Silencing of both ATF4 and PERK inhibits cell cycle progression and promotes the apoptosis of differentiating chondrocytes

In the current study, we demonstrate that the silencing of protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) and activating transcription factor 6 (ATF4) (using small interfering RNA expression constructs) inhibits the chondrocyte cell cycle and proliferation in vitro and ex vivo. The silencing of PERK alone using siRNA against PERK (siPERK) led to arrest in the G1 phase, it decreased the number of cells in the S phase, and delayed progressoin to the G2-M phase. Co-transfection with siRNA against ATF (siATF4) led to a more profound inhibitory effect on cell cycle progression. Moreover, transfection with siPERK was associated with enhanced endoplasmic reticulum (ER) stress-induced apoptosis during bone morphogenetic protein 2 (BMP2)-induced chondrogenesis, and transfection with siATF4 exacerbated ER stress-related cell death. Data from flow cytometry (FCM), immunohistochemistry and TUNEL assays supported these findings in vitro and ex vivo. As shown by our results, the combined effect of the silencing of ATF4 and PERK led to the activation of an ER stress-specific caspase cascade in the cartilage tissue. On the whole, these findings reveal a new crucial combined effect of the silencing of PERK and ATF4 in modulating ER stress-mediated apoptosis during chondrocyte differentiation and proliferation.