NF-kappaB activation limits airway branching through inhibition of Sp1-mediated fibroblast growth factor-10 expression

Abstract

Bronchopulmonary dysplasia (BPD) is a frequent complication of preterm birth. This chronic lung disease results from arrested saccular airway development and is most common in infants exposed to inflammatory stimuli. In experimental models, inflammation inhibits expression of fibroblast growth factor-10 (FGF-10) and impairs epithelial-mesenchymal interactions during lung development; however, the mechanisms connecting inflammatory signaling with reduced growth factor expression are not yet understood. In this study we found that soluble inflammatory mediators present in tracheal fluid from preterm infants can prevent saccular airway branching. In addition, LPS treatment led to local production of mediators that inhibited airway branching and FGF-10 expression in LPS-resistant C.C3-Tlr4(Lpsd)/J fetal mouse lung explants. Both direct NF-κB activation and inflammatory cytokines (IL-1β and TNF-α) that activate NF-κB reduced FGF-10 expression, whereas chemokines that signal via other inflammatory pathways had no effect. Mutational analysis of the FGF-10 promoter failed to identify genetic elements required for direct NF-κB-mediated FGF-10 inhibition. Instead, NF-κB activation appeared to interfere with the normal stimulation of FGF-10 expression by Sp1. Chromatin immunoprecipitation and nuclear coimmunoprecipitation studies demonstrated that the RelA subunit of NF-κB and Sp1 physically interact at the FGF-10 promoter. These findings indicate that inflammatory signaling through NF-κB disrupts the normal expression of FGF-10 in fetal lung mesenchyme by interfering with the transcriptional machinery critical for lung morphogenesis.

FIGURE 1

Tracheal aspirate fluid from newborn patients exposed to chorioamnionitis inhibits airway branching. Tracheal fluid was aspirated from intubated extremely preterm infants following delivery and added to saccular stage fetal mouse lung explants. A–F, Brightfield images of control mouse lung explants (A, D) and explants cultured with tracheal aspirate fluid from a patient born preterm owing to maternal pre-eclampsia (B, E) or maternal chorioamnionitis (C, F) (original magnification ×25). Higher magnification images are shown in D–F (original magnification ×200). Arrows indicate saccular airways. G, The number of new saccular airway branches that formed during the culture period was lower when tracheal fluid from chorioamnionitis patients was added to the media. *p < 0.05; n = nine chorioamnionitis samples, seven pre-eclampsia samples, and six others. H, Concentrations of IL-1β, TNF-α, and IL-8 were measured in each patient sample. Elevated cytokine levels were detected in chorioamnionitis samples. Scale bar indicates mean concentration. ^#p < 0.05 compared with chorioamnionitis; n = nine chorioamnionitis samples, seven pre-eclampsia samples, and six others).

FIGURE 2

LPS-conditioned media disrupt saccular airway branching in TLR4 mutant explants. Media from control and LPS-treated BALB/cJ explants were added to LPS-resistant C.C3-Tlr4^Lpsd/J explants. A–F, Brightfield images of C.C3-Tlr4^Lpsd/J explants cultured with control-conditioned media (A, D), LPS-conditioned media (B, E), or LPS-conditioned media with the NF-κB inhibitor parthenolide (1 μM; C, F) (original magnification ×25). Higher-magnification images are shown in D–F (original magnification ×200). Arrows indicate saccular airways. G, LPS-conditioned media inhibited formation of new saccular airways in C.C3-Tlr4^Lpsd/J explants. *p < 0.001; n = 7. H, LPS-conditioned media inhibited FGF-10 expression in C.C3-Tlr4^Lpsd/J explants, as measured by real-time PCR. *p < 0.05; n = 9.

FIGURE 3

NF-κB activation inhibits FGF-10 gene expression. A, Primary fetal mouse lung mesenchymal cells were treated with LPS (250 ng/ml), IL-1β (10 ng/ml), TNF-α (10 ng/ml), MIP-1α (100 ng/ml), or MCP-1 (100 ng/ml). Following 4 h of treatment, RNA was isolated and FGF-10 expression measured by real-time PCR. *p < 0.05; n = 12. B, Time course of changes in FGF-10 gene expression following treatment of primary fetal mouse lung mesenchymal cells with LPS (solid line), TNF-α (dashed line), or IL-1β (dotted line). *p < 0.05; n = 4 for IL-1β and TNF-α, n = 30 for LPS. C, IL-1β and TNF-α inhibit FGF-10 reporter activity. CHO cells transfected with FGF-10 luciferase were treated with IL-1β or TNF-α in the absence (white bars) or presence (gray bars) of parthenolide (10 μm) or the IKKβ inhibitor BMS-345541 (1 μM). *p < 0.05; n = 6. D, Overexpression of caIKK inhibited FGF-10 reporter activity (white bar). Expressing a dominantnegative isoform of IκB increased FGF-10 reporter activity (gray bar). *p < 0.05; n = 6. E, Chemical MAPK inhibitors did not prevent IL-1β–mediated inhibition of FGF-10 reporter activity. *p < 0.05; n = 6. DN-IκB, dominant-negative isoform of IκB; IKK inh, IKK inhibitor; parth, parthenolide.

FIGURE 4

NF-κB inhibits FGF-10 promoter activity via a noncanonical interaction near the FGF-10 transcriptional start site. A, Schematic diagram showing predicted NF-κB binding sites (●) along the FGF-10 promoter. Serial truncations of the FGF-10 promoter were generated as indicated. The FGF-10-D construct contained only a single predicted NF-κB binding site. B and C, Deletion of all but a 350-kb upstream region of the FGF-10 promoter retained sequences required for inhibition by IL-1β (B) or caIKK (C). *p < 0.05; n = 4. D, Deletion of the predicted NF-κB binding site in the FGF-10-D construct (ΔκB) did not prevent inhibition by caIKK. *p < 0.05; n = 5.

FIGURE 5

NF-κB activation interferes with Sp1-mediated FGF-10 expression. A, Schematic diagram showing location of GC boxes predicted to bind Sp1 (black boxes) in relation to the predicted NF-κB binding site (●) and transcriptional start site in the FGF-10 promoter. B, Sp1 immunolocalization in fetal mouse lung. Cells within E16 fetal lung mesenchyme showed variable expression of Sp1 (green). Airway epithelia indicated by E-cadherin staining (red). Nuclei labeled with DAPI (blue) (original magnification ×400). C, Sp1 increased FGF-10 reporter activity. Increasing amounts of Sp1 cDNA were cotransfected into CHO cells along with the FGF-10 luciferase reporter. *p < 0.05; n = 4. D, NF-κB prevents Sp1-mediated FGF-10 expression. CHO cells were cotransfected with FGF-10 luciferase, Sp1 (white bars), and either RelA (dark gray bars) or caIKK (light gray bars). Experiments were repeated with the FGF-10-D truncated promoter with similar results (right side of graph). *p < 0.05; n = 6. E, LPS increases recruitment of RelA to the FGF-10 promoter. Primary fetal mouse lung mesenchymal cells were treated with LPS, and transcription factor binding to the FGF-10 promoter was measured by chromatin immunoprecipitation. A small amount of RelA–FGF-10 interaction was detected in control cells, with increased signal in LPS-treated cells. Sp1 appears to bind the FGF-10 promoter both in control and in LPS-treated samples. E, Sp1 and RelA interact by coimmunoprecipitation. Sp1 was immunoprecipitated from control and IL-1β–treated CHO cell nuclear lysates. Samples were immunoblotted with Abs against RelA, demonstrating the presence of RelA in Sp1 immunoprecipitates.