Different oxidative stress response in keratinocytes and fibroblasts of reconstructed skin exposed to non extreme daily-ultraviolet radiation

Abstract

Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as "daily UV radiation" (DUVR) with a higher UVA (320-400 nm) to UVB (280-320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure.

Figure 1. Tissular and cellular effects of simulated UV daylight on human reconstructed skin.

(a): DUVR spectrum was recorded with a calibrated Macam spectroradiometer. Hatched area represents UVA portion in DUVR spectrum. (b): Sham irradiated (control), 7 J/cm² and 13 J/cm² DUVR exposed samples were taken for classical histology and for vimentin (vimentin: green labelling, nuclei counterstaining: red labelling) and p53 immunolabelling at 48 h. Exposure to 7 J/cm² DUVR did not induce changes compared to the control sample. In contrast, 13 J/cm² DUVR led to the disappearance of superficial dermal fibroblasts and to the induction of p53 positive keratinocytes (arrows). (c) Samples were sham irradiated (control) or exposed to 7 J/cm² and 13 J/cm² DUVR once a day for 5 consecutive days. They were sampled for histology and vimnetin immunostaining 72 h after the last exposure. Compared to controls, samples treated with repeated doses of DUVR showed drastic alterations in epidermis (decreased thickness and death of suprabasal keratinocytes) and dermis (decrease in the number of fibroblasts). Arrows indicate living fibroblasts, brackets show living keratinocytes. (d): MMP-1 ELISA assay : the amount of MMP-1 produced in the culture medium was measured after acute or repeated exposures (once a day for 5 consecutive days) to DUVR at the doses indicated. Asteriks indicate significant different values. Bar = 50 µm.

Figure 2. ROS assay in reconstructed skin exposed to DUVR.

Left pannel - Levels of DCFH-DA fluorescence in reconstructed skin after DUVR exposure. Right pannel - Sections of reconstructed skin after DCFH-DA probe incorporation and DUVR exposure. Bracket and arrows indicated the fluorescent keratinocytes and fibroblasts respectively in DUVR-exposed samples. None of them were detected in non-exposed reconstructed skin. Dotted line delimitates epidermis and dermis.

Figure 3. Distribution, type and mean of gene modulation after DUVR exposure of human reconstructed skin.

2, 6 and 24 hours after DUVR exposure, mRNA levels of 24 oxidative stress markers were quantified by QPCR in fibroblasts (F) and keratinocytes (K) of reconstructed skin separately, in three independent experiments per time point. (a) Number and type of modulation of significantly modulated genes. (b) Means and confidence intervals of the log of modulation ratios. At each time point, gene modulation ratios (mRNA amount in exposed sample to mean mRNA amount in the 3 control samples) were calculated for each marker. Means and confidence intervals of the 24 log₁₀ of modulation ratio were calculated. Values higher or lower than 0, correspond to induction or repression of mRNA amount, respectively.

Figure 4. Modulation of mRNA and protein levels of Nrf2 target genes in reconstructed skin exposed to DUVR.

(a): 2 and 6 hours after DUVR exposure (7 or 13 J/cm²), mRNA levels of HO-1, TXNR, NQO1, γ GCS-L and γ GCS-H were quantified by QPCR in fibroblasts (F) and keratinocytes (K). Each histogram bar shows the mean value ± standard error of mean (SEM) of normalized mRNA amount (n = 3). mRNA amount in sham-exposed samples was adjusted to the 1 value. Indicated values correspond to significant modulations (*, p<0.05). (b): 6 and 24 hours after 7 J/cm² DUVR, HO-1 and TXNR protein levels, respectively present in whole cell and cytosolic extracts, were determined by western blot in fibroblasts (F) and keratinocytes (K) of reconstructed skin. GAPDH levels were used to normalize data. Positive control: normal human melanocytes treated with 20 µM forskolin.

Figure 5. Effects of DUVR on mRNA levels of sestrins in human reconstructed skin.

At 2 hours and 6 hours after DUVR exposure, mRNA levels of sestrins genes i.e. SESN1-T1, SESN1-T2, SESN3, SESN2 were quantified by QPCR in fibroblasts (F) and keratinocytes (K) of reconstructed skin, in sham or DUVR exposed samples (7 or 13 J/cm²). Each histogram bar shows the mean value ± SEM of normalized mRNA amount (n = 3). mRNA amount of each marker in sham-exposed samples was adjusted to the 1 value. Indicated values correspond to means of mRNA amount in DUVR exposed samples statistically different from means of mRNA amount in control samples (*, Student't test, p<0.05).

Figure 6. Effects of DUVR on mRNA levels of metallothionein subunits in human reconstructed skin.

At 6 hours after DUVR exposure, mRNA levels of metallothionein genes i.e. MT1G, MT1X, MT1E and MT2A were quantified by QPCR in fibroblasts (F) and keratinocytes (K) of reconstructed skin, in sham or DUVR exposed samples (7 or 13 J/cm²). Each histogram bar shows the mean value ± SEM of normalized mRNA amount (n = 3). mRNA amount of each marker in sham-exposed samples was adjusted to the 1 value. Indicated values correspond to means of mRNA amount in DUVR exposed samples statistically different from means of mRNA amount in control samples (*, Student't test, p<0.05).

Figure 7. Effects of DUVR on MSRA mRNA and protein levels in human reconstructed skin.

(a) 2, 6 and 24 hours after DUVR exposure (7 or 13 J/cm²), mRNA levels of MSRA were quantified by QPCR in fibroblasts (F) and keratinocytes (K) of reconstructed skin. Each histogram bar shows the mean value ± SEM of normalized mRNA amount (n = 3). mRNA amount of sham-exposed samples was adjusted to the 1 value. Indicated values correspond to significant modulations (see Material and Methods, * Student't test, p<0.05). (b) 6 and 24 hours after 7 J/cm² DUVR, levels of MSRA protein was determined in whole cell extracts of epidermal keratinocytes of reconstructed skin. Levels of GAPDH were used to normalize data. The arrow indicates the band corresponding to MSRA.