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Store Operated Ca 2+ Entry Suppressed TGFβ1‐ Smad3 Signaling Pathway in Glomerular Mesangial Cells
Author(s) -
Chaudhari Sarika,
Li Weizu,
Ma Rong
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1224.28
Subject(s) - smad , microbiology and biotechnology , chemistry , western blot , transforming growth factor , signal transduction , mesangial cell , phosphorylation , extracellular matrix , medicine , endocrinology , biology , kidney , gene , biochemistry
Excessive expression of extracellular matrix (ECM) proteins in glomerulus is one of the consistent pathological changes seen in kidney diseases, such as diabetic nephropathy. Transforming growth factor‐ β1 (TGFβ1)‐Smad3 pathway plays a critical role in ECM protein expression and renal fibrosis. Receptor binding of TGFβ1 leads to phosphorylation and subsequent translocation of Smad3 into the nucleus regulating transcription of target genes directly or indirectly. The Orai1‐mediated store operated Ca 2+ 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. We have previously demonstrated that SOCE in MCs suppressed ECM protein expression in vitro and in vivo. However, the downstream mechanism underlying the SOCE effect is not known. 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 phosphorylation and translocation of Smad3 was examined in presence and absence of thapsigargin (TG) (1 μM), a classical activator of store‐operated Ca 2+ 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 La 3+ (5 μM) and GSK‐7975A (10 μM), both of which are selective blockers of store‐operated Ca 2+ 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, and thus could be a potential therapeutic target of kidney disease with glomerular fibrosis. Support or Funding Information UNTHSC Institutional Seed Grant, UNTHSC Doctoral Student Bridge Grant, Sigma Xi Grants ‐in‐Aid research grant.