Title : Circadian rhythms modulation of senescent skin fibroblasts and impact on the biological functions
Introduction: A wide range of behavioral processes and physiological functions in the body are varying in a highly controlled and periodic manner in function of day time. This oscillatory system, called circadian rhythms is highly synchronized and allows organisms to adapt to their environment. Molecular effectors known as clock genes (e.g.: Clock, Bmal1, Per1, Cry1) have been identified and allow the control of those rhythms. As a barrier between external environment and the body, skin takes a major part in protecting homeostasis against environmental variations such as temperature and UV radiation. In this respect, studies of circadian rhythms in skin are particularly relevant to evaluate how disrupted clock signaling may influence skin troubles. It has already been shown that disturbance of biological clock is influencing skin diseases like psoriasis or atopic dermatitis and that biological clock is evolving with age. The purpose of this study was to understand the link between age, rupture of circadian rhythms and skin essential functions.
Methods: Fibroblasts of 3 young healthy donors (27-33 years old) were cultured and senescence was induced by H202 during 2 hours. Circadian gene expression was synchronized by a short dexamethasone treatment. Cells were monitored across a kinetic of 83 hourswith sampling time points during day and night. mRNAwere extracted and a reverse transcription step converted mRNA to cDNA. cDNA have been used in the Biomark system which allows high throughput real-time qPCR (96 samples across 96 genes). In addition to clock genes, other genes were selected to cover different skin cell functions such as elasticity, structure, barrier function and immunity. Expression was normalized thanks to 4 housekeeping genes. Results were expressed in fold induction using ΔΔCt where Ct corresponds to the number of cycles required to generate a signal above predefined threshold.
Results: Observation of gene expression across the time revealed 2 main phases (before and after 53 hours of experiment) in young cells. These two phases were less pronounced for senescent cells. This may be due, at least in part, to a difference in cell proliferation capacity. The analysis of clock genes (PER2, PER3, NR1D1) showed circadian rhythms in fibroblasts. Some functional genes(for example COL7A1, LOXL1, TNC)were expressed in a rhythmic manner and some other genes did not show expression following cycles. COL7A1 (type VII collagen), LOXL1 (lysil oxydase) and TNC (Tenascine C) appeared less expressed, while MMP1 (metalloprotease1) is overexpressed in senescent cells compared to young cells all along the kinetic. Finally, rhythmic expression of COL7A1 and LOXL1 is lost with senescence.
Conclusion: The model of young and senescent fibroblasts coupled to targeted transcriptomic analysis is an interesting tool to study aging and understand how signaling and molecular pathways are modulated. In senescent fibroblasts, we showed firstly a modification of rhythmic cycles and secondly, a disturbance of structure molecules essential to tissue cohesion. The results of circadian rhythms on fibroblasts allow considering the use of cosmetic active ingredients at specific time periods to repair homeostasis in skin.