P0028THE ACTIVATION OF P75NTR RECEPTOR SIGNALING PROMOTES EMT IN ADPKD CELL LINE WT9-12

Abstract

Abstract Background and Aims ADPKD cystic epithelial cells have been shown to display an undifferentiated phenotype and to undergo epithelial to mesenchymal transition (EMT) changes as part of their phenotype. Thus, in ADPKD kidneys there is upregulation of EMT-related genes and increased fibrosis that support the proliferation of the cyst-lining cells and the progression of ADPKD. In our previous study, we provided that TGFβ1-induced p75NTR receptor activation promotes EMT in renal tubular cells, HK2. p75NTR is the common receptor for all the neurotrophins and their precursors. Since its identification and cloning in the 1980s, p75NTR has been reported to be involved in different and often contradictory functions such as survival, apoptosis, differentiation, proliferation and migration, depending on the cell type and cellular context. Furthermore, p75NTR is known to be overexpressed in some human cancer, where it participates in the stimulation of tumor growth and dissemination. p75NTR, through a neurotrophin-dependent mechanism, dramatically enhances migration and invasion of melanoma and glioma cells. Therefore, starting from these pieces of evidence and since WT9-12 cells exhibit higher constitutive expression of p75NTR and TGFβ1 than that of HK-2, the aim of this study is to carry out in vitro studies to investigate whether p75NTR is involved in EMT of cyst-lining cells and in renal fibrosis in ADPKD. Method Immortalized tubular renal cells homozygous for a truncating PKD1 mutation (WT9-12); Western Blot (WB); Real-Time PCR; Scratch and Trans-well migration assays; Immunofluorescence (IF); RNA interference (RNAi); Transfection assay. Results WB and IF assays showed that in WT9-12 cells TGFβ1 (5ng/ml) increased protein expression of p75NTR and of mesenchymal markers Fibronectin, N-Cadherin, α-SMA, Vimentin while reduced the expression of the epithelial marker E-Cadherin. This biological effect is significantly reversed in cells transfected with RNAi-p75NTR (Figure 1). Functional studies revealed that increased p75NTR protein expression was given to greater p75NTR mRNA stability. Time course assays, performed in cells transfected with RNAi-p75NTR and TGFβ1-treated, demonstrated downregulation of pERK1/2 and pJNK, p75NTR downstream pathways and involved in the non-canonical TGFβ1 pathway promoting pro-fibrotic genes transcription Figure 2A). Results of the analysis of SMAD pathway showed that SMAD3 was not significantly activated in cells untreated with TGFβ1. Moreover, selective ERK1/2 inhibition with PD98059 (10mM) reduced significantly the levels of mesenchymal markers N-Cadherin and α-SMA respect to the controls of untreated cells. Interestingly, in TGFβ1-treated cells in the presence of PD98059 the reduction of mesenchymal markers expression was minor respect to TGFβ1-untreated cells. These findings emphasize the importance of the ERK1/2 pathway in ADPKD. Our results revealed that the ERK1/2 pathway also plays a pivotal role in non-SMAD pathways downstream of TGFβ1 in fibrotic processes in ADPKD. Finally, Scratch and Trans-well migration assays showed a reduced cell motility in p75NTR-knocked-down cells (Figure 2B). Conclusion Although more basic studies are clearly needed to understand the mechanism of action of p75NTR, our results emphasized the crucial role of p75NTR in EMT changes in ADPKD cells which may contribute to the cystic phenotype and to fibrotic process.