背景:越来越多的证据表明计时系统具有重要的生理作用,被称为昼夜节律时钟(CC),不仅调节我们的睡眠-清醒节律,还调节外周组织中的许多其他细胞过程。在各种细胞类型中都显示出环境应激源,包括紫外线B辐射(UV-B),调节调节CC(CCG)的基因的表达,并且这些CCG调节UV-B诱导的细胞损伤的易感性。这项初步研究的目的是进一步了解CCs在UV-B诱导的皮肤癌光发生中的作用。
方法:应用RT-PCR,我们分析了在有和没有1,25-二羟维生素D(D3)和/或UV-B的HaCaT细胞中几个时间点(0-60h)的两个核心CCGs(脑和肌肉ARNT样1(Bmal1)和Per2(Per2))的表达,并进行了cosinor分析以评估这些条件对昼夜节律的影响,并进行了扩展的随机混合效应线性建模,接下来,我们研究了这两个基因在角质形成细胞中的表达,这些角质形成细胞代表了皮肤光致癌的不同阶段,比较正常(正常人表皮角质形成细胞-NHEK;p53野生型),癌前病变(HaCaT角质形成细胞;p53突变状态),和恶性(鳞状细胞癌SCL-1;p53无效状态)角质形成细胞在相同条件下12小时后。
结果:我们证明在HaCaT细胞中,Bmal1表现出强烈的昼夜节律,而Per2的证据有限。这两个基因的整体表达,但尤其是Bmal1,在UV-B处理后增加,而Per2在D3后显示出抑制的总体表达。UVB和1,25(OH)2D3均表明Bmal1存在明显的相移(顶相p<0.05),而对振幅没有具体影响可以证明。当我们比较不同的治疗方式(UV-B和/或D3)或细胞类型(NHEK,HaCaT,和SCL-1细胞)。
结论:比较代表皮肤光致癌不同阶段的表皮角质形成细胞,我们为在人体皮肤中独立运行的计时系统提供了进一步的证据,受UV-B调节,并在皮肤光致癌过程中受到干扰。我们的发现,这种昼夜节律模式通过用UV-B治疗而发生了差异改变,与用D3处理相比,不支持以下假设:这些CCGs的表达可能通过UV-B诱导的维生素D合成来调节,但可能通过生物钟引入维生素D的新的光保护特性。
BACKGROUND: Increasing evidence points at an important physiological role of the timekeeping system, known as the circadian clock (CC), regulating not only our sleep-awake rhythm but additionally many other cellular processes in peripheral tissues. It was shown in various cell types that environmental stressors, including ultraviolet B radiation (UV-B), modulate the expression of genes that regulate the CC (CCGs) and that these CCGs modulate susceptibility for UV-B-induced cellular damage. It was the aim of this pilot
study to gain further insights into the CCs\' putative role for UV-B-induced photocarcinogenesis of skin cancer.
METHODS: Applying RT-PCR, we analyzed the expression of two core CCGs (brain and muscle ARNT-like 1 (Bmal1) and Period-2 (Per2)) over several time points (0-60 h) in HaCaT cells with and without 1,25-dihydroxyvitamin D (D3) and/or UV-B and conducted a cosinor analysis to evaluate the effects of those conditions on the circadian rhythm and an extended mixed-effects linear modeling to account for both fixed effects of experimental conditions and random inter-individual variability. Next, we investigated the expression of these two genes in keratinocytes representing different stages of skin photocarcinogenesis, comparing normal (Normal Human Epidermal Keratinocytes-NHEK; p53 wild type), precancerous (HaCaT keratinocytes; mutated p53 status), and malignant (Squamous Cell Carcinoma SCL-1; p53 null status) keratinocytes after 12 h under the same conditions.
RESULTS: We demonstrated that in HaCaT cells, Bmal1 showed a robust circadian rhythm, while the evidence for Per2 was limited. Overall expression of both genes, but especially for Bmal1, was increased following UV-B treatment, while Per2 showed a suppressed overall expression following D3. Both UVB and 1,25(OH)2D3 suggested a significant phase shift for Bmal1 (p < 0.05 for the acrophase), while no specific effect on the amplitude could be evidenced. Differential effects on the expression of BMAL1 and Per2 were found when we compared different treatment modalities (UV-B and/or D3) or cell types (NHEK, HaCaT, and SCL-1 cells).
CONCLUSIONS: Comparing epidermal keratinocytes representing different stages of skin photocarcinogenesis, we provide further evidence for an independently operating timekeeping system in human skin, which is regulated by UV-B and disturbed during skin photocarcinogenesis. Our finding that this pattern of circadian rhythm was differentially altered by treatment with UV-B, as compared with treatment with D3, does not support the hypothesis that the expression of these CCGs may be regulated via UV-B-induced synthesis of vitamin D but might be introducing a novel photoprotective property of vitamin D through the circadian clock.