Ras activation modulates methylglyoxal-induced mesangial cell apoptosis through superoxide production.
OBJECTIVE
While previous studies have demonstrated that diabetic nephropathy is attributable to glucose-derived dicarbonyl compounds, methylglyoxal (MGO)-inducing apoptosis in renal mesangial cells, the molecular mechanism of upper stream redox signaling modulation, has not been fully elucidated.


METHODS
Rat mesangial cells pretreated with or without superoxide dismutase, diphenyloniodium, SB203580, and manumycin A were cultured in methylglyoxal stress-induced apoptosis. Signaling protein expression, flow cytometry, and morphological features of apoptotic cell death were assessed.


RESULTS
Methylglyoxal decreased cell viability in mesangial cells. Superoxide mediated methylglyoxal-induced caspase 3 cleavage. Pretreatment with diphenyloniodium, SB203580, and manumycin A reduced methylglyoxal augmentation of superoxide synthesis and caspase-3 activation. Methylglyoxal rapidly enhanced Ras activation and progressively increased cytosolic P38 and nuclear c-Jun activation. Scavenging of superoxide by superoxide dismutase or diphenyloniodium, inhibiting P38 by SB203580, and inhibiting Ras with manumycin A successfully reduced the promoting effect of methylglyoxal on P38 and c-Jun phosphorylation (activation). Furthermore, pretreatment with superoxide dismutase, diphenyloniodium, SB203580, and manumycin A significantly attenuated methylglyoxal induction of apoptosis on the basis of Annexin-V assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) staining.


CONCLUSIONS
This study has shown that methylglyoxal increased Ras modulation of superoxide-mediated P38 activation and c-Jun activation, which resulted in increased apoptosis.
Huang
Wei Jan
WJ
Chiayi Campus, Chang Gung Institute of Technology, Pu-tzu City, Chiayi, Taiwan.
Tung
Chun Wu
CW
Ho
Cheng
C
Yang
Jen Tsung
JT
Chen
Min Li
ML
Chang
Pey Jium
PJ
Lee
Pei Hsien
PH
Lin
Chun Liang
CL
Wang
Jeng Yi
JY
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002395:Catecholamines / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D007093:Imidazoles / Q000494:pharmacology; D048288:Imidazolines / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology; D053284:Polyunsaturated Alkamides / Q000494:pharmacology; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011765:Pyruvaldehyde / Q000494:pharmacology*; D051381:Rats; D013481:Superoxides / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D002395:Catecholamines; D007093:Imidazoles; D048288:Imidazolines; D011090:Polyenes; D053284:Polyunsaturated Alkamides; D016755:Proto-Oncogene Proteins c-jun; D011725:Pyridines; D013481:Superoxides; D011765:Pyruvaldehyde; C011775:(3,4-dihydroxyphenylamino)-2-imidazoline; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D018631:ras Proteins; C054474:manumycin; C093642:SB 203580