Transforming growth factor-β (TGFβ) is a key mediator of extracellular matrix (ECM) accumulation in sclerotic kidney diseases such as diabetic nephropathy. One of the main target cells for TGFβ in the kidney are glomerular mesangial cells, which respond by increasing expression of ECM proteins, such as collagens, laminin and fibronectin, while suppressing the expression of ECM-degrading proteases and increasing the synthesis of ECM protease inhibitors, including plasminogen activator inhibitor-1. Previous studies have shown that exposure of mesangial cells to chronic high-glucose conditions, such as those seen in diabetes, increases ECM deposition in a mechanism involving glucose-mediated up-regulation of TGFβ expression. Naturally occurring inhibitors of this TGFβ-dependent fibrotic response include decorin, a small leucine-rich proteoglycan. While the mechanism by which TGFβ stimulates gene expression via the Smad signal-transduction pathway is becoming clear, the precise mechanism by which decorin may impinge upon TGFβ activity remains to be established. In this study, for the first time we provide evidence that decorin can disrupt glucose- and TGFβ/Smad-dependent transcriptional events in human mesangial cells through a mechanism that involves an increase in Ca2+ signalling, the activation of Ca2+/calmodulin-dependent protein kinase II and ensuing phosphorylation of Smad2 at Ser-240. We show that decorin also induces Ser-240 phospho-Smad hetero-oligomerization with Smad4 and the nuclear localization of this complex independently of TGFβ receptor activation. Thus, in human mesangial cells, the mechanism of decorin-mediated inhibition of TGFβ signalling may involve activation of Ca2+ signalling, the subsequent phosphorylation of Smad2 at a key regulatory site, and the sequestration of Smad4 in the nucleus.
Abbreviations used: Cam kinase II, Ca2+/calmodulin-dependent protein kinase II; CTGF, connective-tissue growth factor; ECM, extracellular matrix; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, haemagglutinin; HMC, human mesangial cell; PAI-1, plasminogen activator inhibitor-1; TGFβ, transforming growth factor-β; THMC, transformed HMC; RT-PCR, reverse transcriptase PCR.