Differential behavior of mesangial cells derived from 12/15-lipoxygenase knockout mice relative to control mice

Background. The 12/15-lipoxygenase (12/15-LO) enzyme has been implicated in the pathogenesis of diabetic nephropathy since lipoxygenase products induce cellular hypertrophy and extracellular matrix deposition in mesangial cells. In this study, in order to determine the potential in vivo functional role of 12/15-LO in kidney disease, we compared mouse mesangial cells (MMCs) derived from 12/15-LO knockout mice with those from genetic control wild-type mice. Methods. MMCs were isolated from wild-type and 12/15-LO knockout mice. Cellular growth, activation of mitogen-activated protein kinases (MAPKs), transcription factors, superoxide levels, and fibronectin expression were compared in the two cell types. Results. Levels of the 12/15-LO product and protein were lower in MMC from 12/15-LO knockout relative to wild-type. MMCs from 12/15-LO knockout mice grew slower than wild-type cells, and also showed lower rates of tritiated thymidine and leucine incorporation (21% and 15% of wild-type, respectively, P < 0.001). Levels of superoxide and the matrix protein fibronectin were also lower in 12/15-LO knockout mice cells. Serum and angiotensin II (Ang II)-stimulated activities of p38 or ERK1/2 MAPKs, and cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) transcription factor were lower in 12/15-LO knockout relative to wild-type cells. In addition, DNA binding and transcriptional activities of activated protein-1 (AP-1) and CREB were lower in 12/15-LO knockout cells. Furthermore, stable 12/15-LO overexpression in MMC led to reciprocal increase in p38 MAPK activation and fibronectin expression. Conclusion. The differential activation of oxidant stress, specific signaling pathways, transcription factors, and growth and matrix genes may lead to reduced growth and growth factor responses in 12/15-LO knockout versus wild-type MMCs. These results provide ex vivo functional evidence for the first time that 12/15-LO activation plays a key role in mesangial cell responses associated with renal diseases such as diabetic nephropathy.