4-Hydroxynonenal Contributes to Fibroblast Senescence in Skin Photoaging Evoked by UV-A Radiation

Abstract

Solar ultraviolet A (UV-A) radiation promotes a huge variety of damages on connective tissues and dermal fibroblasts, including cellular senescence, a major contributor of skin photoaging. The mechanisms of skin photoaging evoked by UV-A partly involve the generation of reactive oxygen species and lipid peroxidation. We previously reported that 4-hydroxynonenal (HNE), a lipid peroxidation-derived aldehyde, forms adducts on elastin in the skins of UV-A irradiated hairless mice, possibly contributing to actinic elastosis. In the present study, we investigated whether and how HNE promotes fibroblast senescence in skin photoaging. Dermal fibroblasts of skins from UV-A-exposed hairless mice exhibited an increased number of γH2AX foci characteristic of cell senescence, together with an accumulation of HNE adducts partly colocalizing with the cytoskeletal protein vimentin. Murine fibroblasts exposed to UV-A radiation (two cycles of 15 J/cm²), or HNE (30 µM, 4 h), exhibited senescence patterns characterized by an increased γH2AX foci expression, an accumulation of acetylated proteins, and a decreased expression of the sirtuin SIRT1. HNE adducts were detected on vimentin in cultured fibroblasts irradiated by UV-A or incubated with HNE. The HNE scavenger carnosine prevented both vimentin modification and fibroblast senescence evoked by HNE in vitro and in the skins of UV-A-exposed mice. Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in fibroblast senescence, and confirm the protective effect of carnosine in skin photoaging.

Keywords: 4-hydroxynonenal; UV-A; carnosine; fibroblasts; photoaging; senescence; skin; vimentin.

Figure 1

Expression of HNE adducts in fibroblasts in the skins of UV-A-exposed hairless mice. Immunofluorescence pictures (and higher magnification ×3 of each staining on the right of each picture) showing the presence of vimentin in fibroblasts (red), HNE adducts (green), and the merge (yellow). Upper left panel, control (untreated); Upper right panel, UV-A irradiated (20 J/cm²/d up to 600 J/cm²); lower left panel, propylene glycol (PG)-treated UV-A irradiated; lower right panel, carnosine (1% in PG)-treated/UV-A irradiated, as indicated in [32]. Scale bar, 20 µm, magnification 5 µm. Nuclei were stained with DAPI (blue). These data are representative of five separate experiments.

Figure 2

Expression of γH2AX in the skins of UV-A-exposed hairless mice. (A), immunofluorescence and confocal imaging of γH2AX (green) in skin fibroblasts from hairless mice untreated (control), UV-A-exposed (UV-A), UV-A-exposed/propylene glycol (UV-A+PG), and UV-A-exposed + carnosine (1% in PG). Nuclei were stained with DAPI (blue). Scale bar, 20 µm. Inserts indicate the area selected for higher magnification ×4, right panels. White arrows indicate the γH2AX positive nuclei. (B), statistical quantification of the percentage of γH2AX positive cells in the dermis area. The data are expressed as medians ± interquartile range. Statistical significance was assessed using the nonparametric Mann-Whitney U test (* p < 0.05).

Figure 3

Expression of γ-H2AX evoked by UV-A or HNE in fibroblasts nuclei. Fibroblasts were exposed to UV-A (two cycles of irradiation, each up to 15 J/cm² in HBSS medium) or HNE (30 µM, 4 h in HBSS medium), ± carnosine (100 µM). (A), Cell viability evaluated by the MTT test, in fibroblasts exposed to UV-A or HNE, ± carnosine. (B), expression of γH2AX and protective effect of carnosine evaluated by western blot of fibroblasts exposed to UV-A or HNE, as described in 3A. Right panel, statistical quantification of γH2AX, expression, data are represented by means ± SEM of four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. (C), immunofluorescence and confocal imaging of γH2AX foci in fibroblasts treated by UV-A (upper panels) or HNE (lower panels) ± carnosine. Nuclei were stained with DAPI. Scale bar, 20 µm.

Figure 4

SIRT1 expression and accumulation of acetylated and ubiquitinated proteins in UV-A- and HNE-treated fibroblasts. (A), SIRT1 expression evaluated by western blot in fibroblasts exposed to UV-A or HNE, and protection by carnosine, using β-actin as control. Left panel, effect of HNE; right panel, effect of UV-A. On the right of each western blot picture, statistical quantification of SIRT1 in UV-A- or HNE-treated cells vs control untreated fibroblasts. (B), Western blot experiments showing the accumulation of acetylated (upper panel), and high molecular weight polyubiquitinated proteins (lower panel), in fibroblasts stimulated by HNE or UV-A, and protective effect of carnosine. Right panel, statistical quantification of acetylated and ubiquitinated vs control untreated fibroblasts. These results are a mean of four separate experiments and are expressed as means ± SEM. Statistical analysis was assessed using a Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

HNE adduct formation on vimentin in fibroblasts exposed to HNE. Cultured fibroblasts were exposed to UV-A (two cycles of irradiation, each up to 15 J/cm² in HBSS medium) or HNE (30 µM, 4 h in HBSS medium), ± carnosine (100 µM). Western blot analyses were carried out on cells incubated with HNE as described above, followed by 24 h incubation at 37 °C in fresh RPMI medium supplemented with 1% FBS as indicated in the Method section. (A), Representative confocal imaging pictures showing the presence of HNE adducts (green, lower panel) on vimentin (red, middle panel), and the merge (yellow, upper panel), in fibroblasts incubated with HNE, and protective effect of carnosine. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. In insert, original 63× confocal image. (B), Detection of HNE adducts on vimentin immunoprecipitates from fibroblasts incubated with HNE and protective effect of carnosine (mean of two separate experiments) (C), Expression of vimentin in fibroblasts incubated with HNE and protective effect of carnosine. These results are the mean of three separate experiments and are expressed as means ± SEM. Statistical analysis was assessed using a Student t-test. * p < 0.05.

Figure 6

HNE adduct formation on vimentin in UV-A-exposed fibroblasts. (A) representative confocal imaging pictures showing the presence of HNE adducts (green, lower panels) on vimentin filaments (red, middle panels), and the merge (yellow, upper panel), in fibroblasts exposed to UV-A ± carnosine. White arrows indicate the colocalization areas (merge pictures). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (B), Expression of vimentin in fibroblasts exposed to UV-A, and effect of carnosine. These results are the mean of three separate experiments and are expressed as means ± SEM. Statistical analysis was assessed using a Student t-test. ns, non-significant.