Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells

Junichi Hirahashi, Atsushi Takayanagi, Keiichi Hishikawa, Osamu Takase, Akihiro Chikaraishi, Matsuhiko Hayashi, Nobuyoshi Shimizu, Takao Saruta

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Background. Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-κB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-κB affects TNF-α- induced apoptosis in rat mesangial cells (MCs). Methods. To specifically inhibit NF-κB activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κB. Electrophoretic mobility shift assay was performed to evaluate NF-κB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results. TNF-α-induced NF-κB activation was blocked by overexpression of IκBΔN. Overexpression of IκBΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α- induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion. Overexpression of IκBΔN potentiates TNF-α- induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκBΔN to induce apoptosis in MCs under inflammatory conditions.

Original languageEnglish
Pages (from-to)959-968
Number of pages10
JournalKidney International
Volume57
Issue number3
DOIs
Publication statusPublished - 2000

Fingerprint

Mesangial Cells
NF-kappa B
Tumor Necrosis Factor-alpha
Apoptosis
bcl-2-Associated X Protein
Caspase 3
Caspase 8
I-kappa B Proteins
Enzyme-Linked Immunosorbent Assay
Bisbenzimidazole
Caspase Inhibitors
Electrophoretic Mobility Shift Assay
DNA Fragmentation
Caspases
Glomerulonephritis
Adenoviridae
Transfection
Western Blotting
Phosphorylation
Staining and Labeling

Keywords

  • Apoptosis
  • Caspase-3
  • Cell death
  • Glomerulonephritis
  • IκB
  • Mesangial cells
  • NF-κB
  • TNF-α

ASJC Scopus subject areas

  • Nephrology

Cite this

Hirahashi, J., Takayanagi, A., Hishikawa, K., Takase, O., Chikaraishi, A., Hayashi, M., ... Saruta, T. (2000). Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells. Kidney International, 57(3), 959-968. https://doi.org/10.1046/j.1523-1755.2000.00924.x

Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells. / Hirahashi, Junichi; Takayanagi, Atsushi; Hishikawa, Keiichi; Takase, Osamu; Chikaraishi, Akihiro; Hayashi, Matsuhiko; Shimizu, Nobuyoshi; Saruta, Takao.

In: Kidney International, Vol. 57, No. 3, 2000, p. 959-968.

Research output: Contribution to journalArticle

Hirahashi, J, Takayanagi, A, Hishikawa, K, Takase, O, Chikaraishi, A, Hayashi, M, Shimizu, N & Saruta, T 2000, 'Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells', Kidney International, vol. 57, no. 3, pp. 959-968. https://doi.org/10.1046/j.1523-1755.2000.00924.x
Hirahashi, Junichi ; Takayanagi, Atsushi ; Hishikawa, Keiichi ; Takase, Osamu ; Chikaraishi, Akihiro ; Hayashi, Matsuhiko ; Shimizu, Nobuyoshi ; Saruta, Takao. / Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells. In: Kidney International. 2000 ; Vol. 57, No. 3. pp. 959-968.
@article{d9c416e9cde54bcd87d48062aaf6c1a1,
title = "Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells",
abstract = "Background. Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-κB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-κB affects TNF-α- induced apoptosis in rat mesangial cells (MCs). Methods. To specifically inhibit NF-κB activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κB. Electrophoretic mobility shift assay was performed to evaluate NF-κB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results. TNF-α-induced NF-κB activation was blocked by overexpression of IκBΔN. Overexpression of IκBΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α- induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion. Overexpression of IκBΔN potentiates TNF-α- induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκBΔN to induce apoptosis in MCs under inflammatory conditions.",
keywords = "Apoptosis, Caspase-3, Cell death, Glomerulonephritis, IκB, Mesangial cells, NF-κB, TNF-α",
author = "Junichi Hirahashi and Atsushi Takayanagi and Keiichi Hishikawa and Osamu Takase and Akihiro Chikaraishi and Matsuhiko Hayashi and Nobuyoshi Shimizu and Takao Saruta",
year = "2000",
doi = "10.1046/j.1523-1755.2000.00924.x",
language = "English",
volume = "57",
pages = "959--968",
journal = "Kidney International",
issn = "0085-2538",
publisher = "Nature Publishing Group",
number = "3",

}

TY - JOUR

T1 - Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells

AU - Hirahashi, Junichi

AU - Takayanagi, Atsushi

AU - Hishikawa, Keiichi

AU - Takase, Osamu

AU - Chikaraishi, Akihiro

AU - Hayashi, Matsuhiko

AU - Shimizu, Nobuyoshi

AU - Saruta, Takao

PY - 2000

Y1 - 2000

N2 - Background. Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-κB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-κB affects TNF-α- induced apoptosis in rat mesangial cells (MCs). Methods. To specifically inhibit NF-κB activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κB. Electrophoretic mobility shift assay was performed to evaluate NF-κB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results. TNF-α-induced NF-κB activation was blocked by overexpression of IκBΔN. Overexpression of IκBΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α- induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion. Overexpression of IκBΔN potentiates TNF-α- induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκBΔN to induce apoptosis in MCs under inflammatory conditions.

AB - Background. Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-κB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-κB affects TNF-α- induced apoptosis in rat mesangial cells (MCs). Methods. To specifically inhibit NF-κB activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κB. Electrophoretic mobility shift assay was performed to evaluate NF-κB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results. TNF-α-induced NF-κB activation was blocked by overexpression of IκBΔN. Overexpression of IκBΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α- induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion. Overexpression of IκBΔN potentiates TNF-α- induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκBΔN to induce apoptosis in MCs under inflammatory conditions.

KW - Apoptosis

KW - Caspase-3

KW - Cell death

KW - Glomerulonephritis

KW - IκB

KW - Mesangial cells

KW - NF-κB

KW - TNF-α

UR - http://www.scopus.com/inward/record.url?scp=0033935099&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033935099&partnerID=8YFLogxK

U2 - 10.1046/j.1523-1755.2000.00924.x

DO - 10.1046/j.1523-1755.2000.00924.x

M3 - Article

C2 - 10720949

AN - SCOPUS:0033935099

VL - 57

SP - 959

EP - 968

JO - Kidney International

JF - Kidney International

SN - 0085-2538

IS - 3

ER -