Molecular Biology of the Ribonuclease A Gene Superfamily

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During this reporting period, I joined with collaborators at NCI Frederick and reported that the eosinophil-derived neurotoxin (EDN) an eosinophil granule-derived secretory protein with ribonuclease and antiviral activity, activated myeloid DCs by triggering the Toll-like receptor (TLR)2-myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses (J Exp Med, 2008). We have also continued our work on the transcriptional regulation of the genes encoding human EDN and ECP. Augmented expression of both GATA-1 and GATA-2 is detected in eosinophil promyelocyte HL-60 clone 15 cells in response to biochemical differentiation with butyric acid. Ablation of one or both consensus GATA binding sites in the extended 1000 base pair (bp) 5 promoter of EDN results in a profound reduction in reporter activity, while ablation of similar consensus GATA sites in the 5 promoter of ECP has no effect. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicated that GATA-1 and GATA-2 proteins bind to the functional GATA sequences in the EDN promoter. Furthermore, although RNA silencing of GATA-1 alone had no impact on EDN expression, silencing of GATA-2 resulted in reduced expression of EDN and of GATA-1. Taken together, our data indicate that GATA-2 plays a major role in promoting transcription of EDN, both directly via interactions with the promoter and indirectly, via its ability to regulate expression of GATA-1 (Qiu et al., manuscript in review). I have also authored two invited, peer-reviewed manuscripts covering the field of RNase A biology, one entitled "RNase A ribonucleases and host defense: an evolving story" (J. Leukoc. Biol. 2008; 83: 1079-1087) and "Eosinophil-derived neurotoxin / RNase 2: connecting the past, the present and the future" (Current Pharm Biotechnol 2008; 9: 135-140). I have also been invited to join the Editorial Board of Journal of Biological Chemistry (July 2008 - 2013) largely due to expertise on ribonuclease chemistry and biochemistry.