20-Hydroxyecdysone indirectly regulates Hemolin gene expression in Hyalophora cecropia

Development and innate immune defence are two vital processes that have been demonstrated to use the same or similar molecules and signalling pathways in insects. Hemolin is a moth haemolymph protein belonging to the immunoglobulin superfamily. It is strongly induced upon bacterial infection. However, recent studies indicate a developmental regulation of hemolin. We show that the steroid hormone 20-hydroxyecdysone (20E) can activate the expression of Hyalophora cecropia Hemolin (HcHemolin) in the fat body of diapausing pupae. Using the protein synthesis inhibitor cycloheximide we demonstrate that Hemolin up-regulation by 20E requires ongoing protein synthesis. Moreover, 20E enhances transcription of the Hemolin gene in response to bacteria. Comparing the upstream regions of Manduca sexta Hemolin (MsHemolin) and HcHemolin, we identified four putative regulatory sites. Two are putative hormone response elements (HREs), one with an imperfect inverted repeat (HRE-IR) and one with a monomeric site (HRE-M). An additional monomeric hormone receptor site (MRE) is present only in HcHemolin. The third conserved motif is similar to the interferon (IFN) regulatory factor binding element (IRF-E) and IFN-stimulated response element (ISRE). The fourth conserved element is a kappaB motif situated between the Cap-site and the TATA-box. Finally, by electrophoresis mobility shift assay we demonstrate that the HRE-IR forms specific complexes with nuclear extract proteins of normal pupae that increase after 20E stimulation.