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. 2024 Jan 14;16(2):254.
doi: 10.3390/nu16020254.

Differential Regulation of Circadian Clock Genes by UV-B Radiation and 1,25-Dihydroxyvitamin D: A Pilot Study during Different Stages of Skin Photocarcinogenesis

Leandros Lamnis  1 , Christoforos Christofi  1 , Alexandra Stark  1 , Heike Palm  1 , Klaus Roemer  2 , Thomas Vogt  1 , Jörg Reichrath  1
Affiliations

Differential Regulation of Circadian Clock Genes by UV-B Radiation and 1,25-Dihydroxyvitamin D: A Pilot Study during Different Stages of Skin Photocarcinogenesis

Leandros Lamnis et al. Nutrients. .

Abstract

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.

Keywords: circadian clock; circadian rhythmicity; photocarcinogenesis; skin; skin cancer; skin photocarcinogenesis; ultraviolet radiation; vitamin D; vitamin D receptor; vitamin D receptor signaling; vitamin D signaling.

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Conflict of interest statement

Prof. Reichrath is member of the Arnold Rikli-Award Jury of the Jörg Wolff Foundation. The Saarland University, together with Prof. Reichrath as one of several responsible group leaders, has received a research grant from the Jörg Wolff Foundation, Stuttgart, Germany.

Figures

Figure 1
Figure 1
Cosinor plot with the fitting curve, for −ΔCt values of Bmal1, normalized to GADPH. A significant effect of both UVB (p = 0.001) and D3 (p = 0.001) on the acrophase indicated a phase shift, meaning the timing of the peak high of the oscillation curve happened significantly later in comparison with that of the control curve. A significant effect in the amplitude was not suggested under either condition. As shown in the illustration, both a relevant phase shift and an amplitude decrease under UVB could be assessed, while for D3, the effects seemed rather insignificant, closely following the estimated rhythmic pattern of the control.
Figure 2
Figure 2
Circadian rhythm of relative mRNA gene expression of Bmal1 (A,C) and Per2 (B,D) in HaCaT keratinocytes (−ΔCt values normalized to the GADPH house-keeping gene). Mixed-effects cosinor plot for Bmal1 (A) and Per2 (B). The curve represents the best-fit sinusoidal curve that models the circadian rhythmicity of gene expression for Bmal1 and Per2. It was based on the combined cos(2π × Time/24) and sin(2π × Time/24) terms from the mixed-effects model, reflecting the predicted rhythmic pattern over time. The horizontal dashed line indicates the mesor, which is the midline estimating statistic of rhythm. The mesor represents the average level of the rhythmic function over a complete cycle, serving as a baseline around which the oscillations occur. There was a significant increase in Bmal1 expression (p = 0.021) after UVB treatment, as well as a decrease in Per2 (p < 0.018) after D3 supplementation, as indicated by the asterisks and double arrows. While UVB did not significantly increase Per2 expression, its combination with UVB mediated a significantly (p = 0.038) opposite (increasing) effect to that of D3 alone. This could reflect a probable stronger increasing effect of UVB, which could not be found in this specific model, or a more complex interplay between UVB and D3. In (C,D) the illustration of analysis of overall gene expression irrespective of the time factor (two-way repeated measures ANOVA). UV-B treatment increased the overall expression of both Bmal1 and Per2 (p < 0.001 (C,D)). Treatment with 1,25(OH)2D3, on the other hand, showed a significant effect on non-UVB-treated samples but not in UVB-treated ones. The decreasing effect of lone D3 treatment found in our MELM analysis was here absent. Different effects of UVB and 1,25(OH)2D3 in both cases speak nevertheless against the 1,25(OH)2D3 being a mediator of UVB-induced effects (n: p > 0.05, *: p < 0.05, ***: p < 0.001).
Figure 3
Figure 3
Relative mRNA gene expression of BMAL1 (A,B) and Per2 (C,D) 24 h following treatment in NHEK, HaCaT, and SCL-1 cells normalized to GADPH. The mean expression ± SD (in this instance, fold ratio 2−ΔΔCt relative to the control condition of HaCaT for a better representation of the results) is illustrated for the different treatment conditions (A,C). For both BMAL1 (A) and Per2 (C) we saw a significant effect (p < 0.001) of UVB. For BMAL1 we also saw a differential effect of UVB between HaCaT/NHEK (p < 0.001; upregulation) and SCL-1 (downregulation), which was not evident for Per2. Additionally, an illustration of the overall expression of HaCaT, NHEK, and SCL-1 (B: BMAL1, D:Per2) indicated a significant differential expression of BMAL1 between HaCaT/SCL-1 (p = 0.003) and NHEK/SCL-1 (p < 0.001) but no difference between HaCaT/NHEK, as well no differential expression regarding Per2.
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