Pediatr Nephrol 15: 290C301, 2000. by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 M) and GSK-7975A (10 M), both of which are selective blockers of store-operated Ca2+ channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+ channels, significantly augmented TGF-1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the Srebf1 TGF-1/Smad3 signaling pathway. < 0.001 vs. NT and HCl; **< 0.01 vs. TGF-1 and TGF-1 + DMSO (= no. of independent experiments). Open in a separate window Fig. 5. SOCE decreased TGF-1-stimulated nuclear translocation of Smad3 in human MCs. < 0.001 vs. NT and HCl; *< 0.05 vs. TGF-1, TGF1 + DMSO, and TGF-1 + TG + La3+ (= no. of independent experiments). < 0.01 compared with NT group; ?< 0.01 compared with groups of TGF-1, TGF-1 + DMSO, and TGF-1 + TG + GSK. The numbers under each bar (and of the experiment, and the mice were euthanized on < 0.05 was considered statistically significant. Statistical analysis was performed using SigmaStat (Jandel Scientific, San Rafael, CA). RESULTS SOCE activation did not alter the amount of secreted TGF-1 by human MCs. MCs are known to synthesize and secrete TGF-1 (23, 58, 62). To study whether Ca2+ entry via store-operated Ca2+ channels, i.e., SOCE, affected secretion of TGF-1 by MCs, we activated store-operated Ca2+ channels by treating the cells with 1 M TG for 8 and 15 h. ELISA assay showed that TG treatment for both time periods did not significantly change the concentration of TGF-1 in the cell culture media (Fig. 1). These results indicate that SOCE did not affect the amount of secreted TGF-1 protein by MCs. Open in a separate window Fig. 1. Effect of store-operated Ca2+ entry (SOCE) on transforming growth factor-1 (TGF-1) secretion in cultured human mesangial cells (MCs). ELISA, showing TGF-1 concentration in culture media. Confluent human MCs were incubated with serum-free DMEM for 72 h. One group was without any treatment (NT), Panaxtriol and the other groups were treated with DMSO (1:1,000) or TG (1 M) for 8 and 15 h before collection of media. DMSO and thapsigargin (TG) were present in the media throughout the period of treatment; = no. of independent experiments. SOCE inhibited TGF-1-induced phosphorylation of Smad3. A variety of stimuli such as angiotensin II, high glucose, advanced glycosylation end products, and reactive oxygen species activate TGF-1 to regulate expression of matrix proteins by MCs (16, 22, 53). One of the major intracellular downstream pathways mediating this effect has been demonstrated to be via the activation of Smad proteins, particularly Smad3 (14, 25, 27, Panaxtriol 30, 31, 48). In agreement with those studies, we also found that administration of TGF-1 (5 ng/ml), but not its vehicle control (HCl), for 15 h induced a robust increase in the content of phospho-Smad3, the active form of Smad3, in human MCs (Fig. 2, and and after transfection cells were treated with TGF-1 (5 ng/ml) in the presence or absence of TG (1 M) for 15 h, -tubulin was used as the loading control. L, protein ladder. < 0.01 and ***< 0.001 vs. UT; *< 0.05 vs. TGF-1, TGF-1 + Scr, and TGF-1 + siOrai1 + TG; #< 0.05 vs. TGF-1 + siOrai1 + TG; ##< 0.01 vs. TGF1 + siOrai1 (= no. of independent Panaxtriol experiments). To support these findings, we next overexpressed Orai1 protein in human MCs with FLAG-Orai1. The expressed Orai1 band was detected at 80 kDa (Fig. 4and after transfection, cells were treated with TGF-1 (5 ng/ml) in the presence or absence of TG (1 M) for 15 h. UT, cells without transfection or treatment; DMSO (1:1,000), vehicle control for TG. -Tubulin was used as the loading control. < 0.01 vs. UT; *< 0.05 vs. TGF-1, TGF-1 + DMSO, and TGF-1 + Orai1 + TG (= no. of independent experiments). < 0.01 compared with both Untrans and YFP groups; = no. of cells analyzed in each group. SOCE decreased TGF-1-mediated nuclear translocation of Smad3 in human MCs. Activation of Smad3 by TGF-1 involves its phosphorylation and subsequent.
