Thrombin inhibits aquaporin 4 expression through protein kinase C-dependent pathway in cultured astrocytes.
Aquaporin 4 (AQP4) is a key molecule for maintaining water balance in the central nervous system, and its dysfunction might cause brain edema. However, little is known about the regulation of AQP4 expression. Because thrombin has been implicated in brain edema formation, the purpose of this study is to determine whether thrombin affects expression of AQP4 in astrocytes. Here, the effect of thrombin on AQP4 expression in vitro was evaluated using Western blot analysis and RT-PCR. Meanwhile, we investigated whether the effect of thrombin on AQP4 expression was due to protease-activated receptor 1 (PAR-1). In addition, we examined the role of protein kinase C (PKC) in the effect of thrombin on AQP4 expression using Western blot analysis. We found that thrombin did not affect cell viability at concentrations of 0.05, 0.5, 5, or 50 nM but killed astrocytes at concentrations of 500 nM, with approx 72% of astrocytes surviving at 500 nM thrombin. Our data showed that AQP4 protein expression achieved only 28% of controls in 500 nM thrombin treatment, even if astrocytes survived approx 72% of controls at 500 nM thrombin. Thrombin significantly inhibited AQP4 in a time- and dose dependent manner in vitro (p<0.05). Cathepsin-G, a thrombin PAR-1 inhibitor, reversed significantly (p<0.05) the effect of thrombin on AQP4 mRNA and protein expression in astrocytes. We also observed that PKC inhibitor H-7 or prolonged pretreatment with TPA can rapidly increase AQP4 expression (p<0.05). Thrombin might inhibit AQP4 expression in rat astrocytes, and this effect is possibly mediated by the PKC pathway.
Tang
Yuping
Y
Laboratory of Neurology, Institute of Integrative Medicine, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Cai
Dingfang
D
Chen
Yiping
Y
D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics / Q000378:metabolism*; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D044463:Receptor, PAR-1 / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors
C496032:Aqp4 protein, rat; D051401:Aquaporin 4; D044463:Receptor, PAR-1; D011493:Protein Kinase C; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G; C534097:Ctsg protein, rat; D013917:Thrombin; D010975:Platelet Aggregation Inhibitors