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20-hydroxyecdysone indirectly regulates hemolin gene expression in Hyalophora cecropia
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2005 (English)In: Insect molecular biology (Print), ISSN 0962-1075, E-ISSN 1365-2583, Vol. 14, no 6, 645-652 p.Article in journal (Refereed) Published
Abstract [en]

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 κB 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.

Place, publisher, year, edition, pages
John Wiley & Sons , 2005. Vol. 14, no 6, 645-652 p.
Keyword [en]
20-hydroxyecdysone;Hemolin gene regulation;hormone responsive element;interferon regulatory factor binding element;interferon stimulated response element
National Category
URN: urn:nbn:se:su:diva-23776DOI: 10.1111/j.1365-2583.2005.00593.xOAI: diva2:194460
Part of urn:nbn:se:su:diva-482Available from: 2005-05-04 Created: 2005-05-04 Last updated: 2010-10-27Bibliographically approved
In thesis
1. Hemolin expression during Cecropia development and its effect on malaria parasites
Open this publication in new window or tab >>Hemolin expression during Cecropia development and its effect on malaria parasites
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hemolin is a lepidopteran member of the immunoglobulin superfamily, initially isolated from the giant silkmoth Hyalophora cecropia. Hemolin is also induced by stimulation with microbial cell wall components and was recently shown to be strongly upregulated by baculovirus and double stranded RNA. An interesting characteristic of the protein is that it is not only highly expressed during infection but also during development.

The work presented in this thesis investigated the expression of hemolin during oogenesis and embryogenesis in H. cecropia. Vitellogenic follicles from ovaries were analysed for the presence of the protein by immunohistochemistry in whole-mount preparations and in cryosections. PCR was used to show the presence of Hemolin transcripts throughout vitellogenesis and choriogenesis and in fertilized and unfertilized mature eggs and Western blots showed the protein in unfertilized eggs, yolk cells and embryo.

Injection of the moulting hormone 20-hydroxyecdysone (20E) into hibernating diapausing pupae (low metabolic state), upregulates Hemolin. When diapausing pupae were treated with 20E and the protein synthesis inhibitor cycloheximide, its expression stayed low. This shows that the hormone indirectly regulates Hemolin by some factor(s) induced by 20E. When both bacteria and 20E were injected into diapausing pupae, an enhanced induction of hemolin gene expression occurred. Despite the seemingly indirect 20E regulation, several putative hormone responsive elements were found in the upstream region of the Hemolin (HRE-IR, HRE-M and MRE). When these elements were analysed by gel electrophoresis mobility shift assays (EMSA) to investigate their binding to nuclear factors, all the sites resulted in specific retarded bands. The HRE-IR binding factor was clearly increased by ecdysone. Last but not least we have investigated the effect of Hemolin on development of the malaria parasite Plasmodium falciparum in the midgut of the Anopheles mosquitoes. Hemolin completely inhibits the development of the parasite into its final transmission stage, the sporozoite. A future goal is to generate para-transgenic mosquitoes, enforced by hemolin, to stop malaria transmission. Importantly, hemolin did not affect the mosquito fecundity when fed to the mosquito. We are currently constructing truncated forms of hemolin to gain insight into which parts are important for its effect on the parasite.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi, 2005. 55 p.
National Category
urn:nbn:se:su:diva-482 (URN)91-7155-089-5 (ISBN)
Public defence
2005-05-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Available from: 2005-05-04 Created: 2005-05-04Bibliographically approved

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Assefaw-Redda, Yohannes
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