Adenosine 5'-triphosphate stimulates the increase of TGF-beta1 in rat mesangial cells under high-glucose conditions via reactive oxygen species and ERK1/2.
OBJECTIVE
To investigate the role of adenosine 5'-triphosphate (ATP)-induced generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the production of transforming growth factor-beta1 (TGF-beta1) in cultured rat glomerular mesangial cells under high-glucose conditions.


METHODS
Subconfluent glomerular mesangial cells were serum-starved for 24 h and pretreated with suramin, diphenylenechloride iodonium (DPI) or PD98059 followed by stimulation with a high concentration of glucose (30 mmol/L D-glucose) or ATP (300 micromol/L). Extracellular and total ATP and ROS production were detected using commercially available kits. Phosphorylation of ERK1/2 was evaluated by Western blot. TGF-beta1 mRNA expression was examined by real-time PCR.


RESULTS
Suramin had a dose-dependent inhibitory effect on the generation of ROS induced by high glucose. Extracellular ATP production by mesangial cells increased markedly after a 2-h incubation with high glucose. ROS production was upregulated in mesangial cells after 5 min incubation with 300 micromol/L ATP and was sustained for 120 min. ERK1/2 was significantly activated after 5 min incubation of mesangial cells with ATP, this activation was partially inhibited by DPI. The effects of high glucose on TGF-beta1 mRNA were markedly inhibited by suramin, DPI or PD98059.


CONCLUSIONS
Our results suggest that a high concentration of glucose increases the extracellular levels of ATP in mesangial cells within a short time-frame. ATP, in turn, activates ERK1/2, an effect which is at least partially dependent on ROS, which results in the upregulation of TGF-beta1.
Qu
Lin-ping
LP
Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai, China.
Xue
Hong
H
Yuan
Ping
P
Zhou
Li
L
Yao
Tai
T
Huang
Yu
Y
Lu
Li-min
LM
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002460:Cell Line; D005419:Flavonoids / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D050527:Mesangial Cells / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009861:Onium Compounds / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013498:Suramin / Q000494:pharmacology; D013873:Thionucleotides / Q000378:metabolism* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
D005419:Flavonoids; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D013873:Thionucleotides; D053773:Transforming Growth Factor beta1; C014308:adenosine 5'-phosphorothioate; D000249:Adenosine Monophosphate; D013498:Suramin; C007517:diphenyleneiodonium; D019001:Nitric Oxide Synthase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D005947:Glucose; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one