Nuclear factor κB mediates suppression of canonical transient receptor potential 6 expression by reactive oxygen species and protein kinase C in kidney cells

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Abstract

This study was carried out to explore the molecular mechanism for down-regulation of TRPC6 expression in the reactive oxygen species (ROS)/PKC signaling in kidney cells. In cultured human mesangial cells, H 2O2 and TNF-α inhibited TRPC6 mRNA expression in a time-dependent manner. Inhibition of NF-κB reversed both H 2O2- and phorbol 12-myristate 13-acetate (PMA)-induced decrease in TRPC6 protein expression. Activation of NF-κB by knocking down IκBα using siRNA could mimic the suppressive effect of ROS/PKC on TRPC6. a Ca2+ imaging study showed that activation and inhibition of NF-κB significantly decreased and increased the TRPC6-mediated Ca 2+ entry, respectively. Further experiments showed that PMA, but not its inactive analog 4α-phorbol 12, 13-didecanoate (4α-PDD), caused phosphorylation of IκBα and stimulated the nuclear translocation of NF-κB p50 and p65 subunits. The PMA-dependent IκBα phosphorylation was significantly inhibited by Gö6976. Electrophoretic mobility shift assay revealed that PMA stimulated DNA binding activity of NF-κB. Furthermore, specific knockdown of p65, but not p50, prevented an H2O2 inhibitory effect onTRPC6protein expression, suggesting p65 as a predominant NF-κB subunit repressing TRPC6. In agreement with a major role of p65, chromatin immunoprecipitation assays showed that PMA treatment induced p65 binding to the TRPC6 promoter. Moreover, PMA treatment increased the association of p65 with histone deacetylase (HDAC) and decreased histone acetylation at the TRPC6 promoter. Consistently, knockdown of HDAC2 by siRNA or inhibition of HDAC with trichostatin A prevented a H 2O2-induced decrease in TRPC6 mRNA and protein expressions, respectively. Taken together, our findings imply an important role of NF-κB in a negative regulation of TRPC6 expression at the gene transcription level in kidney cells.

Original languageEnglish
Pages (from-to)12852-12865
Number of pages14
JournalJournal of Biological Chemistry
Volume288
Issue number18
DOIs
StatePublished - 3 May 2013

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Protein Kinase C
Reactive Oxygen Species
Acetates
Kidney
Phosphorylation
Histone Deacetylases
Small Interfering RNA
Assays
trichostatin A
Chemical activation
Acetylation
Electrophoretic mobility
Messenger RNA
Mesangial Cells
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
Transcription
Histones
Chromatin
phorbol-12-myristate

Cite this

@article{ccf62f075f77447594522db5861443f8,
title = "Nuclear factor κB mediates suppression of canonical transient receptor potential 6 expression by reactive oxygen species and protein kinase C in kidney cells",
abstract = "This study was carried out to explore the molecular mechanism for down-regulation of TRPC6 expression in the reactive oxygen species (ROS)/PKC signaling in kidney cells. In cultured human mesangial cells, H 2O2 and TNF-α inhibited TRPC6 mRNA expression in a time-dependent manner. Inhibition of NF-κB reversed both H 2O2- and phorbol 12-myristate 13-acetate (PMA)-induced decrease in TRPC6 protein expression. Activation of NF-κB by knocking down IκBα using siRNA could mimic the suppressive effect of ROS/PKC on TRPC6. a Ca2+ imaging study showed that activation and inhibition of NF-κB significantly decreased and increased the TRPC6-mediated Ca 2+ entry, respectively. Further experiments showed that PMA, but not its inactive analog 4α-phorbol 12, 13-didecanoate (4α-PDD), caused phosphorylation of IκBα and stimulated the nuclear translocation of NF-κB p50 and p65 subunits. The PMA-dependent IκBα phosphorylation was significantly inhibited by G{\"o}6976. Electrophoretic mobility shift assay revealed that PMA stimulated DNA binding activity of NF-κB. Furthermore, specific knockdown of p65, but not p50, prevented an H2O2 inhibitory effect onTRPC6protein expression, suggesting p65 as a predominant NF-κB subunit repressing TRPC6. In agreement with a major role of p65, chromatin immunoprecipitation assays showed that PMA treatment induced p65 binding to the TRPC6 promoter. Moreover, PMA treatment increased the association of p65 with histone deacetylase (HDAC) and decreased histone acetylation at the TRPC6 promoter. Consistently, knockdown of HDAC2 by siRNA or inhibition of HDAC with trichostatin A prevented a H 2O2-induced decrease in TRPC6 mRNA and protein expressions, respectively. Taken together, our findings imply an important role of NF-κB in a negative regulation of TRPC6 expression at the gene transcription level in kidney cells.",
author = "Yanxia Wang and Min Ding and Sarika Chaudhari and Yanfeng Ding and Yuan, {Joseph P.} and Stankowska, {Dorota Luiza} and Shaoqing He and Raghu Krishnamoorthy and Cunningham, {Joseph Thomas} and Rong Ma",
year = "2013",
month = "5",
day = "3",
doi = "10.1074/jbc.M112.410357",
language = "English",
volume = "288",
pages = "12852--12865",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "18",

}

TY - JOUR

T1 - Nuclear factor κB mediates suppression of canonical transient receptor potential 6 expression by reactive oxygen species and protein kinase C in kidney cells

AU - Wang, Yanxia

AU - Ding, Min

AU - Chaudhari, Sarika

AU - Ding, Yanfeng

AU - Yuan, Joseph P.

AU - Stankowska, Dorota Luiza

AU - He, Shaoqing

AU - Krishnamoorthy, Raghu

AU - Cunningham, Joseph Thomas

AU - Ma, Rong

PY - 2013/5/3

Y1 - 2013/5/3

N2 - This study was carried out to explore the molecular mechanism for down-regulation of TRPC6 expression in the reactive oxygen species (ROS)/PKC signaling in kidney cells. In cultured human mesangial cells, H 2O2 and TNF-α inhibited TRPC6 mRNA expression in a time-dependent manner. Inhibition of NF-κB reversed both H 2O2- and phorbol 12-myristate 13-acetate (PMA)-induced decrease in TRPC6 protein expression. Activation of NF-κB by knocking down IκBα using siRNA could mimic the suppressive effect of ROS/PKC on TRPC6. a Ca2+ imaging study showed that activation and inhibition of NF-κB significantly decreased and increased the TRPC6-mediated Ca 2+ entry, respectively. Further experiments showed that PMA, but not its inactive analog 4α-phorbol 12, 13-didecanoate (4α-PDD), caused phosphorylation of IκBα and stimulated the nuclear translocation of NF-κB p50 and p65 subunits. The PMA-dependent IκBα phosphorylation was significantly inhibited by Gö6976. Electrophoretic mobility shift assay revealed that PMA stimulated DNA binding activity of NF-κB. Furthermore, specific knockdown of p65, but not p50, prevented an H2O2 inhibitory effect onTRPC6protein expression, suggesting p65 as a predominant NF-κB subunit repressing TRPC6. In agreement with a major role of p65, chromatin immunoprecipitation assays showed that PMA treatment induced p65 binding to the TRPC6 promoter. Moreover, PMA treatment increased the association of p65 with histone deacetylase (HDAC) and decreased histone acetylation at the TRPC6 promoter. Consistently, knockdown of HDAC2 by siRNA or inhibition of HDAC with trichostatin A prevented a H 2O2-induced decrease in TRPC6 mRNA and protein expressions, respectively. Taken together, our findings imply an important role of NF-κB in a negative regulation of TRPC6 expression at the gene transcription level in kidney cells.

AB - This study was carried out to explore the molecular mechanism for down-regulation of TRPC6 expression in the reactive oxygen species (ROS)/PKC signaling in kidney cells. In cultured human mesangial cells, H 2O2 and TNF-α inhibited TRPC6 mRNA expression in a time-dependent manner. Inhibition of NF-κB reversed both H 2O2- and phorbol 12-myristate 13-acetate (PMA)-induced decrease in TRPC6 protein expression. Activation of NF-κB by knocking down IκBα using siRNA could mimic the suppressive effect of ROS/PKC on TRPC6. a Ca2+ imaging study showed that activation and inhibition of NF-κB significantly decreased and increased the TRPC6-mediated Ca 2+ entry, respectively. Further experiments showed that PMA, but not its inactive analog 4α-phorbol 12, 13-didecanoate (4α-PDD), caused phosphorylation of IκBα and stimulated the nuclear translocation of NF-κB p50 and p65 subunits. The PMA-dependent IκBα phosphorylation was significantly inhibited by Gö6976. Electrophoretic mobility shift assay revealed that PMA stimulated DNA binding activity of NF-κB. Furthermore, specific knockdown of p65, but not p50, prevented an H2O2 inhibitory effect onTRPC6protein expression, suggesting p65 as a predominant NF-κB subunit repressing TRPC6. In agreement with a major role of p65, chromatin immunoprecipitation assays showed that PMA treatment induced p65 binding to the TRPC6 promoter. Moreover, PMA treatment increased the association of p65 with histone deacetylase (HDAC) and decreased histone acetylation at the TRPC6 promoter. Consistently, knockdown of HDAC2 by siRNA or inhibition of HDAC with trichostatin A prevented a H 2O2-induced decrease in TRPC6 mRNA and protein expressions, respectively. Taken together, our findings imply an important role of NF-κB in a negative regulation of TRPC6 expression at the gene transcription level in kidney cells.

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U2 - 10.1074/jbc.M112.410357

DO - 10.1074/jbc.M112.410357

M3 - Article

VL - 288

SP - 12852

EP - 12865

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 18

ER -