BDNF/TrkB axis activation promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis

Abstract

Background: Neurotrophins (NT) belongs to a family of growth factors which promotes neurons survival and differentiation. Increasing evidence show that NT and their receptor are expressed in lung tissues suggesting a possible role in lung health and disease. Here we investigated the expression and functional role of the TrkB/BDNF axis in idiopathic pulmonary fibrotic lung (myo)fibroblasts.

Methods: Lung fibroblast were isolated from IPF patients and characterized for the expression of mesenchymal markers in comparison to normal lung fibroblasts isolated from non-IPF controls.

Results: BDNF treatment promoted mesenchymal differentiation and this effect was counteracted by the TrkB inhibitor K252a. In this regard, we showed that K252a treatment was able to control the expression of transcription factors involved in epithelial to mesenchymal transition (EMT). Accordingly, K252a treatment reduced matrix metalloproteinase-9 enzyme activity and E-cadherin expression while increased cytoplasmic β-catenin expression.

Conclusions: Our results suggest that BDNF/TrkB axis plays a role in EMT promoting the acquisition of (myo)fibroblast cell phenotype in IPF. Targeting BDNF/TrkB seems to represent a viable approach in order to prevent EMT dependent lung fibrosis.

Keywords: BDNF; EMT; Idiopathic pulmonary fibrosis; Lung; TrkB.

Fig. 1

Analysis of mesenchymal and epithelial markers in representative idiopathic pulmonary fibrosis (IPF/UIP1 and 2) and normal fibroblast (FibroN2) cell cultures using flow cytometry. a Analysis of mesenchymal and epithelial markers in representative idiopathic pulmonary fibrosis (IPF/UIP1 and 2) and normal fibroblast (FibroN2) cell cultures using flow cytometry. b Panel B represents the mean values ± SD of the results obtained in the two normal fibroblast cell cultures considered as controls in comparison with the five fibroblast cell cultures obtained from IPF patients. * p < 0.01 versus normal fibroblast cell cultures. Negative sample was generated with the corresponding isotope (negative)

Fig. 2

K252a by BDNF/TrkB axis modulate the mesenchymal and epithelial markers in fibroblast cell line. Representative immunofluorescence pictures showing that BDNF binding to TrKB receptor up-regulates the expression of mesenchymal markers. These effects are inhibited by the specific TrkB receptor selective inhibitor K252a

Fig. 3

Cell signaling in idiopathic pulmonary fibrosis. Western blot analysis of the expression of mesenchymal marchers in homogenates of fibroblast cell cultures obtained from normal and IPF subjects. As noticeable, the expression of mesenchymal marker protein was decreased in the presence of the TrkB receptor blocker K252a in IPF fibroblast homogenates (*p < 0.001; **p < 0.05; ^***p < 0,01 vs BDNF treated and controls) (a). Contrarily, the presence of the BDNF increased α-actin expression in normal fibroblast (*p < 0.001; **p < 0.01 vs controls) (b). The addition of TrkB receptor selective inhibitor K252a reduces the phosphorylation of AKT in IPF fibroblasts (*p < 0.05; **p < 0.001 vs K252a treated cells) (c). This effect was slightly noticeable in normal fibroblasts (*p < 0.01; **p < 0.05 vs K252a treated cells) (d). Results were statistically evaluated by using Student’s t test

Fig. 4

Effect of the TrkB receptor blocker K252a on fibroblast cell growth. a K252a induced a decrease in cell growth in comparison to cell line treated with BDNF and no treated cells after 120 h (C). The number of viable cells present in a cell suspension was obtained by tryplan-blue staining. Histogram at the bottom represents the number of fibroblast cell viability obtained in the Trypan blue exclusion test after 24 h of treatment with indirubin. The asterisk indicates significant difference compared to the BDNF treated cell and control group (*p < 0.05, ** p < 0.01, ANOVA followed by Tukey’s test). Each bar represents the mean ± SD of three independent experiments. b Migration ability of fibroblast cell lines determined by wound healing assay. Cell lines treated with BDNF have a higher motility into the wounded area, in comparison those treated with TrkB inhibitor K252a (black rectangles). Results were statistically evaluated by Students t test (*p < 0.05 K252a treated cells vs controls, **p < 0.01 BDNF treated cells vs controls). The amount of migrated cells was measured by ImageJ software (version 1.32j)

Fig. 5

BDNF/TrKB axis activation on transcriptional factors and ECM markers. a mRNA levels of Zeb, Snail and Twist changes in the presence of BDNF or TrkB receptor blocker K252a. Note the decrease in the mRNA expression in comparison to BDNF treated cell lines. b Zymography assay. Enzyme activity of MPP9 was reduced after k252a treatment. c BDNF effects on cytoplasmic and nuclear expression of the β-catenin protein. Increase of the β-catenin cytoplasmic expression induced by BDNF was inhibited blocking TrKB receptor by K252a (*p = 0.003). The nuclear expression of β-catenin was increased in the presence of BDNF. K252a reverted this effect (**p = 0.0007). UDR: densitometric relative unit