Overexpression of truncated I kappa B alpha potentiates TNF-alpha-induced apoptosis in mesangial cells.
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-alpha (TNF-alpha) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-kappa B), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-kappa B affects TNF-alpha-induced apoptosis in rat mesangial cells (MCs).


METHODS
To specifically inhibit NF-kappa B activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa B alpha (AdexI kappa B delta N) that lacks the phosphorylation sites essential for the activation of NF-kappa B. Electrophoretic mobility shift assay was performed to evaluate NF-kappa B 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-alpha-induced NF-kappa B activation was blocked by overexpression of I kappa B delta N. Overexpression of I kappa B delta N potentiated TNF-alpha-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of I kappa B delta N augmented TNF-alpha-induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression.


CONCLUSIONS
Overexpression of I kappa B delta N potentiates TNF-alpha-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 AdexI kappa B delta N to induce apoptosis in MCs under inflammatory conditions.
Hirahashi
J
J
Department of Internal Medicine, and Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Takayanagi
A
A
Hishikawa
K
K
Takase
O
O
Chikaraishi
A
A
Hayashi
M
M
Shimizu
N
N
Saruta
T
T
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000502:physiology; D002478:Cells, Cultured; D005822:Genetic Vectors; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000737:chemistry / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D020672:I-kappa B Proteins; D016328:NF-kappa B; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases