TRIB3 Mediates Fibroblast Activation and Fibrosis though Interaction with ATF4 in IPF

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by fibroblast activation, excessive deposition of extracellular matrix, and progressive scarring; the pathogenesis remains elusive. The present study explored the role of Tribbles pseudokinase 3 (TRIB3), a well-known stress and metabolic sensor, in IPF. TRIB3 is down-regulated in the lungs of IPF patients in comparison to control subjects. Deficiency of TRIB3 markedly inhibited A549 epithelial cells' proliferation and migration, significantly reducing wound healing. Conversely, overexpression of TRIB3 promoted A549 cell proliferation and transmigration while it inhibited its apoptosis. Meanwhile, overexpressed TRIB3 inhibited fibroblast activation and decreased ECM synthesis and deposition in MRC5 cells. TRIB3 attenuated pulmonary fibrosis by negative regulation of ATF4, while TRIB3 expression markedly inhibited ATF4 promoter-driven transcription activity and down-regulated ATF4 expression. A co-culture system showed that TRIB3 is important to maintain the normal epithelial-mesenchymal crosstalk and regulate fibroblast activation. Taken together, our data suggested that an axis of TRIB3-ATF4 is a key mediator in IPF which might be a potential target for fibroproliferative lung disease treatment.

Keywords: ATF4; TRIB3; epithelial cell; fibroblast activation; idiopathic pulmonary fibrosis.

Figure 1

Down-regulation of TRIB3 in IPF lung. (A) TRIB3 was down-regulated in IPF patients, which was profoundly validated from the Gene Expression Omnibus (GEO) RNA-seq dataset (GSE32537). Statistical analysis of the microarray was performed by R package “limma”. (B) TRIB3 was negatively correlated with COL1A1, COL1A2, FN1, and ACTA2. Data were obtained from GSE32537 (Control = 50, IPF = 119) in the public database Gene Expression Omnibus (GEO). Spearson rank analysis was used to analyze the correlation between TRIB3 and FN1, ACTA2, COL1A1, and COL1A2 expression. (C) TRIB3 was down-regulated from patients with IPF in transcription level. The statistical test used was the Mann–Whitney U test for comparisons between two groups. * p < 0.05. (D) Western blot analysis showed reduced TRIB3 expression in IPF lungs (N = 4) compared with healthy lungs (N = 4). The statistical test used was the Mann–Whitney U test for comparisons between two groups. * p < 0.05.

Figure 2

TRIB3 deficiency inhibited A549 cell proliferation, migration, and profibrotic gene expressions. (A) The effect of TRIB3 silencing on the viability of A549 cells was assessed by CCK8 assay. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01. (B) The effect of TRIB3 silencing on the colony formation of A549 cells (N = 3 independent experiments). (C) TRIB3-shRNA inhibited the migration of A549 cells. (D) Gene expression analysis by quantitative PCR (qPCR) demonstrates a significant decrease in ACTA2, COL1A1, and FN1 transcript levels in TRIB3-silenced normal epithelial cells. GAPDH was used as an internal control. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01, **** p < 0.0001. (E) TRIB3-shRNA decreased the protein level of TRIB3, α-SMA, E-Cadherin, and β-actin (N = 3 independent experiments). β-actin was used as an internal control. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. * p < 0.05 and *** p < 0.001.

Figure 3

TRIB3 expression promoted A549 cell proliferation, migration, and profibrotic genes expression. (A) The effect of TRIB3 overexpression on the viability of A549 cells was assessed by CCK8 assay (N = 4 independent experiments). Significant differences between groups were evaluated by two-way ANOVA with Šídák’s multiple comparisons test for pairwise comparisons. * p < 0.05. (B) The effect of TRIB3 overexpression on the colony formation of A549 cells (N = 3 independent experiments). The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. * p < 0.05. (C) The EdU proliferation assay was used to measure A549 cell proliferation. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001. (D) Crystal violet staining of A549 cell is shown for the transwell assay (N = 3 independent experiments). The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001. (E) Cell apoptosis measured by cytometry, with quantification analysis (N = 3 independent experiments). (F) Gene expression analysis by qPCR demonstrated a significant increase in TRIB3, ACTA2, and VIM transcript levels in TRIB3-overexpressed normal epithelial cells. GAPDH was used as an internal control. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. * p < 0.05 and **** p < 0.0001 vs. control. (G) Western blot analysis showed that the expression of α-SMA, vimentin, E-Cadherin, and p-Smad3 in A549 cells increased after overexpressing TRIB3 (N = 3 independent experiments). For western blot, GAPDH was used as an internal control.

Figure 4

TRIB3 inhibits lung fibroblast activation. (A) Gene expression analysis by qPCR demonstrates a significant decrease in ACTA2, COL1A1, and FN1 transcript levels in TRIB3-overexpressing normal lung fibroblasts. GAPDH was used as an internal control. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (B) Western blot analysis showed αSMA, collagen I, and fibronectin elevation in TRIB3-overexpressed lung fibroblasts (N = 3 independent experiments). β-actin was used as an internal control. (C) The activation of fibroblasts was examined based on fibroblast contraction in 3D collagen matrices (N = 3 independent experiments). We used ImageJ software to measure the area of gels. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001. (D) MRC5 cells were subjected to immunofluorescence with a vimentin antibody. Scale bars, 20 μm. ImageJ software was used to quantify the fluorescence intensity. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01.

Figure 5

TRIB3 interacts with ATF4 and negatively regulates ATF4. (A) Immunoprecipitation for TRIB3 in MRC5 cells. TRIB3 interacts with ATF4 by silver staining. (B) Co-IP of flag-tagged TRIB3 and HA-tagged ATF4 protein from the transfected cell lysates. (C,D) Overexpression of TRIB3 decreased the expression of ATF4 both at RNA and protein levels (N = 3 independent experiments). GAPDH was used as an internal control for qPCR, and β-actin was used as an internal control for western blot. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01. (E) Relative expression of ATF4 promoter-driven luciferase reporters in TRIB3-overexpressing cells (N = 3 independent experiments). Results were normalized to Renilla luciferase activity and expressed as relative luciferase units. Significant differences between groups were evaluated by two-way ANOVA with Šídák’s multiple comparisons test for pairwise comparisons. *** p < 0.001. ns, no significance.

Figure 6

ATF4 promotes lung fibroblast activation. (A) qPCR analysis of ATF4-overexpressing normal fibroblasts (pcDNA3.1+HA-ATF4) shows enhanced profibrotic transcript levels compared with normal lung fibroblasts transfected with an empty vector plasmid. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (B) HA-tagged ATF4, collagen I, α-SMA, fibronectin, and β-actin proteins were detected by western blot (N = 3 independent experiments). (C) The activation of fibroblasts was examined based on fibroblast contraction in 3D collagen matrices (N = 3 independent experiments). We used ImageJ software to measure the area of gels. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001. (D) MRC5 cells were subjected to immunofluorescence with a vimentin antibody. Scale bars, 20 μm. ImageJ software was used to quantify the fluorescence intensity. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001.

Figure 7

TRIB3 attenuates lung fibrosis by negative regulation of ATF4. (A) Schematic of transfection of A549 cells before CM collection and transfer. CM from pcDNA3.1 or pcDNA3.1+Flag-TRIB3-transfected cells was collected 72 h after transfection and transferred to human lung fibroblast (MRC5) for an additional 72 h. (B) qPCR analysis showing that CM from pcDNA3.1+ Flag-TRIB3-transfected cells reduced the mRNA level of ATF4 and profibrotic genes in MRC5 cells. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. (C) Western blot analysis showing CM from pcDNA3.1+ Flag-TRIB3-transfected cells reduced the expression of ATF4, α-SMA, Collagen I, Fibronectin, and vimentin. Significant differences between groups were evaluated by two-way ANOVA with Šídák’s multiple comparisons test for pairwise comparisons. * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Figure 8

Antifibrotic mechanism of TRIB3 mediation of pulmonary fibrosis through inhibition of lung fibroblast activation. TRIB3 inhibits the transformation of pulmonary fibroblasts into myofibroblasts through interaction with ATF4 and inhibiting its expression.