|
Background: We investigated the role of c-Jun NH2- terminal kinase (JNK), a member of the mitogen-activated protein kinase family, in the expression of angiotensin II (Ang II)-induced monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-1 (TGF-1), and in the production of fibronectin (FN), by human mesangial cells (HMCs).
Methods: JNK activation in cultured human mesangial cells was determined by Western blotting with an antibody against the phosphorylated Ser63 residue of c-Jun. Binding of the activator protein (AP-1) to the MCP-1 AP-1 motif was detected via the electrophoretic mobility shift assay (EMSA). The transient luciferase reporter was used to examine MCP-1 promoter activity; an RNase protection assay and ELISA were used respectively to detect the expression of MCP-1 mRNA and production of MCP-1, TGF-¦Â and FN.
Results: Anthra (1,9-cd) pyrazol-6(2H)-one (SP600125), a pharmacological inhibitor of JNK, almost completely abolished Ang II-induced Ser63 phosphorylation of c-Jun at concentrations of 5-20 ¦Ìmol/L: JNK activity was reduced by 75% with 10 ¦Ìmol/L SP600125, and by 90% with 20 ¦Ìmol/L. Ang II increased AP-1 binding to the MCP-1 AP-1 motif in a time-dependent manner, as detected by EMSA, while SP600125 effectively blocked this increased AP-1 binding in a concentration-dependent manner. Treatment with 100 nmol/L Ang II led to a steady increase in MCP-1 mRNA expression, and to an enhanced production of MCP-1, TGF-¦Â and FN. These effects were blocked by SP60025 in a dose-dependent manner. SP600125 also reduced MCP-1 mRNA stability: the half-life of MCP-1 mRNA was approximately 5 hours in cells treated with Ang II only, but was reduced to 2 hours when treated with a combination of Ang II and SP600125.
Conclusions: These results show that the JNK/AP-1 pathway is involved in the expression of MCP-1 and TGF-¦Â, and in extracellular matrix production. JNK is an important therapeutic target for glomerulonephritis and glomerulosclerosis.
World J Pediatr 2010;6(2):169-176
|
