Effects of advanced glycation end products on the expressions and activity of cathepsin D in ultraviolet A-irradiated human dermal fibroblasts

Abstract

Objective To investigate the effects of advanced glycation end products(AGE)on the expressions and activity of cathepsin D(CatD)in ultraviolet A(UVA)-irradiated human dermal fibroblasts. Methods Human dermal fibroblasts were isolated and harvested from the circumcised foreskin of children, and subjected to a primary culture. CCK-8 assay was performed to screen non-cytotoxic concentrations of AGE-bovine serum albumin(BSA). Some fibroblasts were incubated with 50, 100 and 300 mg/L AGE-BSA separately for 24 hours, with untreated cells as the control group. Then, reverse transcription(RT)-PCR, Western-blot analysis and a fluorimetric assay were performed to measure the mRNA and protein expressions as well as activity of CatD, respectively. Some fibroblasts were classified into six groups: control group receiving no treatment, AGE-BSA group and BSA group treated with the highest non-cytotoxic concentration of AGE-BSA and the same concentration of BSA respectively for 24 hours, UVA group irradiated by 10 J/cm2 UVA, UVA-AGE-BSA group and UVA-BSA group treated with AGE-BSA and BSA at the above non-cytotoxic concentration respectively for 24 hours both before and after UVA radiation at 10 J/cm2. After the treatments, RT-PCR, Western-blot analysis and a fluorimetric assay were conducted to detect mRNA and protein expressions and activity of CatD respectively. Results AGE-BSA of 50-200 mg/L exhibited no obvious influence on cellular proliferation of fibroblasts. The fibroblasts incubated with AGE-BSA of 50, 100 and 200 mg/L showed a significant increase in the mRNA expression(0.267 ± 0.007, 0.348 ± 0.007, and 0.418 ± 0.006 respectively), protein expression(1.403 ± 0.181, 2.233 ± 0.090 and 2.477 ± 0.111 respectively), and activity(1.760 ± 0.080, 2.330 ± 0.060 and 2.890 ± 0.080 respectively)of CatD compared with the control group(mRNA: 0.161 ± 0.006; protein: 0.903 ± 0.200; activity: 1.100 ± 0.090, all P < 0.05). AGE-BSA increased CatD expressions and activity in a dose-dependent manner. The mRNA and protein expressions as well as activity of CatD were significantly higher in the UVA group than in the control group(mRNA expression: 0.480 ± 0.005 vs. 0.155 ± 0.005; protein expression: 2.583 ± 0.199 vs. 0.920 ± 0.235; activity: 2.970 ± 0.110 vs. 1.110 ± 0.040, all P < 0.05), but significantly lower in the UVA-AGE-BSA group than in the UVA group(mRNA expression: 0.394 ± 0.008 vs. 0.480 ± 0.005; protein expression: 2.070 ± 0.125 vs. 2.583 ± 0.199; activity: 2.560 ± 0.060 vs. 2.970 ± 0.110, all P < 0.05). Conclusion AGEs could increase CatD expressions and activity in human dermal fibroblasts not receiving UVA irradiation, but inhibit their increase in UVA-induced human dermal fibroblasts. Key words: Fibroblasts; Glycosylation end products, advanced; Ultraviolet rays; Cathepsin D