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First published online 25 January 2005
doi: 10.1242/jcs.01651

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Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-ß1 signaling pathway

¹ Laboratory of Biochemistry and Cellular Biology, Department of Biology, University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur, Belgium
² Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement – EA 3106 – Université Paris 7, France
³ Eppendorf Array Technologies, Rue du Séminaire, 12, 5000 Namur, Belgium

View larger version (58K): [in a new window]   Fig. 1. Exposure to a series of 10 stresses to UVB at 250 mJ/cm2 or 72 hours of stimulation with TGF-ß1 do not lead to apoptosis. (A) Cytotoxicity at 24 hours after 10 exposures to UVB. Doses of UVB ranged from 0 to 500 mJ/cm2 with 2 stresses per day for 5 days. Results are expressed as percentage of cell survival compared to day 0 (d0, 100%) before any stress. Results are given as mean ± s.d. of three independent experiments. Statistical analysis was carried out with Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01. (B) No activation of caspase-3 after 10 exposures to UVB at 250 mJ/cm2 or 72 hours of stimulation with TGF-ß1. Semi-quantitative confocal microscopy of skin HDFs that were seeded on glass cover slides at 16, 40 or 64 hours after the last of a series of 10 exposures to UVB at 250 mJ/cm2 (micrographs 6, 8, 10). Control cells submitted to the same culture conditions but not exposed to UVB were checked (micrographs 5, 7, 9). Cells exposed to etoposide 25 µM for 16 hours were used as positive controls (micrograph 2). Cells incubated for 16 hours in BME were used as negative controls (micrograph 1). At 24 hours after seeding, the activation of caspase-3 was detected using a specific anti-active caspase-3 antibody (green). The nuclei were stained with TO-PRO-3 (blue). No pro-apoptotic effect was found after 72 hours of stimulation with 5 ng/ml of TGF-ß1 (micrograph 4) compared with the controls (micrograph 3). (C) No cleavage of PARP after a series of 10 exposures of skin HDFs to UVB at 250 mJ/cm2 or 72 hours of stimulation with TGF-ß1. Skin HDFs were submitted to a series of 10 UVB exposures at 250 mJ/cm2. Proteins were extracted at 4, 16, 40 or 64 hours after the last stress. Control cells submitted to the same culture conditions but not exposed to UVB were checked. Cells exposed to cytotoxic concentrations of t-BHP or not were used as respective positive (CTL +) and negative (CTL –) controls. Total cell extracts were analyzed by western blotting with an anti-PARP-1 antibody. The full length PARP-1 protein (116 kDa) and the fragment resulting for PARP cleavage (85 kDa) are indicated. -tubulin protein level was used as a reference. No cleavage of PARP was observed after 72 hours of stimulation with 5 ng/ml TGF-ß1 (TGF) or not (CTL).

View larger version (36K): [in a new window]   Fig. 2. Effect of a series of 10 exposures of HDFs at early cumulative population doublings (CPDs) to UVB at 250 mJ/cm2 on SA ß-gal activity, common 4,977 bp mitochondrial DNA deletion, incorporation of [3H]thymidine and the protein levels of p53, p21WAF-1 and p16INK-4A. (A) Proportion of cells positive for the SA ß-gal activity at 72 hours after 10 exposures to UVB at 250 mJ/cm2. Cells at early CPDs before exposure to UVB (d0), incubated 10 times in a thin layer of PBS without (CTL) or with UVB exposure (UVB) and cells at late CPDs (±90% of their proliferative life span) were examined. Results are given as mean ± s.d. of 3 independent experiments. (B) Detection of the common 4,977 bp mitochondrial DNA deletion by nested PCR after 10 exposures of UVB at 250 mJ/cm2. (a) Detection of a conserved 247 bp fragment of the mitochondrial DNA in cells incubated 10 times in a thin layer of PBS exposed to UVB (UVB) or not (CTL). Cells at day 0 (d0, before any incubation in PBS) were also examined. (b) Detection of the deletion by nested PCR. The 4,977 bp deletion is detected (404 bp product) in UVB-exposed cells (UVB) and not in CTL or d0 cells. (C) Effect of repeated UVB stresses on the incorporation of [3H]thymidine. Skin HDFs at early CPDs were incubated 10 times in a thin layer of PBS exposed to UVB (250 mJ/cm2) or not (CTL). They were trypsinised after 24, 48 or 72 hours after the last stress and incubated for 48 hours with [3H]thymidine at 1 µCi. Results are given as mean ± s.d. of three independent experiments. (D) (a) Analysis of p53, p21WAF-1 and p16INK-4 protein level by western blotting. Proteins were extracted at 72 hours after the last stress. -tubulin protein was used as reference level. (b) Quantification of the protein level. The results are expressed as 100% of the protein level in control cells (CTL). Results are given as mean ± s.d. of three independent experiments. Statistical analysis was carried out with the Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01; **, 0.01>P>0.001; ***, P<0.001.

View larger version (22K): [in a new window]   Fig. 3. Effects in HDFs at early cumulative population doublings (CPDs) to 10 exposures of UVB at 250 mJ/cm2 per exposure on the steady-state mRNA level of senescence- and cell cycle-associated genes, and the level of intracellular and extracellular protein level of apo J. (A) Steady-state mRNA level of apolipoprotein J (apo J), fibronectin (fibro), osteonectin (osteo), SM22, p21WAF-1, p53, c-fos and c-jun. Total RNA was extracted at 72 hours after the last stress. The GAPDH steady-state mRNA level was used as reference in the real-time RT-PCR. (a) The results obtained from the UVB-treated cells are expressed as percentage of the steady-state mRNA level of the respective mRNA species in control cells (CTL). (b) The results obtained with senescent cells (late CPDs) are compared with cells at early CPDs. Results are given as mean ± s.d. of three independent experiments. (B) Analysis of intracellular (a) and extracellular (b) protein level of apo J by western blotting. Proteins were extracted at 72 hours after the last stress. -tubulin protein was used as reference level to estimate the intracellular level of apo J while the same volume of collected medium was used to determine the extracellular levels. Statistical analysis was carried out with the Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01; **, 0.01>P>0.001; ***, P<0.001.

View larger version (19K): [in a new window]   Fig. 4. Effects of retrovirally transfected apo J against UVB-induced SIPS. (A) Overexpression of human apo J after retrovirally-mediated overexpression in skin HDFs. (a) Overexpression at mRNA level of both native and tagged (V5/His) apo J compared to empty PLXSN vector. Real time RT-PCR was used to detect apo J mRNA. (b) Overexpression of protein level of both native and tagged (V5/His) apo J compared to empty pLXSN vector (western blotting). (B-D) Effects of retrovirally transfected apo J against SIPS induced by 10 subcytotoxic exposures to 250 mJ/cm2 UVB in skin HDFs. SA ß-gal activity (B), incorporation of [3H]thymidine (C) and relative steady-state mRNA level of fibronectin (D) were studied as described in Materials and Methods. Skin HDFs transfected with the empty pLXSN vector, with pLXSN/apo J vector or pLXSN/apo J-V5/His vector were exposed (gray columns) or not (white columns) to UVB. Results are given as mean ± s.d. of three independent experiments except (D) where pools of RNA were obtained from extracts of RNA from three independent experiments. Statistical analysis was carried out with the Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01; **, 0.01>P>0.001; ***, P<0.001.

View larger version (69K): [in a new window]   Fig. 5. Oxidized protein patterns in SIPS and replicative senescence. SIPS was induced by 10 exposures of AG04431 HDFs to UVB at 250 mJ/cm2, five exposures of WI-38 HDFs to t-BHP (Dierick et al., 2002), and a single exposure of IMR-90 HDFs to H2O2 (Frippiat et al., 2001). Procedures for detecting oxidized proteins are described in Materials and Methods. (a) Lane 1: control AGO4431 HDFs at early cumulative population doublings (CPDs). Lanes 2, 3: AGO4431 HDFs exposed to a series of 10 UVB stresses and proteins extracted at 24 and 72 hours, respectively, after the last stress. Lane 4: control WI-38 cells at early cumulative population doublings. Lanes 5, 6: WI-38 HDFs exposed to a series of 5 t-BHP stresses and proteins extracted at 24 and 72 hours, respectively, after the last stress. Lane 7: control IMR-90 cells at early at early cumulative population doublings. Lanes 8, 9, 10: H2O2 stressed IMR-90 cells, protein were extracted at 6, 24 and 72 hours, respectively, after the stress. Lanes 11, 12: WI-38 cells at early and late CPDs respectively. MW: molecular weights. (b) -tubulin was used as reference protein. The arrows indicate the most evident modifications of the oxidized protein patterns.

View larger version (24K): [in a new window]   Fig. 6. Role of TGF-ß1 in UVB-induced SIPS. (A) Steady-state mRNA level of TGF-ß1 in skin HDFs exposed (UVB) or not (CTL) to 10 repeated subcytotoxic doses of UVB at 250 mJ/cm2. Total RNA samples were extracted at 24, 48 or 72 hours after the last stress. Results were obtained by real-time RT-PCR with GAPDH mRNA as reference. The steady-state mRNA level of TGF-ß1 in control cells after 24 hours was considered as 100%. Results are given as mean ± s.d. of three independent experiments. (B) Steady-state mRNA level of TGF-ß2 and TGF-ß3 at 72 hours after a series of 10 exposures to UVB at 250 mJ/cm2. Results are given as mean ± s.d. of three independent experiments. (Ca) Effects of the stimulation of skin HDFs with TGF-ß1 at 1, 5 and 10 ng/ml on the percentage of cells positive for SA ß-gal activity after 72 hours of stimulation. Results are given as mean ± s.d. of three independent experiments. (Cb) Effect of anti-TGFß-1 receptor II and anti-TGF-ß1-neutralizing antibodies on the proportion of cells positive for SA ß-gal activity at 72 hours after exposure of skin HDFs to 10 subcytotoxic stress with 250 mJ/cm2 UVB. Control (CTL) cells had no UVB exposure. The concentration and incubation conditions of antibodies are given in the Materials and Methods. (D) Steady-state mRNA level of apolipoprotein J (apo J), fibronectin (fibro), osteonectin (osteo), SM22, p21WAF-1, p53 and TGF-ß1 after 72 hours of stimulation with TGF-ß1 at 5 ng/ml. Total RNA was extracted at 72 hours after the last stress. The GAPDH steady-state mRNA level was considered as reference level with real time RT-PCR. The results are expressed as 100% for the steady-state mRNA level in control cells (CTL) not exposed to UVB. Results are given as mean ± s.d. of three independent experiments. Statistical analysis was carried out with the Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01; **, 0.01>P>0.001; ***, P<0.001.

View larger version (69K): [in a new window]   Fig. 7. A series of 10 exposures of skin HDFs to UVB at 250 mJ/cm2 increases sharply the abundance of both latent and active forms of TGF-ß1. Skin HDFs were given a series of 10 exposures to UVB at 250 mJ/cm2 and seeded on glass cover slides at 16, 40 or 64 h after the last stress. Control cells were submitted to the same culture conditions but not exposed to UVB. At 24 hours after seeding, the activation of TGF-ß1 was investigated by immunofluorescence using antibodies recognizing the latent form of TGF-ß1 (LAP) (green; left images) and the active TGF-ß1 (red; middle images) using semi-quantitative confocal microscopy. (Right images) Superimposition of phase contrast and fluorescence with green and red emission.

View larger version (26K): [in a new window]   Fig. 8. Effects of anti-TGF-ß1 neutralizing antibody (3 µg/ml) on the steady-state mRNA level of apolipoprotein J (A), osteonectin (B), SM22 (C), TGF-ß1 (D), fibronectin (E) and p21WAF-1 (F) at 72 hours after a series of 10 exposures of skin HDFs to UVB at 250 mJ/cm2. Anti-TGF-ß1 CTL and CTL cells had no UVB exposure and were incubated with or without anti-TGF-ß1 antibodies, respectively. Statistical analysis was carried out with the Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01.