The Ultraviolet Irradiation of Keratinocytes Induces Ectopic Expression of LINE-1 Retrotransposon Machinery and Leads to Cellular Senescence

Abstract

Retrotransposons have played an important role in evolution through their transposable activity. The largest and the only currently active human group of mobile DNAs are the LINE-1 retrotransposons. The ectopic expression of LINE-1 has been correlated with genomic instability. Narrow-band ultraviolet B (NB-UVB) and broad-band ultraviolet B (BB-UVB) phototherapy is commonly used for the treatment of dermatological diseases. UVB exposure is carcinogenic and can lead, in keratinocytes, to genomic instability. We hypothesize that LINE-1 reactivation occurs at a high rate in response to UVB exposure on the skin, which significantly contributes to genomic instability and DNA damage leading to cellular senescence and photoaging. Immortalized N/TERT1 and HaCaT human keratinocyte cell lines were irradiated in vitro with either NB-UVB or BB-UVB. Using immunofluorescence and Western blotting, we confirmed UVB-induced protein expression of LINE-1. Using RT-qPCR, we measured the mRNA expression of LINE-1 and senescence markers that were upregulated after several NB-UVB exposures. Selected miRNAs that are known to bind LINE-1 mRNA were measured using RT-qPCR, and the expression of miR-16 was downregulated with UVB exposure. Our findings demonstrate that UVB irradiation induces LINE-1 reactivation and DNA damage in normal keratinocytes along with the associated upregulation of cellular senescence markers and change in miR-16 expression.

Keywords: BB-UVB; HaCaT; N/TERT1; NB-UVB; cellular senescence; genomic instability; keratinocytes; long interspersed nucleotide element-1 (LINE-1); miR-16; microRNAs; phototherapy.

Figure A1

UV irradiation decreases cell proliferation and increases cell diameter. (A) The proliferation curve of N/TERT1 cells at 1, 2, 3, and 4 days following 6 UV irradiations with either NB-UVB or BB-UVB as compared to an unirradiated control sample. (B) Cell diameter of N/TERT1 or cells after 6 UV irradiations (50–70 cells/condition). The photos were taken on an Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969) (scale bar 10 μm). Significance for N/TERT1 samples was calculated by a two-way ANOVA test and corrected for multiple comparisons using Dunnett’s test (both the day of measurement and the UV radiation were significant as compared with Day 1 and the control, respectively, p < 0.05).

Figure 1

UV irradiation induces DNA damage. (A) The expression of γH2AX (green) in N/TERT1 keratinocytes as shown by immunofluorescence staining following 24 h of UV irradiation with either NB-UVB or BB-UVB as compared to unirradiated control samples (n = 3 with 500 cells/condition). The three patterns of γH2AX staining correspond to the number of double-stranded DNA breaks, i.e., type 1 expression <10 nuclear foci (low-level DNA damage), type 2 expression >10 nuclear foci (high-level DNA damage), and type 3 pan-nuclear expression (pre-apoptotic state). The photos were taken on an Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969) (scale bar 10 μm). Significance was calculated using a mixed-effects model to analyze the data, and for multiple comparisons correction, Dunnett’s test was applied. (B) Nuclear size counts of NTERT cells after 6 UV irradiations (n = 3 with 50–69 cells/condition). Significance was calculated using the one-way ANOVA test and was corrected for multiple comparisons using Dunnett’s test. (**** p value < 0.0001, ** p value < 0.0021, * p value < 0.05).

Figure 2

UV irradiation decreases cell proliferation and increases cell diameter. (A) The proliferation curve of HaCaT (n = 3) cells at 1, 2, 3, and 4 days following 6 UV irradiations with either NB-UVB or BB-UVB as compared to unirradiated control samples. (B) Cell diameter of HaCaT (n = 3) cells after 6 UV irradiations (50–70 cells/condition). The photos were taken on an Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969) (scale bar 10 μm). Significance was calculated using a mixed-effects model to analyze the data, and for multiple comparisons correction, Tukey’s test was performed. (**** p value < 0.0001, ** p value < 0.0021, * p value < 0.05).

Figure 3

LINE-1 protein expression on HaCaT and N/TERT1 cells after 6 UV exposures with immunofluorescence. The expression of ORF1 proteins of LINE1 elements (red) as counterstained by DAPI (blue) in HaCaT (A,B) and N/TERT1 (C,D) keratinocytes as shown by immunofluorescence staining after 24 h of 6 UV irradiations with either NB-UVB or BB-UVB as compared to unirradiated control samples. The photos were taken on an Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969) (scale bar 20 μm). Significance was calculated by a one-way ANOVA test and was corrected for multiple comparisons using Dunnett’s test. (*** p value < 0.0002, ** p value < 0.0021).

Figure 4

LINE-1 protein and mRNA expression in HaCaT and N/TERT1 cells after 6 UV exposures. (A) Measurement of ORF1 protein of LINE-1 elements using Western blotting in N/TERT1 cells. The mRNA expression of ORF2 protein of LINE-1 elements in N/TERT1 cells (n = 6) (B) and HaCaT cells (n = 3) (C) as normalized by the mRNA expression of GAPDH. Kruskal–Wallis nonparametric test was performed, and the statistical significance was corrected for multiple comparisons using Dunn’s test (** p value < 0.0021, * p value < 0.05).

Figure 5

Time course of LINE-1 protein expression in N/TERT1 cells following 6 UV exposures with NB-UVB. (A) Immunofluorescence visualization of the expression of ORF1 protein (green) counterstained with DAPI (blue) in N/TERT1 cells (n = 5) after 6 irradiations with NB-UVB at different time points (0, 1, 3, 6, 16, and 24 h) (B) Quantification of LINE-1 expression at different time points compared with the expression immediately after NB-UVB exposure. The photos were taken on an Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969) (scale bar 20 μm). Significance was calculated by a one-way ANOVA test corrected for multiple comparisons using Dunnett’s test. (**** p value < 0.0001, * p value < 0.05).

Figure 6

The mRNA expression of senescence markers following multiple UV exposures. The mRNA expression of senescence markers in N/TERT1 cells (n = 4) (A–E) and HaCaT cells (n = 3) (F–J) following 6 UV irradiations. Significance was calculated by the Kruskal–Wallis nonparametric test using the uncorrected Dunn’s test (** p value < 0.0021, * p value < 0.05).

Figure 7

The expression of regulatory RNAs of LINE-1 reactivation. (A) LINE-1 mRNA sequence and its microRNAs binding sites. The microRNAs selected are those from the cross-search between microRNAs dysregulated upon UV irradiation and the ones that are able to target LINE-1 mRNA. (B–I) The expression of selected miRNAs in N/TERT1 cells following 6 repeated UV exposures (n = 4). Significance was calculated by one-way ANOVA using Fisher’s LSD test (* p value < 0.05).