ATG5 regulates type Ⅱ collagen expression by modulation of autophagy

To explore the regulative effect of autophagy-related protein ATG5 on the expression of type Ⅱ collagen in human chondrocytes through affecting autophagy function.

Transcriptome data of 13 human cartilage tissue samples were downloaded from GEO database to analyze the correlation between ATG5 and COL2A1 expression. Western blotting and immunofluorescence assay were used to detect the expression of type Ⅱ collagen（Collagen Ⅱ, COL2）in human chondrocytes C28/I2 after rapamycin（Rapa）and bafilomycin A1（BafA1）treatment. Human COL2A1 promoter plasmid was constructed, and dual-luciferase reporter assay was applied to detect the effect of overexpression of ATG5 on COL2A1 promoter activity. mRFP-GFP-LC3 autophagy dual fluorescent virus was used to dynamically monitor the changes in autophagic flux of C28/I2 cells with overexpression and knockdown of ATG5. Western blotting was used to detect the effects of Rapa and BafA1 on COL2, LC3Ⅱ/LC3Ⅰ expression levels after overexpression or knockdown of ATG5.

The results of correlation analysis showed a positive correlation between the mRNA expression of ATG5 and COL2A1 in human chondrocytes samples. Rapa and BafA1 significantly promoted and inhibited the expression of COL2 in C28/I2 cells（P<0.05）, respectively. The human COL2A1 promoter plasmid was successfully constructed, and the transcriptional activity of the COL2A1 promoter was up-regulated by the overexpression of ATG5（P<0.01）. Treatment with autophagy dual-fluorescence virus resulted in LC3-GFP fluorescence quenching after overexpression of ATG5（P<0.01）, suggesting autophagy activation; and a greater fluorescence intensity of LC3-GFP was observed after knockdown of ATG5（P<0.05）, demonstrating autophagy inhibition. Rapa obviously promoted the expression of COL2 and LC3Ⅱ/LCⅠ, which were up-regulated by ATG5（P<0.05）, but BafA1 showed the opposite effect of Rapa（P<0.05）.

Overexpression of ATG5 up-regulates the expression of type Ⅱ collagen, and the regulation of ATG5 on the expression of type Ⅱ collagen depends on autophagy in chondrocytes.