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Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis

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ABSTRACT

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

No MeSH data available.


Autophagy is activated by 20E.(a,b) qRT-PCR analysis of BmATG8 (a) and BmATG1 (b) in midgut cells after administration of 20E; (c,d) Western blot analysis of BmAtg8–PE (c) and acid phosphatase activity (d) in midgut of larvae injected with the hormone; (e) Western blot analysis of p-Bm4ebp1 in hormone-treated larvae. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).
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f3: Autophagy is activated by 20E.(a,b) qRT-PCR analysis of BmATG8 (a) and BmATG1 (b) in midgut cells after administration of 20E; (c,d) Western blot analysis of BmAtg8–PE (c) and acid phosphatase activity (d) in midgut of larvae injected with the hormone; (e) Western blot analysis of p-Bm4ebp1 in hormone-treated larvae. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).

Mentions: qRT-PCR analysis detected overexpression of the BmATG8 (Fig. 3a) and BmATG1 (Fig. 3b) genes following 20E administration, and a strong increase in BmAtg8–PE was observed after the hormone treatment (Fig. 3c). The rise in acid phosphatase activity confirmed full induction of the autophagic process (Fig. 3d). It was previously demonstrated that 20E inhibits the protein Tor (target of rapamycin) both in Drosophila and B. mori fat body, thus eliciting the autophagic process933. However, previous studies have also reported that 20E activates autophagy directly, affecting the expression of specific ATG genes (such as ATG1), and thus independently of inhibition of the Tor pathway9.


Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis
Autophagy is activated by 20E.(a,b) qRT-PCR analysis of BmATG8 (a) and BmATG1 (b) in midgut cells after administration of 20E; (c,d) Western blot analysis of BmAtg8–PE (c) and acid phosphatase activity (d) in midgut of larvae injected with the hormone; (e) Western blot analysis of p-Bm4ebp1 in hormone-treated larvae. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016986&req=5

f3: Autophagy is activated by 20E.(a,b) qRT-PCR analysis of BmATG8 (a) and BmATG1 (b) in midgut cells after administration of 20E; (c,d) Western blot analysis of BmAtg8–PE (c) and acid phosphatase activity (d) in midgut of larvae injected with the hormone; (e) Western blot analysis of p-Bm4ebp1 in hormone-treated larvae. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).
Mentions: qRT-PCR analysis detected overexpression of the BmATG8 (Fig. 3a) and BmATG1 (Fig. 3b) genes following 20E administration, and a strong increase in BmAtg8–PE was observed after the hormone treatment (Fig. 3c). The rise in acid phosphatase activity confirmed full induction of the autophagic process (Fig. 3d). It was previously demonstrated that 20E inhibits the protein Tor (target of rapamycin) both in Drosophila and B. mori fat body, thus eliciting the autophagic process933. However, previous studies have also reported that 20E activates autophagy directly, affecting the expression of specific ATG genes (such as ATG1), and thus independently of inhibition of the Tor pathway9.

View Article: PubMed Central - PubMed

ABSTRACT

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

No MeSH data available.