Regulation of TGF β1 signaling pathway by Store Operated Calcium Entry in Mesangial Cells: A mechanism for controlling Extracellular Matrix Protein Expression in Kidney




Wang, Yanxia
Chaudhari, Sarika
Li, Weizu
Ma, Rong


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Excessive extracellular matrix (ECM) proteins accumulation in glomerulus is one of the consistent pathological changes seen in kidney diseases, such as diabetic nephropathy. The Orai1-mediated store operated Ca2+ entry (SOCE) is associated with many physiological and pathological processes in a variety of cells, including glomerular mesangial cells (MCs) which are a major source of ECM proteins. Previously, we demonstrated in vivo and in vitro that SOCE is enhanced in MCs in diabetes. Also, in cultured human MCs, activation of store–operated Ca2+ channels significantly decreased fibronectin protein expression and collagen IV mRNA expression while inhibition of the channels significantly increased the expression of fibronectin and collagen IV. In vivo knockdown of Orai1 in MCs in mice using the targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV. However, the downstream mechanism underlying the SOCE effect is not known. Transforming growth factor- β1 (TGFβ1)-Smad3 pathway plays a critical role in ECM protein expression and renal fibrosis. The present study was conducted to test the hypothesis that SOCE suppressed ECM protein expression by inhibiting TGF β1-Smad3 pathway in MCs. In cultured human MCs, TGFβ1- induced activation of Smad3 in terms of its phosphorylation and translocation was examined in presence and absence of thapsigargin (TG, 1 µM), a classical activator of store-operated Ca2+ channel. We found that treatment with TGFβ1 (5 ng/ml for 15 hours) significantly increased the expression level of Phospho-Smad3 (p-Smad3) evaluated by Western blot. However, this response was markedly inhibited by TG treatment. Consistently, immunocytochemistry and Western blot showed that TGF β1 significantly increased the expression of nuclear Smad3. Again, this TGFβ1-induced nuclear translocation of Smad3 was prevented by pre-treatment with TG. Importantly, the TG effect was reversed by La3+ (5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+ channel. Furthermore, knockdown of Orai1 using siRNA approach significantly augmented TGFβ1-induced p-Smad3 expression. Taken together, our results indicate that SOCE in MCs negatively regulates the TGFβ1/Smad3 signaling pathway which may in turn suppress the ECM proteins and thus could be a potential therapeutic target of kidney disease with glomerular fibrosis like diabetic nephropathy.