Synergistic interplay of UV radiation and urban particulate matter induces impairment of autophagy and alters cellular fate in senescence-prone human dermal fibroblasts

Abstract

Skin aging is a complex process influenced by intrinsic factors and environmental stressors, including ultraviolet (UV) radiation and air pollution, among others. In this study, we investigated the effects of UVA and UVB radiation, combined with urban particulate matter (UPM), on human dermal fibroblasts (HDF). We show here that treatment of HDF with a subcytotoxic dose of UVA/UVB results in a series of events leading to mitochondrial dysfunction, increased ROS levels, and DNA damage. These effects are known to trigger either cellular senescence or cell death, depending on the cells' ability to clear damage by activating autophagy. Whereas UPM treatment in isolation did not affect proliferation or survival of HDF, of note, simultaneous UPM treatment of UV-irradiated cells selectively inhibited autophagic flux, thereby changing cell fate of a fraction of the cell population from senescence to apoptotic cell death. Our findings highlight the synergistic effects of UV radiation and UPM on skin aging, emphasizing the need to consider these factors in assessing the impact of environmental stressors on human health and opening opportunities for developing comprehensive approaches to protect and preserve skin integrity in the face of growing environmental challenges.

Keywords: UV; air pollution; apoptosis; autophagy impairment; mitochondrial dysfunction; senescence; skin aging.

FIGURE 1

UV + UPM treatment in HFF‐2 causes stress‐induced premature senescence. (a) Schematic representation of the UV + UPM treatment. (b) Growth curve showing cumulative population doublings (cPDL) for each treatment. (c) Representative pictures of SA‐β‐galactosidase assay staining at day 9. (d) Percentage of positive SA‐β‐galactosidase cells. (e) Heatmap representing RT‐qPCR data showing mRNA expression of p21, LaminB1, IL‐1, and MMP‐1 on days 4 and day 9. (f) Composite of western blot pictures showing pRb, p53, p53ph (Serin15), p21, LaminB1, and GAPDH on days 4 and 9. Data represents mean values ± SD, n = 3. For statistical analysis one‐way ANOVA was used. In all graphics, ns: non‐significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

UV + UPM‐treated cells show dysfunctional mitochondria, autophagic flux impairment, and increased apoptosis. (a) Representative pictures from Complex V immunofluorescence on day 4. (b) Measurement of mitochondrial fragments per cell on day 4. (c) Representative western blot pictures showing MFN1, FIS1, and GAPDH on day 4. (d) Densitometry of western blot pictures showing FIS1 on day 4. (e) Densitometry of western blot pictures showing MFN1 on day 4. (f) Fluorescence intensity from CM‐H₂XRos FACS at day 4. (f) JC‐1 FACS data showing mitochondrial membrane potential on day Data represents mean values ± SD, n = 3. For statistical analysis one‐way ANOVA was used. (g) JC1 FACS data showing mitochondrial membrane potential on Day 4. In all graphics, ns: non‐significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

UV + UPM treatment causes DNA damage, autophagic flux impairment and increases apoptosis. (a) Representative pictures from γ‐H2AX immunofluorescence on day 4. (b) Fluorescence intensity from γ‐H2AX immunofluorescence on day 4. (c) Quantification of γ‐H2AX foci per nucleus on day 4. (d) Representative pictures from LC3‐GFP HDF cells subjected to UV, UV + UPM +/− bafilomycin at day 4. (e) Quantification of LC3 puncta/cell from LC3‐GFP HDF subjected to UPM, UV, UV + UPM +/− bafilomycin at day 4. (f) Composite of western blot pictures showing LC3 A/B and GAPDH in HDF posttreatment +/− bafilomycin at day 4. (g) Densitometry of LC3‐II on day 4. (h) Fluorescence intensity from active caspase 3 immunofluorescence at day 4. Data represents mean values ± SD, N = 3. For statistical analysis one‐way ANOVA was used. In all graphics ns: non‐significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.