To investigate the regulatory role of cathepsin D (CatD) in the degradation of intracellular advanced glycation end products (AGEs) endocytosed by human dermal fibroblasts (HDFs) .

Cultured HDFs were treated with 1 μmol/L CA074Me (an inhibitor of CatB and CatL) , 75 μmol/L pepstatin A (an inhibitor of CatD) and 1 μmol/L MG-132 (an inhibitor of 20S proteasome) separately for 4 hours, and then cell counting kit 8 (CCK8) assay and fluorometric assay were performed to determine the cellular viability and protease activity, respectively. The cells in the CA074Me group, pepstatin A group and MG-132 group were additionally treated with AGE-bovine serum albumin (BSA) for 8 hours, and the cells in the blank control group were treated with phosphate-buffered saline (PBS) alone. After 8-hour cultivation, the cells in the above groups were subsequently reincubated with fresh culture medium containing the corresponding inhibitors for 24 hours. Then, flow cytometry was performed to assess the mean fluorescence intensity of intracellular AGE-BSA at different time points. Some other HDFs were treated with 37.5, 75 and 150 μmol/L pepstatin A and PBS separately for 4 hours, and then the cells in the 4 groups were treated with 200 mg/L AGE-BSA for 8 hours, followed by the removal of AGE-BSA from the medium and the treatment with 37.5, 75 and 150 μmol/L pepstatin A and PBS respectively. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the mean concentration of intracellular AGE-BSA at different time points, and the degradation rate of AGE was calculated. Some HDFs were divided into 3 groups: blank control group receiving no treatment, NC group transfected with an empty vector, and CatD group transfected with a CatD-overexpressing lentiviral vector. Fluorescence microscopy was conducted to estimate the transfection efficiency. Reverse transcription-PCR, Western blot analysis and fluorometric assay were performed to determine the mRNA and protein expression, and activity of CatD respectively. Then, the cells in the above 3 groups were incubated with AGE-BSA for 8 hours, followed by the removal of AGE-BSA from the medium and the treatment with fresh culture medium. The detection methods were same as the above experiment, and the degradation rate was calculated.

The cellular proliferative activity in the 1-μmol/L CA074Me group, 75-μmol/L pepstatin A group and 1-μmol/L MG-132 group was more than 90%, and there was no significant difference between the 3 groups and the control group (100%, F=1.525, P > 0.05) . Twenty-four hours after the removal of AGE-BSA from the medium, the fluorescence intensities of intracellular AGE-BSA in the CA074Me+AGE-BSA group (275.00 ± 10.15) and MG-132+AGE-BSA group (259.00 ± 11.14) significantly decreased compared with those at the 8-hour time point (295.00 ± 6.56 and 285.67 ± 8.74 respectively; paired t test, t=4.778, 6.154 respectively, both P < 0.05) , while no significant difference was observed in the fluorescence intensities of intracellular AGE-BSA in the pepstatin A+AGE-BSA group between the 8-hour time point and 32-hour time point (P > 0.05) . The degradation rates of intracellular AGE-BSA within 24 hours in the 37.5, 75 and 150 μmol/L pepstatin A groups were 9.64% ± 1.27%, 5.62% ± 0.47% and 3.21% ± 0.73% respectively; there were significant differences among the 3 groups (F=45.876, P < 0.05) , and the degradation rate significantly decreased along with the increase of pepstatin A concentration (P < 0.05) . Fluorescence microscopy showed no fluorescent cells in the blank control group, while the NC group and CatD group both showed a high proportion (> 80%) of fluorescent cells. The mRNA and protein expression as well as the activity of CatD were significantly higher in the CatD group than in the blank control group and NC group (all P < 0.05) . The CatD+AGE-BSA group showed a significantly higher degradation rate of intracellular AGE-BSA within 24 hours compared with the AGE-BSA group and NC+AGE-BSA group (both P < 0.05) .

CatD can promote the degradation of intracellular AGE-BSA endocytosed by HDFs.

Cathepsin D; Fibroblasts; Glycosylation end products, advanced; Degradation