Downregulation of integrin-linked kinase mRNA expression by nitric oxide in rat glomerular mesangial cells

Inflammatory glomerular diseases are accompanied by changes in the expressi on patterns of growth factors, mediators and matrix-associated proteins in mesangial cells and by the production of nitric oxide via the cytokine-indu cible form of the nitric oxide synthase. Nitric oxide has been shown to act potently on gone transcription. To identify genes that are differentially expressed by endogenously produced nitric oxide, we forced rat mesangial ce lls to produce high amounts of nitric oxide by exposure to inflammatory cyt okines and compared the mRNA expression patterns of cytokine-stimulated mes angial cells with cells that were additionally treated with the nitric oxid e synthase inhibitor L-NMMA to block endogenous NO synthesis. We used a mod ification of a low stringency RT-PCR approach designated as RNA arbitrarily -primed polymerase chain reaction (RAP-PCR). In this way, we identified amo ng others the integrin-linked kinase (ILK) as an NO-regulated gene. The NO- mediated changes in the mRNA and protein expression patterns of ILK were co mpared to that of "secreted protein acidic and rich in cystein" (SPARC), a gene that was identified as NO-regulated in the same set of experiments (Wa lpen et al., J. Am. Soc. Nephrol., 11, 468-476). ILK and SPARC mRNA levels by were downregulated by cytokines via endogenously produced nitric oxide i n a comparable manner as verified by Northern blot analysis. In contrast, c ytokine-induced NO production or administration of exogenous NO-donors stro ngly reduced SPARC protein levels without altering ILK protein content in m esangial cells over a period up to 72 hours. Blocking de novo protein synth esis showed a short half-life of SPARC (<2 hours) whereas ILK-protein was s table over a period of 7 hours, indicating that NO-mediated reduction of IL K mRNA levels does not influence protein content of ILK in mesangial cells under the time limitations given under cell culture conditions. However, a role for cytokines/NO in ILK-long-term regulation in chronic inflammatory d iseases that may influence phenotypic responses such as apoptosis or cell p roliferation remains to be elucidated. (C) 2001 Elsevier Science Inc. All r ights reserved.