Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB.
The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVbetagal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5'-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVbetagal was dependent on the -161- to -96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVbetagal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-kappaB at position -113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-kappaB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVbetagal and an adenoviral vector expressing IkappaBalpha resulted in suppression of adCMVbetagal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-kappaB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-kappaB.
Borgland
S L
SL
Department of Medicine, University of Calgary, Calgary, Alberta, Canada.
Bowen
G P
GP
Wong
N C
NC
Libermann
T A
TA
Muruve
D A
DA
D000260:Adenoviruses, Human / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000235:genetics*; D005786:Gene Expression Regulation; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D007371:Interferon-gamma / Q000235:genetics*; D051379:Mice; D008811:Mice, Inbred DBA; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D011401:Promoter Regions, Genetic; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D015683:Gene Products, gag; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma