TY - JOUR
T1 - Negative regulation of smad1 pathway and collagen IV expression by store-operated Ca2+ entry in glomerular mesangial cells
AU - Wu, Peiwen
AU - Ren, Yuezhong
AU - Ma, Yuhong
AU - Wang, Yanxia
AU - Jiang, Hui
AU - Chaudhari, Sarika
AU - Davis, Mark E.
AU - Zuckerman, Jonathan E.
AU - Ma, Rong
N1 - Funding Information:
We thank the Harry S. Moss Heart Trust for supporting this study and Dr. Xia Lin at the Baylor College of Medicine for providing Flag-tagged Smad1 expression plasmid.This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5RO1-DK-079968-01 (to R. Ma), by a Grant-in-Aid from the American Heart Association Southwestern Affiliate (16GRNT27780043, to R. Ma), by the National Natural Science Foundation of China (81400805, to P. Wu), and by the Natural Science Foundation of Fujian Province (2016J01449, to P. Wu).
Publisher Copyright:
© 2017 the American Physiological Society.
PY - 2017/6/4
Y1 - 2017/6/4
N2 - 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.
AB - 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.
KW - Extracellular matrix
KW - Store-operated calcium entry
UR - http://www.scopus.com/inward/record.url?scp=85020458539&partnerID=8YFLogxK
U2 - 10.1152/ajprenal.00642.2016
DO - 10.1152/ajprenal.00642.2016
M3 - Article
C2 - 28298362
AN - SCOPUS:85020458539
SN - 0363-6127
VL - 312
SP - F1090-F1100
JO - American Journal of Physiology - Renal Physiology
JF - American Journal of Physiology - Renal Physiology
IS - 6
ER -