The proteasome is an integral part of solar ultraviolet a radiation-induced gene expression

Abstract

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

Figures

FIGURE 1.

Effect of UVA treatment on protein carbonyl formation and proteasome activity in dermal fibroblast cells. Cells were cultured and exposed to UVA as described under “Experimental Procedures.” Three hours later the cells were harvested, lysed, and analyzed for the protein carbonyl levels (A) by enzyme-linked immunosorbent assay (data are the means ± S.E.; n = 4; *, p < 0.05 versus nonirradiated, ANOVA, Bonferroni's multiple comparison test) or the 20 S proteasome activity (B) (data are the means ± S.E.; n = 4; *, p < 0.05 versus nonirradiated, ANOVA, Bonferroni's Multiple Comparison test). The same lysates were analyzed for the proteasome content (C) by an immunoblot (see the upper panel) and quantified amount (see the lower panel). Immunodetection of proteasomal subunits was by employing a polyclonal anti-proteasome antibody. MCA, 7-amino-4-methylcoumarin.

FIGURE 2.

Effect of UVA treatment on protein carbonyl formation and proteasome content in vivo. Human skin was UVA exposed in vivo, and biopsies were taken 1 h after irradiation or sham treatment. Biopsies were split into half. One part was used for biochemical analysis. After homogenization in PBS, the homogenate was analyzed by enzyme-linked immunosorbent assay for protein oxidation (A) or proteasome activity (B) (data are the means ± S.E.; controls in one experiment were set to 100%. n = 4; *, p < 0.05 versus nonirradiated, Student's t test). The other part was cut into 5-μm-thick sections and used for histochemical analysis. The protein carbonyl formation was measured after DNP hydrazine reaction using a rabbit polyclonal anti-DNP (dinitrophenyl) primary antibody and TRITC-labeled secondary antibody (C), whereas the proteasome content was detected using a rabbit polyclonal anti-20 S primary antibody and fluorescein isothiocyanate (FITC)-labeled secondary antibody (D). In both panels 4′,6-diamidino-2-phenylindole (DAPI) staining was used to identify cell nuclei. Quantification was done separately for the epidermal and dermal layers of the skin (data are the means ± S.E.; n = 4; *, p < 0.05 versus nonirradiated, ANOVA, Bonferroni's multiple comparison test).

FIGURE 3.

UVA treatment of dermal fibroblast cells causes singlet oxygen formation and consequently inhibits proteolysis and proteasome activity. Fibroblasts were UVA treated as described under “Experimental Procedures” with the exception that NaN3 as a potential 1O2 quencher was present. Panel A demonstrates the proteasomal activity after UVA treatment. Data are the means ± S.E. (n = 3). *, p < 0.05 versus 0J/cm2, ANOVA, Bonferroni's multiple comparison test. To mimic the 1O2 generation of UVA, we used the singlet oxygen donor NDPO2 and tested the effect on the proteasomal activity (B). Data are the means ± S.E. (n = 3). *, p < 0.05 versus untreated, Student's t test. 20 S proteasome was isolated as described before (14), and the effects of NDPO2 were tested on its activity (C). Control experiments were performed with solutions of pre-decomposed NDPO2 containing the decomposition product, NDP. Data are the means ± S.E. (n = 3). In panel D the effect of UVA on the isolated 20 S proteasome was tested. UVA was used here in significantly higher doses compared with cell treatment. Data are the means ± S.E. (n = 3) ANOVA, Bonferroni's multiple comparison test. The effect of UVA on the overall protein degradation in fibroblasts after UVA irradiation was tested (E). The release of trichloroacetic acid-soluble radioactivity within 3 h from the intracellular protein pool was used as a measure of proteolysis. The data represent the means ± S.E. of three independent experiments, each with six independent measurements (*, p < 0.05 versus 0J/cm2, Student's t test). MCA, 7-amino-4-methylcoumarin.

FIGURE 4.

UVA treatment of dermal fibroblasts causes protein aggregation. Detergent solubility of cellular proteins was measured using liquid scintillation counting after [35S]methionine/cysteine labeling and UVA treatment in the absence or presence of the singlet oxygen quencher sodium azide (20 mm). The data represent the means ± S.E. of three independent experiments (*, p < 0.05 versus nonirradiated, ANOVA, Bonferroni's multiple comparison test). Detergent-soluble (A) proteins and detergent-insoluble aggregates (B) were determined without and after 30J/cm2 UVA treatment.

FIGURE 5.

UVA induced MMP-1 and TIMP-1 expressions in fibroblasts after UVA treatment. The dose dependence of MMP-1 mRNA (A) and TIMP-1 mRNA (B) expressions 3 h post-irradiation are shown (n = 3; *, p < 0.05 versus nonirradiated, ANOVA, Bonferroni's multiple comparison test. Data are the means ± S.E.).

FIGURE 6.

Cross-linked protein aggregates inhibit the proteasome activity and cause an increase in MMP-1 mRNA expression and a decrease in TIMP-1 mRNA expression. Fibroblasts were treated with artificial lipofuscin as described under “Experimental Procedures.” Panel A shows the autofluorescence image after 2 weeks of incubation of fibroblasts with 0.6 mg of lipofuscin, whereas in panel B the quantification of autofluorescence using fluorometry with 360-nm excitation and 590-nm emission is demonstrated. Data are the means ± S.E., (n = 3). *, p < 0.05 versus untreated, Students t test. C, the proteasome activity without and with lipofuscin treatment of the cells was measured as described under “Experimental Procedures.” Panel D shows the relative MMP-1 mRNA level compared with 18 S rRNA levels, and panel E shows the relative TIMP-1 mRNA level compared with 18 S rRNA levels (n = 3, *, p < 0.05 versus untreated, Student's t test. Data are the means ± S.E.).

FIGURE 7.

Proteasome inhibition increases MMP-1 mRNA levels. Fibroblasts were treated with lactacystin in panels A–C. Panel A shows the proteasome activity inhibition by 20 μm lactacystin. n = 3; *, p < 0.05 versus untreated. Panel B shows the MMP-1 mRNA expression without and with lactacystin. n = 3; *, p < 0.05 versus untreated, whereas panel C demonstrates the TIMP-1 mRNA expression without and with lactacystin. n = 3; *, p < 0.05 versus untreated. Student's t test. Data are the means ± S.E.

FIGURE 8.

Proteasome overexpression attenuates UVA-induced protein aggregation and rise in MMP-1 mRNA levels. A cell clone overexpressing the β5-subunit of the proteasome or the vector control was exposed to UVA. A, the proteasome activity after β5-subunit overexpression was measured; n = 3; *, p < 0.05 versus vector control. B, viability was measured under control conditions (gray columns) or after UVA treatment (black columns) in untransfected cells, vector control, and β5-overexpressing cells. C, protein aggregation (levels of detergent-insoluble aggregates, right panel) and levels of detergent-soluble proteins (left panel) were determined as in Fig. 4; n = 3; *, p < 0.05 versus nonirradiated. Panel D demonstrates the MMP-1 mRNA levels in vector control and β5-overexpressing cells after UVA treatment (black columns) or untreated cells (gray columns) after proteasome overexpression. n = 3; *, p < 0.05 versus nonirradiated vector cells; &, p < 0.05 versus irradiated vector cells, ANOVA, Bonferroni's multiple comparison test. Data are the means ± S.E.

FIGURE 9.

Effect of MsrA overexpression on UVA-induced outcomes. MsrA-transfected cells were used for the experiment, and vector-transfected cells were used as controls. All experiments were performed in triplicate. *, p < 0.05, versus nonirradiated vector; &, p < 0.05, versus 30 J/cm2 irradiated vector, ANOVA, Bonferroni's multiple comparison test. Data are the means ± S.E. Panel A shows the effect of UVA on viability in vector and MsrA-transfected fibroblasts. B, protein aggregation (levels of detergent-insoluble aggregates, right panel) and levels of detergent-soluble proteins (left panel) were determined as in Fig. 4; n = 3; *, p < 0.05 versus nonirradiated. The proteasome activity after UVA treatment in vector and MsrA-overexpressing cells is demonstrated in panel C, whereas panel D shows MMP-1 mRNA levels after UVA treatment in vector and MsrA-overexpressing cells; &, p < 0.05 versus irradiated cells in the first and second columns.

FIGURE 10.

C-Jun expression, phosphorylation, and AP-1 activation are results of proteasome inhibition. Fibroblasts were irradiated with 30 J/cm2, treated with lipofuscin (LF) or lactacystin (LC), or used without treatment (C) as described under “Experimental Procedures.” Cells were harvested and lysed, and proteins were analyzed by immunoblotting with anti-c-Jun, anti-Pi-c-Jun, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies giving the products with the sizes of 40, 47, and 37 kDa, respectively (A). Immunoblotting for glyceraldehyde-3-phosphate dehydrogenase (bottom panel) showed equal loading of the proteins in each lane. The results of quantitative analysis of these immunoblots are depicted in the columns in the lower part of the panel. Amounts were quantified in relation to glyceraldehyde-3-phosphate dehydrogenase by densitometry. B, the Cignal AP1-GFP reporter assay measures the activation of AP-1 (see “Experimental Procedures”). After UVA and lactacystin treatments, bright field and fluorescent images were taken of the cultures. Clearly the green florescence in response to AP-1 activation after UVA and lactacystin is visible. The effect of JNK inhibitors (JNKi I and SP600125 or JNKi II) on lactacystin-induced MMP-1 mRNA level increase is shown in panel C; n = 3; *, p < 0.05 versus control cells without lactacystin treatment; &, p < 0.05 versus only lactacystin-treated cells, ANOVA, Bonferroni's multiple comparison test.