Abstract
Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.
Highlights
The epithelial to mesenchymal transition (EMT) is a fundamental process of the embryogenesis and morphogenesis, wherein epithelial cells gradually lose their characteristic features and functionality acquiring a mesenchymal cell phenotype [1]
In the present study, we investigated the role of some prototypal P1 (A1, A2A) and P2 (P2Y1, P2Y11, P2X7) purine receptors in TGF-β1-driven EMT in Madin Darby canine kidney (MDCK) cells by focusing on the underlying mechanisms linked to their antifibrotic effects
We first determined the optimum concentration and the time of exposure of TGF-β1 able to induce, in MDCK cells, the expression of EMT-related genes, ZEB1, Slug, Fibronectin, and α-SMA, which are associated to profibrotic processes, contributing to the loss of epithelial cell polarity and the acquisition of a fibroblastoid phenotype
Summary
The epithelial to mesenchymal transition (EMT) is a fundamental process of the embryogenesis and morphogenesis, wherein epithelial cells gradually lose their characteristic features and functionality acquiring a mesenchymal cell phenotype [1]. EMT reactivation in adults seems to play a pivotal role in tissue repair (wound healing and/or scar formation) through the production of proinflammatory cytokines. It has been considered as a crucial link between inflammation and the progression of organ fibrosis and cancer [2]. The contribution of EMT to tissue fibrosis in vivo is not univocal and it should be better defined [3,4,5,6], the proinflammatory cytokine TGF-β1 has been widely considered as one of the most potent activators of the experimentally induced EMT for its profibrotic effect in many cell types. Upon the binding to its receptor, TGF-β1 is able to induce EMT either via canonical pathways involving Smad2/3/4. Nieto et al [15]
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