Fig. 8.
Role of JunB in A2B receptor-dependent VEGF production. (A) Effect of NECA on JunB binding to the VEGF promoter. ChIP analysis was performed using an antibody specific for JunB (IP: JunB) and IgG as a nonspecific control (IP: ns). Specific primer sets were used for the promoter region harboring the AP-1 site (−1140 to −733). JunB binding to the VEGF promoter region between −1140 and −733 was detected and was enhanced in LLC cells treated with 10 µM NECA for 3 hours. (B) Effects of AP-1 site mutation and dominant negative JunB on VEGF reporter activity. The role of JunB in A2B receptor-dependent regulation of the VEGF gene promoter was studied in LLC cells transiently cotransfected with VEGF luciferase reporter together with plasmid encoding DNJunB or with an empty vector (mock). A set of LLC cells was also transfected with VEGF luciferase reporter containing the mutated AP-1 site at −1093 (indicated on graph as mutAP-1). Twenty-four hours after transfections, cells were incubated in the absence (basal) or presence of 10 μM NECA for an additional 6 hours. Values are presented as mean ± S.E.M. (n = 3). Asterisks indicate statistical difference (**P < 0.01) compared with basal levels, and a dagger indicates the statistical difference (†P < 0.05) compared with mock controls by one-way ANOVA with Bonferroni post-test. (C) Expression of JunB and DNJunB in LLC cells stably transfected with plasmid encoding DNJunB or with an empty vector (mock). Western blot analysis was performed using antibodies against a common epitope in the sequences of JunB and DNJunB. Arrows indicate positions of JunB and DNJunB. Immunostaining of β-tubulin was used as a loading control. (D) Effect of the expression of dominant negative JunB on VEGF secretion. The role of JunB in A2B receptor-dependent regulation of VEGF secretion was studied in LLC cells stably transfected with plasmid encoding DNJunB or with an empty vector (mock) by measuring VEGF release from cells incubated in the absence (basal) or presence of 10 μM NECA for 6 hours. Values are presented as mean ± S.E.M. (n = 3). Asterisks indicate statistical difference (*P < 0.05; ***P < 0.001) compared with basal levels; daggers indicate statistical difference (††P < 0.01) compared with mock controls by one-way ANOVA with Bonferroni post test. (E) Efficacy of JunB silencing after stable lentiviral transfection of LLC cells with different shRNA constructs (shRNA1-5) compared with shRNA nontargeting (NT) and empty vector (EV) controls. Upper panels show representative Western blots of basal levels of targeted JunB protein and β-tubulin loading controls. The lower panel is a graphic representation of the data expressed as relative density of JunB bands normalized to corresponding β-tubulin bands and NT control. (F) Real-time reverse-transcription polymerase chain reaction analysis of JunB mRNA levels in LLC cells stably transfected with lentiviral vectors encoding shRNA5 or nontargeting shRNA (NT control). Cells were incubated in the absence (basal) or presence of 10 μM NECA for 60 minutes. Values are normalized to β-actin mRNA expression and expressed as the average of two determinations made in triplicate. (G) Effect of JunB knockdown on VEGF secretion. The role of JunB in A2B receptor-dependent regulation of the VEGF secretion was studied in LLC cells stably expressing JunB silencing shRNA5 or nontargeting shRNA (NT control) incubated in the absence (basal) or presence of 10 μM NECA for 6 hours. Values are presented as mean ± S.E.M. (n = 3). Asterisks indicate statistical difference (*P < 0.05; ***P < 0.001) compared with basal levels; daggers indicate statistical difference (†††P < 0.001) compared with NT controls by one-way ANOVA with Bonferroni post-test.