Background-Marinobufagenin (MBG), a bufadienolide cardiotonic steroid, induces cardiovascular fibrosis. Because levels of MBG in preeclampsia are increased, and anti-MBG monoclonal antibody reduces blood pressure (BP) in a rat model of preeclampsia, we hypothesized that in preeclampsia, elevated MBG levels would be associated with the development of fibrosis in feto-placental circulation and with impairment of vascular relaxation.

Background The bioactive steroid, marinobufagenin (MBG), is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthetized by adrenocortical and placental cells. MBG binding to Na/K-ATPase initiates pro-fibrotic cell signaling, and heightened MBG levels are implicated in the pathogenesis of hypertension, preeclampsia and chronic kidney disease. Steroids are derived from cholesterol through the “traditional” steroidogenesis pathway initiated by enzyme CYP11A1, and via the “acidic” bile acid pathway, which is controlled by enzyme CYP27A1. The mechanism of MBG biosynthesis in mammals however remains unknown. Methods and Results Here we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and MBG levels by 67%, compared to non-treated cells or cells transfected with non-targeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect MBG production in either cell culture, but suppressed production of progesterone 2-fold in human trophoblast cells, and of corticosterone by 90% in rat adrenocortical cells, compared to cells transfected with non-targeting siRNA. In vivo, in a high salt administration experiment, male and female Dahl-S rats became hypertensive after 4 weeks on a high NaCl diet, their plasma MBG levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold and 2.0-fold. Conclusions Therefore, the endogenous steroidal Na/K-ATPase inhibitor, MBG, is synthesized in mammalian placenta and adrenal cortex from cholesterol through the novel “acidic” bile acid pathway. These findings will help to understand the role of MBG in highly prevalent human cardiovascular diseases.

The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB–induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli- stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: ( i ) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, ( ii ) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, ( iii ) transcription coactivator activity, and ( iv ) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.