11. The Role of Glucocorticoid Receptor Signaling in Adeno-Associated Virus 2 Infection

Abstract

We previously observed that purified recombinant glucocorticoid receptor (GR) protein specifically binds to the D-sequence within the inverted terminal repeat of the AAV2 genome because the D-sequence shares partial homology to the consensus half-site of the glucocorticoid receptor-binding element (GRE). The half-site is an essential core element, which has been reported to be sufficient to mediate GR signaling. Substitution of the D-sequence with the authentic GRE further increased AAV2 vector-mediated transgene expression. Based on these observations, we systemically examined whether AAV2 infection involves the GR signaling pathway. Our results showed that following infection by wild-type (WT) AAV2, or transduction with recombinant AAV2 vectors in vitro, GR was activated. The serine phosphorylated form of GR in whole cell lysates was significantly increased in a dose-dependent manner 18 hours post viral infection/vector transduction. Translocation and accumulation of GR in the nucleus was also increased correspondingly. To further corroborate our findings in an in vivo model, C57BL/6 mice were injected via tail-vein with either WT-AAV2 (1×1012 vgs/mouse) or AAV2-EGFP vectors (5×1011 vgs/mouse) and sacrificed 16 hours post virus/vector administration. A significant increase in the serine phosphorylated form of GR was also observed in liver tissues. We also observed that in cell cultures, dexamethasone, a well-known activator of GR, significantly enhanced rAAV2 vector-mediated transgene expression in various GR-positive cell lines, but not in a GR-deficient human osteosarcoma cell line, U2OS. Furthermore, stable transfection of U2OS cells with a GR expression plasmid not only facilitated AAV2 vector transduction, but also restored the ability of dexamethasone to further increase AAV2 vector-mediated transgene expression. Together with our previous data, we propose a model for elucidating the role of glucocorticoid receptor signaling pathway in AAV2 infection/AAV2 vector transduction (Fig. 1Fig. 1). Upon viral transduction or stimulation with glucocorticoids, the cytoplasmic GR is phosphorylated and translocated into the nucleus. Following AAV2 entry into the nucleus, the viral genome is released. Upon viral second-strand DNA synthesis or annealing of the complementary DNA strands, the activated GR binds to the double-stranded D-sequence in the viral inverted terminal repeats and consequently, enhances transgene expression. The activated GR also regulates its downstream pathways, such as NF-kB, which we have previously shown to have a substantial effect on AAV2 vector-mediated transgene expression (Proc Natl Acad Sci USA, 108:3743-3738, 2011). A better understanding of the complex interaction between GR and NF-kB signaling pathways notwithstanding, the availability of the fully functional GRE site-substituted novel AAV vectors to achieve high-efficiency transgene expression has implications in the potential use of these vectors in human gene therapy.View Large Image | Download PowerPoint Slide