Dexamethasone inhibits repair of human airway epithelial cells mediated by glucocorticoid-induced leucine zipper (GILZ)

Abstract

Background: Glucocorticoids (GCs) are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ) is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK)-extracellular-signal-regulated kinase (ERK) signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX) is mediated by GILZ.

Methods: We tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays.

Results: DEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway), proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced.

Conclusions: The inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs.

Figure 1. The effect of DEX on GILZ mRNA and GILZ protein expression in 9HTE cells.

(A) RT-PCR was performed to detect the GILZ mRNA level and showed that DEX induced the expression of GILZ mRNA starting from 6 h until 24 h (for 24 h, 0.553±0.048 in the untreated group compared with 0.658±0.087 in the DEX-treated, *P<0.05, n = 8). (B) 9HTE cells were treated with or without DEX for 6 h, and GILZ was located using immunofluorescence (400×). (C) Western blot was performed to determine the expression levels of GILZ protein from 6 h until 24 h with DEX (for 24 h, 0.092±0.019 in the untreated group compared with 0.693±0.085 in the DEX-treated, **P<0.001, n = 3). β-actin was used as the loading control.

Figure 2. GILZ expression in 9HTE cells transfected with GILZ siRNAs.

(A) Real time-PCR was performed to show transcriptional levels of the GILZ gene 48 h after transfection with GILZ ₁₋₃ siRNAs of cells treated with DEX for 24 h. Non-specific si-RNA was the negative control, and GAPDH si-RNA was the positive control (n = 3). (B) Cellular GILZ protein was collected from 9HTE cells transfected with non-specific si-RNA or GILZ ₃ si-RNA for 48 h. Afterwards, GILZ protein levels were detected by Western blot (0.873±0.109 in the non-specific si-RNA group compared with 0.305±0.065 in the GILZ ₃ si-RNA group, **P<0.001, n = 3), β-actin was used as a loading control. (C) The expression of GILZ protein was detected by immunofluorescence (200×) after 9HTE cells were transfected with non-specific si-RNA or GILZ si-RNA, separately for 48 h.

Figure 3. Differential expressions of components of the MAPK-ERK pathway in non-specific si-RNA, DEX-treated/non-specific si-RNA, and DEX-treated/GILZ si-RNA-transfected 9HTE cells.

Cellular proteins were collected from 9HTE cells transfected with non-specific si-RNA or GILZ si-RNA in the absence or presence of DEX for 24 h. Western blot was performed to detect levels of the phosphorylated forms of Raf-1, Mek1/2, and Erk1/2, and the respective total proteins (p-Raf-1, p-Mek1/2, p-Erk1/2: 0.935±0.056, 0.965±0.042, 0.959±0.052 in the non-specific si-RNA group compared with 0.574±0.143, 0.694±0.145, 0.712±0.066 in the DEX-treated/non-specific si-RNA group, compared with 0.861±0.087, 0.849±0.067, 0.840±0.061 in the DEX-treated/GILZ si-RNA group, n = 3). *Indicates a significant difference (P<0.05), β-actin was used as the loading control.

Figure 4. Silencing of GILZ partially abrogated the effect of DEX on proliferation of 9HTE cells.

(A) 9HTE cells were transfected with non-specific si-RNA or GILZ si-RNA in the absence or presence of DEX. The effect of GILZ knockout on 9HTE cell proliferation was measured via MTT assay, and absorbance was read at 490 nm (0.749±0.057 in the non-specific si-RNA group compared with 0.697±0.057 in the DEX-treated/non-specific si-RNA group, *P<0.05, n = 3). (B) 9HTE cells were labeling with CFSE and the CFSE fluorescence intensity was measured by flow cytometry (6.468±1.463 in the non-specific si-RNA group and 5.233±0.970 in the DEX-treated/GILZ si-RNA group compared with 2.765±0.539 in the DEX-treated/non-specific si-RNA group, *P<0.05, n = 4).

Figure 5. GILZ mediated the inhibiting effect of DEX on migration of 9HTE cells.

(A) Wound sites (area cleared of cells in the center of the scraped area) were observed and photographed. Photographs showed the repair of the wound in the three groups. (0.827±0.080 in the non-specific si-RNA group compared with 0.918±0.045 in the DEX-treated/non-specific si-RNA group, *P<0.05, n = 3). (B) 9HTE cell migration was examined by transwell chamber and counted under a microscope in five randomly chosen fields of each group, three independent experiments (97.066±15.448 in the non-specific si-RNA group and 87.266±12.876 in the DEX-treated/GILZ si-RNA group compared with 69.733±12.572 in the DEX-treated/non-specific si-RNA group, **P<0.001 and *P<0.05, n = 3).