BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation

<p>Autophagy and apoptosis, which could be induced by common stimuli, play crucial roles in development and disease. The functional relationship between autophagy and apoptosis is complex, due to the dual effects of autophagy. In the <i>Bombyx</i> Bm-12 cells, 20-hydroxyecdysone (20E) treatment or starvation-induced cell death, with autophagy preceding apoptosis. In response to 20E or starvation, BmATG8 was rapidly cleaved and conjugated with PE to form BmATG8–PE; subsequently, BmATG5 and BmATG6 were cleaved into BmATG5-tN and BmATG6-C, respectively. Reduction of expression of <i>BmAtg5</i> or <i>BmAtg6</i> by RNAi decreased the proportion of cells undergoing both autophagy and apoptosis after 20E treatment or starvation. Overexpression of <i>BmAtg5</i> or <i>BmAtg6</i> induced autophagy but not apoptosis in the absence of the stimuli, but promoted both autophagy and apoptosis induced by 20E or starvation. Notably, overexpression of cleavage site-deleted <i>BmAtg5</i> or <i>BmAtg6</i> increased autophagy but not apoptosis induced by 20E or starvation, whereas overexpression of <i>BmAtg5-tN</i> and <i>BmAtg6-C</i> was able to directly trigger apoptosis or promote the induced apoptosis. In conclusion, being cleaved into BmATG5-tN and BmATG6-C, BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20E or starvation in <i>Bombyx</i> Bm-12 cells, reflecting that autophagy precedes apoptosis in the midgut during <i>Bombyx</i> metamorphosis.