Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüp-pel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain-and loss-offunction experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.glucocorticoids | skin cancer B iological rhythms regulate cellular and physiological processes ranging from milliseconds to years. A well-studied timing system is the circadian (∼24-h) clockwork that allows organisms to anticipate diurnal variations in environmental conditions such as light, food availability, oxidative stress, pathogen exposure, or temperature. In mammals, the central circadian pacemaker resides in the suprachiasmatic nucleus (SCN), located in the anterior hypothalamus. Oscillations of SCN neurons are cell-autonomous, self-sustained, and synchronized to external time cues (Zeitgebers) such as light (1). The SCN, in turn, synchronizes peripheral clocks by systemic time cues such as neuronal input, hormonal signaling (e.g., cortisol), body temperature, and possibly many others (2). Interestingly, most cells in peripheral tissues also possess cell-autonomous clockworks with a similar molecular makeup to SCN neurons. These peripheral clocks are thought to generate or amplify daytime-dependent physiological and metabolic functions in a tissue-specific manner by circadian regulation of clock-controlled genes (3, 4).On a molecular level, circadian oscillation is generated by interlocked transcriptional-translational feedback loops. The transcription factor dimer CLOCK/BMAL1 drives expression of target genes such as Periods (Per1-3) and Cryptochromes (Cry1-2) by binding to E-box elements in their promoters. The negative feedback is formed by PER/CRY protein complexes that shuttle back into the nucleus, where they block CLOCK/BMAL1-mediated transactivation, thereby inhibiting their own transcr...
To anticipate daily environmental changes, most organisms developed endogenous timing systems, the so-called circadian (∼24 hours) clocks. Circadian clocks exist in most peripheral tissues and govern a huge variety of cellular, metabolic, and physiological processes. Recent studies have suggested daytime-dependent variations in epidermal functions such as barrier recovery and pH homeostasis. However, a local circadian clock in epidermal keratinocytes has not been reported yet, and as such the molecular link between the circadian system and epidermal physiology remains elusive. In this study we describe a functional cell autonomous circadian clock in human adult low calcium temperature (HaCaT) keratinocytes. Using live-cell bioluminescence imaging and mRNA expression time series, we show robust circadian transcription of canonical clock genes in synchronized HaCaT keratinocytes. Genetic and pharmacological perturbation experiments as well as the phase relations between clock gene rhythms confirm that the molecular makeup of the HaCaT keratinocyte clock is very similar to that of other peripheral clocks. Furthermore, temperature was identified to be a potent time cue (Zeitgeber) for the epidermal oscillator. Temperature cycles entrain HaCaT keratinocytes, leading to the identification of rhythmic expression of several genes involved in epidermal physiology such as cholesterol homeostasis and differentiation. Thus, we present HaCaT keratinocytes as an excellent model to study the regulation of keratinocyte physiology by the circadian clock in a simple yet robust in vitro system.
The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms.
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