767. Intact Microtubules Are Required for Ad-Vector Induction of Chemokine Genes in Epithelial Cells

Abstract

Upon entry in epithelial cells, adenovirus vectors (Ad-vectors) activate a cascade of signaling pathways including p38 MAPK (p38) and extracellular-signal regulated kinase (ERK). These signaling events occur within minutes and are triggered directly by the viral capsid. The activation of signaling by Ad-vectors leads to the expression of chemokine inflammatory genes such as IP-10. Shortly following internalization, Ad-vectors interact with microtubules that are required to mediate viral translocation to the nucleus. To better understand the role of microtubules in Ad vector induced signaling and chemokine gene expression, epithelial cells (REC) were treated with the microtubule depolymerizing reagent nocodazole (20mM). Nocodazole substantially reduced transduction of REC cells by a first generation Ad-vector, Adβgal, as determined by β-galactosidase expression 24 hours post-transduction. Vector internalization into REC however was unaffected as measured by flow cytometry confirming that microtubules were required to mediate nuclear translocation of Ad-vectors. To determine the effect of microtubule depolymerization on the induction of chemokine mRNA expression, RANTES and IP-10 gene expression was determined at 6 hours following transduction with Adβgal. Compared to untreated cells, nocodazole blocked the Adβgal induction of RANTES and IP-10 expression. Phosphorylated ERK or p38 was increased by nocodazole in the absence of Ad-vector transduction, a response that was not changed following the addition of Adβgal. Although indirect, this finding suggests that p38 and ERK signaling is linked to microtubules and is required but not sufficient to induce the expression of IP-10 and RANTES. In contrast, experiments with the microtubule-stabilizing agent, paclitaxel, did not affect Adβgal transduction of REC cells. Furthermore, Adβgal induction of p38/ERK phosphorylation and IP-10/RANTES gene expression was unchanged following paclitaxel suggesting that microtubule treadmilling or dynamic instability was not required for Ad-vector induction of host immune responses. These preliminary data suggest that intact microtubules are required for the Ad-vector induction of host inflammatory genes in epithelial cells. Understanding the biology of Ad-vector induced intracellular signaling will contribute significantly to the development of novel recombinant vectors for gene therapy.