The role of the circadian clock in skin and the identity of genes participating in its chronobiology remain largely unknown, leading us to define the circadian transcriptome of mouse skin at two different stages of the hair cycle, telogen and anagen. The circadian transcriptomes of telogen and anagen skin are largely distinct, with the former dominated by genes involved in cell proliferation and metabolism. The expression of many metabolic genes is antiphasic to cell cycle-related genes, the former peaking during the day and the latter at night. Consistently, accumulation of reactive oxygen species, a byproduct of oxidative phosphorylation, and S-phase are antiphasic to each other in telogen skin. Furthermore, the circadian variation in S-phase is controlled by BMAL1 intrinsic to keratinocytes, because keratinocyte-specific deletion of Bmal1 obliterates time-of-day-dependent synchronicity of cell division in the epidermis leading to a constitutively elevated cell proliferation. In agreement with higher cellular susceptibility to UV-induced DNA damage during S-phase, we found that mice are most sensitive to UVB-induced DNA damage in the epidermis at night. Because in the human epidermis maximum numbers of keratinocytes go through S-phase in the late afternoon, we speculate that in humans the circadian clock imposes regulation of epidermal cell proliferation so that skin is at a particularly vulnerable stage during times of maximum UV exposure, thus contributing to the high incidence of human skin cancers.Arntl gene | circadian rhythm | UVB damage | cell cycle T he highly conserved circadian clock regulates organismal adaptation to the light:dark (LD) cycles caused by the rotation of the Earth (1). Located in the suprachiasmatic nucleus of the vertebrate hypothalamus, the central clock is an intrinsic pacemaker with a spontaneous firing rate and a nearly 24-h rhythmic gene expression. This central pacemaker is thought to synchronize peripheral clocks found in the vast majority of tissues and cells. The core circadian clock machinery is composed of the heterodimeric bHLH-PAS transcription factors CLOCK and BMAL1 that bind E-box elements to activate clock-controlled genes, as well as Period (Per1, 2, and 3) and Cryptochrome (Cry1 and 2). PERs and CRYs inhibit CLOCK/BMAL1 activity upon their translocation into the nucleus, thus constituting a negative arm in the circadian feedback loop. CLOCK/ BMAL1 also activate expression of nuclear receptors ROR and REV-ERBα, which in turn respectively activate and inhibit the transcription of Bmal1 and other target genes containing retinoic acid-related orphan receptor response elements.Although the circadian clock is active in most mammalian tissues, the battery of genes under circadian regulation is largely tissue specific (2), suggesting that the circadian clock modulates physiological processes unique to each organ. Here we have focused on the role of the clock within skin, an organ dominated on the one hand by the cycling hair follicles and on the other by the continuously r...

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2−/− mice that Agr2 plays important roles in intestinal homeostasis. Agr2−/− intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.

Summary Using bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, non-invasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio, during the night compared to the day. Furthermore, we demonstrate that single cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that in proliferating stem cells the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity.

The hair follicle (HF) represents a prototypic ectodermal–mesodermal interaction system in which central questions of modern biology can be studied. A unique feature of these stem-cell-rich mini-organs is that they undergo life-long, cyclic transformations between stages of active regeneration (anagen), apoptotic involution (catagen), and relative proliferative quiescence (telogen). Due to the low proliferation rate and small size of the HF during telogen, this stage was conventionally thought of as a stage of dormancy. However, multiple lines of newly emerging evidence show that HFs during telogen are anything but dormant. Here, we emphasize that telogen is a highly energy-efficient default state of the mammalian coat, whose function centres around maintenance of the hair fibre and prompt responses to its loss. While actively retaining hair fibres with minimal energy expenditure, telogen HFs can launch a new regeneration cycle in response to a variety of stimuli originating in their autonomous micro-environment (including its stem cell niche) as well as in their external tissue macro-environment. Regenerative responses of telogen HFs change as a function of time and can be divided into two sub-stages: early ‘refractory’ and late ‘competent’ telogen. These changing activities are reflected in hundreds of dynamically regulated genes in telogen skin, possibly aimed at establishing a fast response-signalling environment to trauma and other disturbances of skin homeostasis. Furthermore, telogen is an interpreter of circadian output in the timing of anagen initiation and the key stage during which the subsequent organ regeneration (anagen) is actively prepared by suppressing molecular brakes on hair growth while activating pro-regenerative signals. Thus, telogen may serve as an excellent model system for dissecting signalling and cellular interactions that precede the active ‘regenerative mode’ of tissue remodeling. This revised understanding of telogen biology also points to intriguing new therapeutic avenues in the management of common human hair growth disorders.