Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB

Abstract

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.

FIG. 1

IP-10 expression in REC cells following infection with adenovirus vectors. (A) REC cells infected with 5 × 10¹⁰ OPU of adCMVβgal per ml express IP-10 protein at 16 h as detected by ELISA (6.1 ± 1.1 versus 50.3 ± 5.7 ng/ml; P < 0.0002). (B) IP-10 mRNA expression detected by RNase protection assay in REC cells following stimulation with IFN-γ. At 6 h, adCMVβgal induces the expression of IP-10 more than stimulation with 1 U/ml but less than stimulation with 10 U of IFN-γ per ml. Bars represent the SD. (C) AdCMVβgal, adCMV-GFP, FG140, and UV-psoralen-inactivated adenovirus (UV/Ps-Ad) induce the expression of IP-10 mRNA in REC cells 6 and 16 h following infection as seen by RNase protection. Vehicle-treated cells do not induce the expression of IP-10.

FIG. 2

Adenovirus vector-induced activation of the IP-10 promoter in REC cells. (A) Organization of the 237-bp fragment upstream of the transcription start site of the mouse IP-10 gene. Positions of the deletional mutants are noted in relation to the potential cis elements involved. (B) Luciferase reporter assay. AdCMVβgal (■) activates the 533-bp fragment of the IP-10 promoter >10-fold more than the vehicle (□)-treated cells. AdCMVβgal-induced luciferase activity is diminished but still significantly greater than with the vehicle in REC cells transfected with IP-10 promoter deletion constructs pGL3–IP-10(−237), pGL3–IP-10(−190), and pGL3–IP-10(−161). Luciferase activity falls to baseline levels in REC cells transfected with the deletional mutant pGL3–IP-10(−96), thus confirming that the minimal elements responsible for IP-10 promoter activation are located between positions −161 and −96 of the IP-10 5′-flanking region. Bars represent the SD.

FIG. 3

EMSA of REC cell nuclear extracts. (A) Nuclear extracts from untreated and vehicle-treated REC cells constitutively form DNA-protein complexes C1 and C2 when incubated with the radiolabeled oligonucleotide, IP-10(−124/−94). IP-10(−124/−94) corresponds to base pairs −124 to −94 of the IP-10 5′-flanking region and contains the NF-κB sequence GGGACTTCC at position −113. AdCMVβgal significantly increases C1 complex 6 and 12 h following infection in REC cells. (B) EMSA using radiolabeled DNA fragment IP-10(−124/−94) and REC nuclear extracts following infection with 5 × 10¹⁰ OPU of various adenovirus vectors per ml. C1 complex is increased by adCMV-GFP, FG140, and UV-psoralen-inactivated adenovirus (UV/Ps-Ad) 6 h following infection, a result similar to the pattern induced by adCMVβgal. C2 complex is minimally or not increased significantly over baseline levels following infection with different adenovirus vectors.

FIG. 4

Competitor EMSA using nuclear extracts from adCMVβgal-infected REC cells and radiolabeled probe IP-10(−124/−94). C1 complex is not competed away by the nonspecific DNA fragment (Oligo 1) or by a DNA fragment containing an alternate NF-κB sequence GGGATGCCC (Oligo 2). DNA-protein interaction is competed for effectively with unlabeled IP-10(−124/−94), confirming the specificity of this interaction.

FIG. 5

Adenovirus vector-induced activation of NF-κB. (A) EMSA using nuclear extracts from adCMVβgal-infected REC cells and radiolabeled probe IP-10(−124/−94). Anti-NF-κB p65 supershifts C1 complex, whereas anti-NF-κB p50 supershifts C2 complex, confirming the activation of p65 primarily by adenovirus vectors. NF-κB p50 is present in the nuclear extracts but is minimally increased by adenovirus vectors compared to untreated or vehicle-treated cells. (B) RNase protection assay of REC cell RNA following infection with 5 × 10¹⁰ OPU of adCMVβgal or adCMV-IκBα per ml. Unlike other adenovirus vectors, adCMV-IκBα does not significantly induce the expression of IP-10 mRNA at 6 and 16 h following infection. Coinfection of adCMVβgal and adCMV-IκBα results in suppression of adCMVβgal-induced expression of IP-10 mRNA at 6 and 16 h, confirming the importance of NF-κB in mediating the transcription of IP-10 after infection with adenovirus vectors.

FIG. 6

RNase protection assay of REC cells infected with adCMVβgal. IP-10 mRNA is induced by adCMVβgal as early as 3 h following infection in the absence of TNF-α and IFN-γ. RNA from DBA/2 mouse liver harvested 16 h following infection with 2 × 10¹¹ OPU of adCMVβgal is used as a positive control for TNF-α and IFN-γ expression.