Negative regulation of smad1 pathway and collagen IV expression by store-operated Ca2+ entry in glomerular mesangial cells

Peiwen Wu, Yuezhong Ren, Yuhong Ma, Yanxia Wang, Hui Jiang, Sarika Chaudhari, Mark E. Davis, Jonathan E. Zuckerman, Rong Ma

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4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)F1090-F1100
JournalAmerican Journal of Physiology - Renal Physiology
Volume312
Issue number6
DOIs
StatePublished - 4 Jun 2017

Fingerprint

Mesangial Cells
Collagen
Smad1 Protein
Small Interfering RNA
Proteins
Glucose
Diabetic Nephropathies
Nanoparticles
Extracellular Matrix
Down-Regulation
Phosphorylation
Pharmacology

Keywords

  • Extracellular matrix
  • Store-operated calcium entry

Cite this

Wu, Peiwen ; Ren, Yuezhong ; Ma, Yuhong ; Wang, Yanxia ; Jiang, Hui ; Chaudhari, Sarika ; Davis, Mark E. ; Zuckerman, Jonathan E. ; Ma, Rong. / Negative regulation of smad1 pathway and collagen IV expression by store-operated Ca2+ entry in glomerular mesangial cells. In: American Journal of Physiology - Renal Physiology. 2017 ; Vol. 312, No. 6. pp. F1090-F1100.
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title = "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.",
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Negative regulation of smad1 pathway and collagen IV expression by store-operated Ca2+ entry in glomerular mesangial cells. / Wu, Peiwen; Ren, Yuezhong; Ma, Yuhong; Wang, Yanxia; Jiang, Hui; Chaudhari, Sarika; Davis, Mark E.; Zuckerman, Jonathan E.; Ma, Rong.

In: American Journal of Physiology - Renal Physiology, Vol. 312, No. 6, 04.06.2017, p. F1090-F1100.

Research output: Contribution to journalArticleResearchpeer-review

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

PY - 2017/6/4

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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

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U2 - 10.1152/ajprenal.00642.2016

DO - 10.1152/ajprenal.00642.2016

M3 - Article

VL - 312

SP - F1090-F1100

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 0363-6127

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