Negative regulation of Smad1 pathway and collagen IV expression by store-operated Ca2+ entry in glomerular mesangial cells

Abstract

Collagen IV (Col IV) is a major component of expanded glomerular extracellular matrix in diabetic nephropathy and Smad1 is a key molecule regulating Col IV expression in mesangial cells (MCs). The present study was conducted to determine if Smad1 pathway and Col IV protein abundance were regulated by store-operated Ca²⁺ entry (SOCE). In cultured human MCs, pharmacological inhibition of SOCE significantly increased the total amount of Smad1 protein. Activation of SOCE blunted high-glucose-increased Smad1 protein content. Treatment of human MCs with ANG II at 1 µM for 15 min, high glucose for 3 days, or TGF-β1 at 5 ng/ml for 30 min increased the level of phosphorylated Smad1. However, the phosphorylation of Smad1 by those stimuli was significantly attenuated by activation of SOCE. Knocking down Smad1 reduced, but expressing Smad1 increased, the amount of Col IV protein. Furthermore, activation of SOCE significantly attenuated high-glucose-induced Col IV protein production, and blockade of SOCE substantially increased the abundance of Col IV. To further verify those in vitro findings, we downregulated SOCE specifically in MCs in mice using small-interfering RNA (siRNA) against Orai1 (the channel protein mediating SOCE) delivered by the targeted nanoparticle delivery system. Immunohistochemical examinations showed that expression of both Smad1 and Col IV proteins was significantly greater in the glomeruli with positively transfected Orai1 siRNA compared with the glomeruli from the mice without Orai1 siRNA treatment. Taken together, our results indicate that SOCE negatively regulates the Smad1 signaling pathway and inhibits Col IV protein production in MCs.

Keywords: extracellular matrix; store-operated calcium entry.

Fig. 1.

Western blot showing effect of store-operated Ca²⁺ entry (SOCE) on Smad1 protein content in human mesangial cells (MCs). A and B: inhibition of SOCE increased Smad1 protein abundance. A: representative Western blots. Cultured human MCs were without treatment (UT) or treated with methanol (Meth) [1:1,000, vehicle control for 2-aminoethyl diphenylborinate (2-APB)] and 2-APB (50 µM) for 2 days. L, protein ladder; TB, tubulin (loading control). B: summary data from the experiments presented in A. *P < 0.05, 2-APB vs. both UT and Meth groups. C and D: high-glucose treatment increased Smad1 protein abundance. C: representative Western blots. Confluent human MCs were cultured in serum-free medium containing normal glucose (NG, 5.6 mM d-glucose + 20 mM mannitol) or high glucose (HG, 25 mM d-glucose) for 1 day (1D) and 3 days (3D). D: summary data from the experiments presented in C. *P < 0.05, compared with NG group at the same time point. n, No. of independent experiments. E and F: activation of SOCE inhibited high-glucose-stimulated Smad1 production. E: representative Western blots. Confluent human MCs were cultured in serum-free medium containing NG (5.6 mM d-glucose + 20 mM mannitol) or HG (25 mM d-glucose) for 3 days. In two groups with high-glucose treatment, DMSO (1:1,000, vehicle control) and thapsigargin (TG, 1 μM) were applied on day 2. F: summary data from the experiments presented in E. P < 0.05, compared with NG group (*) and with both HG and HG + DMSO groups (†).

Fig. 8.

In vivo knockdown of Orai1 in MCs increased glomerular Col IV protein in mice. A: representative images of immunofluorescence staining showing glomerular Col IV in the mouse treated with NP alone (NP-Con) and NP-Cys-siOrai1 (knockdown of Orai1). Col IV is shown as green signals. In the panels of NP-Con, a bright-field image was captured to show glomeruli. In the panels of NP-Cy3-siOrai1, the distribution of NP-Cy3-siOrai1 is indicated by Cy3 signals (red). Arrows indicate glomeruli. Original magnification: ×200. B: ID of Smad1 fluorescence averaged from 4 NP-Con mice and 4 NP-Cy3-siOrai1-treated mice. **P < 0.01 compared with NP-Con. Nos. in parentheses under each bar represent the no. of glomeruli counted from 5 sections/kidney. C and D: Western blot showing Smad1 protein abundance in the cortex of kidney from the mouse treated with NP alone (NP-Con) and NP containing siRNA against Orai1 (NP-siOrai1). C: representative blot. D: summary data. *P < 0.05 compared with NP-Con. n, No. of mice in each group.

Fig. 2.

Western blot showing inhibition of phosphorylation of Smad1 by SOCE in human MCs. A and B: activation of SOCE inhibited ANG II-induced phosphorylation of Smad1. A: representative Western blot showing changes in abundance of phosphorylated Smad1 (p-Smad1) and total Smad1 (T-Smad1) proteins in different treatment groups. Human MCs were without treatment (UT) or treated with ANG II (1 µM) for 15 min in the presence of DMSO (1:1,000, vehicle control for TG) or TG (1 μM). B: summary data from the experiments presented in A showing the ratio of p-Smad1 to T-Smad1 in different groups. P < 0.05 vs. UN (*) and both ANG II and ANG II + DMSO (†). C and D: activation of SOCE inhibited HG-induced phosphorylation of Smad1. C: representative Western blots showing changes in abundance of p-Smad1 and T-Smad1 proteins in different treatment groups. Serum-deprived human MCs were cultured in NG (5.6 mM d-glucose + 20 mM mannitol) or HG (25 mM d-glucose) for 3 days. In two groups with high-glucose treatment, DMSO (1:1,000, vehicle control) and TG (1 μM) were applied on day 2. D: summary data from the experiments presented in C showing the ratio of p-Smad1 to T-Smad1 in different groups. *P < 0.05 compared with both HG and HG + DMSO groups.

Fig. 3.

SOCE inhibited TGF-β1-induced phosphorylation of Smad1 in human MCs. A and B: TGF-β1 phosphorylated Smad1. A: representative Western blot showing changes in abundance of p-Smad1 and T-Smad1 Smad1 in MCs treated with recombinant human TGF-β1 (5 ng/ml) for 0–240 min. B: summary data from the experiments presented in A showing the ratio of p-Smad1 to T-Smad1 at different treatment time periods. *P < 0.05 vs. group of 0 min. C and D: activation of SOCE inhibited TGF-β1-induced phosphorylation of Smad1. C: representative Western blots showing changes in abundance of p-Smad1 and T-Smad1 in different groups. Serum-deprived human MCs were without treatment (UT) or treated with TGF-β1 (5 ng/ml for 30 min) in the presence of DMSO (1:1,000) or TG (1 µM). D: summary data from the experiments presented in C showing the ratio of p-Smad1 to T-Smad1 in different groups. P < 0.05 compared with UT (*) and both TGF-β1 and TGF-β1 + DMSO (†). E and F: inhibition of TG on TGF-β1-induced phosphorylation of Smad1 was due to activation of SOCE. E: representative Western blots showing the abundance of p-Smad1 and T-Smad1 in serum-deprived human MCs treated with TGF-β1 (5 ng/ml for 30 min) in the absence or presence of TG (1 µM) with and without 2-APB (50 µM) or La³⁺ (2 µM). F: summary data from the experiments presented in E showing the ratio of p-Smad1 to T-Smad1 in different groups. P < 0.05 compared with TGF-β1 (*) and TGF-β1 + TG (†). n, No. of independent experiments.

Fig. 4.

Smad1 increased collagen IV (Col IV) protein abundance in human MCs. A: representative Western blot showing Col IV protein content in human MCs without transfection (UTran) and cells transfected with scramble small interfering RNA [siRNA (Scram)] or siRNA against human Smad1 [small interfering RNA against human Smad1 (siSmad1)]. TB was used as a loading control. B: summary data from the experiments presented in A. Col IV abundance in each group was normalized to TB, and the values in each group were further normalized to that of the UTran group. *P < 0.05 compared with both Utran and Scram groups. n, No. of independent experiments. C: representative Western blot from 3 independent experiments showing Smad1 protein content in human MCs without transfection (UTran) and cells transfected with scramble siRNA (Scram) or siSmad1. β-Actin was used as a loading control. D: representative Western blot from 3 independent experiments showing abundance of Smad1 and Col IV proteins in human MCs without transfection (UTran) and the cells transfected with an empty vector (pSHAG) or Flag-tagged Smad1 expresion plasmid (Flag-Smad1). α-TB was used as a loading control.

Fig. 5.

SOCE decreased abundance of Col IV protein produced by human MCs. A and B: effect of activation of SOCE on high-glucose-stimulated Col IV protein production. A: representative Western blots. B: summary data from the experiments presented in A. Human MCs were cultured in medium containing NG (5.6 mM d-glucose + 20 mM mannitol) or HG (25 mM d-glucose) with or without DMSO (1:1,000, vehicle control) and TG (1 μM). MCs were with and without various treatments for 2 days in 0.5% FBS medium. In B, P < 0.05 compared with NG group (*) and both HG and HG + DMSO groups (†). n, No. of independent experiments. C and D: effect of inhibition of SOCE on Col IV protein production in human MCs. C: representative Western blots. D: summary data from the experiments presented in C. UT, untreated cells; Meth, methanol, vehicle control for 2-APB. Serum-starved cells (in 0.5% FBS) were with or without treatments with methanol (1:1,000), or 2-APB (50 μM) or La³⁺ (5 µM), for 2 days before harvest. In D, * denotes P < 0.05 compared with both UT and Meth groups. n, No. of independent experiments.

Fig. 6.

Distribution of nanoparticle (NP)-Cy3-siOrai1 in mouse kidney. A: representative images from 4 mice showing localization of NP-Cy3-siOrai1 (red) in glomeruli (indicated by arrows). Original magnification: ×200. B: localization of NP-Cy3-siOrai1 in MCs (top) but not in podocytes (bottom), representative from 4 mice. MCs were stained with desmin (green), and podocytes were stained with synaptopodin (green). NP-Cy3-siOrai1 was shown as red signals. Original magnification: ×200. C: representative Western blot of renal cortical extracts showing Orai1 protein abundance in the mouse treated with NP alone (NP-Con) and NP containing siRNA against Orai1 (NP-siOrai1). D: summary data. *P < 0.05 compared with NP-Con; n, no. of mice in each group.

Fig. 7.

In vivo knockdown of Orai1 in MCs increased glomerular Smad1 protein in mice. A: representative images of immunofluorescence staining showing glomerular Smad1 in the mouse treated with NP alone (NP-Con) and NP-Cy3-siOrai1 (knockdown of Orai1). Smad1 is shown as green signals. In the panels of NP-Con, a bright-field image was captured to show glomeruli. In the panels of NP-Cy3-siOrai1, the distribution of NP-Cy3-siOrai1 is indicated by Cy3 signals (red). Arrows indicate glomeruli. Original magnification: ×200. B: integrated density (ID) of Smad1 fluorescence averaged from 4 NP-Con mice and 4 NP-Cy3-siOrai1-treated mice. **P < 0.01 compared with NP-Con. Nos. in parentheses under each bar represent the no. of glomeruli counted from 5 sections/kidney. C and D: Western blot showing Smad1 protein abundance in the cortex of kidney from the mouse treated with NP alone (NP-Con) and NP-Cy3-siOrai1 (NP-siOrai). C: representative blot. D: summary data. *P < 0.05 compared with NP-Con. n, No. of mice in each group.