The steroid hormone 20-hydroxyecdysone upregulates calcium release-activated calcium channel modulator 1 expression to induce apoptosis in the midgut of Helicoverpa armigera.

Animal steroid hormones stimulate extracellular Ca2+ influx into cells; however, the mechanism remains unclear. In this study, we determined that the Ca2+ influx induced by steroid hormone 20-hydroxyecdysone (20E) is mediated by the calcium release-activated calcium channel modulator 1 (CRACM1/Orai1). The Orai1 mRNA is highly expressed during midgut programmed cell death in the lepidopteran insect Helicoverpa armigera. 20E upregulated the expression of Orai1 in H. armigera larvae and in an epidermal cell line (HaEpi). Knockdown of Orai1 in HaEpi cells blocked 20E-induced Ca2+ influx, and the inhibitor of inositol 1, 4, 5-trisphosphate receptor (IP3R) Xestospongin (XeC) blocked 20E-induced Ca2+ influx, suggesting that 20E, via Orai1, induces stored-operated Ca2+ influx. Orai1 interacts with stromal interaction molecule 1(Stim1) to exert its function in 20E-induced Ca2+ influx. 20E promotes Orai1 aggregation through G-protein-coupled receptors, phospholipase C gamma 1, and Stim1. Knockdown of Orai1 in the HaEpi cell line repressed apoptosis and maintained autophagy under 20E regulation. Knockdown of Orai1 in larvae delayed pupation, repressed midgut apoptosis, maintained the midgut in an autophagic state, and repressed 20E-pathway gene expression. These results revealed that steroid hormone 20E, via Orai1, induces Ca2+ influx to promote the transition of midgut from autophagy to apoptosis.

Steroid hormone 20-hydroxyecdysone promotes higher calcium mobilization to induce apoptosis.

Calcium ions are essential secondary messengers that regulate diverse cellular processes including gene transcription, cell proliferation, and apoptosis. The steroid hormone 20-hydroxyecdysone (20E) promotes programmed cell death during insect metamorphosis, whereas juvenile hormone (JH) counteracts 20E activity to prevent metamorphosis. Both 20E and JH can induce cellular calcium increase; however, the mechanisms and physiological consequences resulting from calcium increase caused by the two counteracting hormones are unclear. Here, using Helicoverpa armigera epidermal cell line, we show that 20E via a G-protein-coupled receptor induced a major calcium rise in the cells, whereas JH via receptor tyrosine kinase induced a minor calcium increase. The calcium release-activated calcium modulator 1 (Orai1) and transient receptor potential (TRP) channels were necessary for 20E-induced rapid calcium influx. A higher calcium level was maintained in a long time and more genes including Orai1 and TRP channels showed elevated expression after the treatment of 20E than did after JH treatment. Caspase3/7 activation, cell death and pro-apoptotic gene expression were elicited by 20E induction, but not by JH. JH could repress 20E-induced calcium influx, caspase3/7 activation and gene expression. Higher calcium levels induced apoptosis. These results suggest that 20E and JH via different pathways regulate calcium mobilization and homeostasis at different levels, thus inform different gene expression and cellular responses.

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.