Epithelium-Specific Ets-Like Transcription Factor 1, ESE-1, Regulates ICAM-1 Expression in Cultured Lung Epithelial Cell Lines

Abstract

Cystic fibrosis (CF) patients suffer from chronic airway inflammation with excessive neutrophil infiltration. Migration of neutrophils to the lung requires chemokine and cytokine signaling as well as cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), which plays an important role in mediating adhesive interactions between effector and target cells in the immune system. In this study, we investigated the relationship between ICAM-1 and epithelium-specific ETS-like transcription factor 1 (ESE-1) and found that ICAM-1 expression is upregulated in cell lines of CF (IB3-1) as well as non-CF (BEAS-2B and A549) epithelial origin in response to inflammatory cytokine stimulation. Since ESE-1 is highly expressed in A549 cells without stimulation, we examined the effect of ESE-1 knockdown on ICAM-1 expression in these cells. We found that ICAM-1 expression was downregulated when ESE-1 was knocked down in A549 cells. We also tested the effect of ESE-1 knockdown on cell-cell interactions and demonstrate that the knocking down ESE-1 in A549 cells reduce their interactions with HL-60 cells (human promyelocytic leukemia cell line). These results suggest that ESE-1 may play a role in regulating airway inflammation by regulating ICAM-1 expression.

Figure 1

Induction of ESE-1 and ICAM-1 mRNA expression by TNF-α and IL-1β in different cell lines. (a, b) ESE-1 and ICAM-1 mRNA expression in BEAS2B, IB3-1, and A549 cells after stimulation with TNF-α and IL-1β (10 ng/mL each) for 2 hours. The mRNA expression levels were determined by real-time quantitative RT-PCR and the fold of change was based on the mRNA level of non-induced cells. Data were normalized to GAPDH and values were shown in 2^−ΔΔCT as the mean ± SD, n = 3, ^* P < 0.05. Statistics was performed as described in Materials and Methods. (c, d) ESE-1 and ICAM-1 mRNA expression in A549 cells at different time points following cytokine stimulation. Cells were lysed at time points as indicated after TNF-α and IL-1β (10 ng/mL each) stimulation. The fold of change was based on the expression level at time 0 hour. Data were collected and analyzed as described in (a) and (b).

Figure 2

Effects of ESE-1 knockdown in A549 cells on the expression of ICAM-1. (a) Schematic diagrams of the helper-dependent adenovirus vectors (HD-Ad) that were used for the ESE-1 knockdown experiments. ESE-1-RNAi expresses two shRNAs from the murine U6 gene promoter, and C4HSU is used as an empty vector control which does not express any transgene. ITR: inverted terminal repeat; Ψ: packing signal. (b, c) ESE-1 and ICAM-1 mRNA expression in A549 cells 4 and 5 days after transduction with ESE-1-RNAi vector compared to that of C4HSU. Both groups were stimulated with TNF-α and IL-1β at 10 ng/mL each for 2 hours before cell lysis. The mRNA expression levels were normalized to GAPDH and the values were presented in 2^−ΔΔCT as the mean ± SD, n = 3, ^* P < 0.05. Statistics was performed as described in Materials and Methods. (d) A representative western blot analysis of ICAM-1 expression with (+) and without (−) ESE-1-RNAi, compared to that of the C4HSU vector control group on day 4 and day 5 after transduction. Since ICAM-1 levels were very low, subgroup of cells were stimulated with TNF-α and IL-1β (10 ng/mL each) for 16 hours to visualize ICMA-1 protein expression before cell lysis.

Figure 3

Luciferase assay on BEAS2B cells after cotransfection with pcDNA3-ESE-1 and PGL3-ICAM-1 vector. (a) Schematic diagram of pGL3-ICAM-1 promoter reporter plasmid that contains an ICAM-1 promoter sequence expressing the luciferase gene in the pGL3-Basic vector (Promega). (b) Luciferase activity assay. BEAS-2B cells were cotransfected with pGL3-ICAM-1 luciferase reporter plasmid and pcDNA3-ESE-1 or pcDNA3-empty vector [37]. The luciferase activity was measured 24 h after transfection.

Figure 4

Cell adhesion assay of the effect of ESE-1 knocking down on HL-60 binding to A549 cells. (a) Images showing HL-60 cells attached to A549 cells at 40x magnification. (b) Quantification of cell binding. The average number of HL-60 cells per field was compared among groups with ESE-1 knocking down or C4HSU empty vector transfection or nontransfected groups with or without cytokine stimulation (TNF-α and IL-1β at 10 ng/mL each). Cell numbers were counted under a microscope and data from six wells were presented as mean ± SD, ^* P < 0.05.