Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells

The Jagged/Notch pathway has been implicated in TGFβ1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFβ1-stimulated gene responses in human kidney epithelial cells in vitro. TGFβ1 treatment of HK-2 and RPTEC cells for 24 h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of γ-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFβ1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by γ-secretase inhibition, but other TGFβ1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFβ1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFβ1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFβ1, but did not affect α-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFβ1-responsive gene subset. Increased Jagged1 expression upon TGFβ1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFβ1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFβ1 and Notch cascades in kidney epithelia.

Research Highlights
► Jagged/Notch signalling is required for a subset of TGFβ1-regulated EMT genes in kidney epithelial cells.
► Upregulation of Jagged1 and Hes1 precedes changes in EMT genes such as E-cadherin.
► Inhibition of Jagged/Notch signalling prevents TGFβ1-induced changes in cell shape.
► Jagged1 upregulation by TGFβ1 is completely Smad3 dependent, and partially PI3K/ERK dependent.