Growth factor-stimulated DNA synthesis in a variety of cell lines has been shown to be decreased after overnight (or longer) treatment with the 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, the statins. Although this anti-mitogenic effect had been presumed to be the result of the impairment of Ras lipidation, a stable modification (T1/2 approximately 20 h), this study provides new data demonstrating that brief (approximately 1 h) pretreatment of rat vascular smooth muscle cells with 100 microM pravastatin before platelet-derived growth factor-BB (PDGF-BB) stimulation results in attenuation of DNA synthesis through a Ras-independent mechanism. PDGF-BB-stimulated PDGF-beta receptor tyrosine phosphorylation, Ras activity, and mitogen-activated protein/extracellular signal-regulated kinase activity are unaffected by from 10 min to 1 h of pravastatin incubation, while Raf activity is markedly increased after 1 h of pravastatin. Phosphatidylinositol-3 kinase activity and phosphorylation of its downstream effector Akt are decreased after 1 h pravastatin incubation. Rho is stabilized by pravastatin, and ADP-ribosylation of Rho by C3 exoenzyme decreases PDGF-stimulated phosphatidylinositol-3 kinase activity, mimicking the effect of pravastatin on this signaling protein. Levels of the cyclin-dependent kinase inhibitor p27Kip1 are increased when cells were preincubated with pravastatin for 1 h and then exposed to PDGF, and apoptosis is induced by pravastatin incubation times as short as 1 to 4 h. Thus, short-term, high-dose pravastatin inhibits vascular smooth muscle cell growth and induces apoptosis independently of Ras, likely by means of the drug's effect on p27Kip1, mediated by Rho and/or phosphatidylinositol-3 kinase. This work demonstrates for the first time that the statins may be therapeutically useful when applied for short periods of time such that potential toxicity of long-term statin use (such as chronic Ras inhibition) may be avoided, suggesting future therapeutic directions for statin research.

Background. Glomerulonephritis is characterized by the accumulation of extracellular matrix protein within the glomerulus. This process, when allowed to proceed unimpeded, leads to glomerulosclerosis and eventually to cessation of glomerular filtration. There is evidence that protein kinase C (PKC) activation plays an important role in mediating at least some of the effects of TGF-beta in vascular smooth-muscle cells. The current study was undertaken to determine whether PKC activity is required for both TGF-beta and angiotensin II (Ang II) to induce mesangial cell matrix protein secretion.Methods. PKC was inhibited by two separate methods, and [H-3]thymidine incorporation was assessed in both the presence and the absence of PKC inhibition. Conditioned medium from cells stimulated with TGF-beta or Ang II was collected and analysed for secreted matrix proteins and sulphated proteins by SDS-polyacrylamide gel electrophoresis and western blotting.Results. Twenty-four-hour incubation of rat mesangial cells with phorbol-12-myristate-13-acetate (PMA) reduced total PKC activity to basal levels. Both TGF-beta and Ang II were mitogenic in mesangial cells, and chronic PMA pre-incubation inhibited this DNA synthesis. TGF-beta- and Ang-II-induced sulphated protein secretion into conditioned medium was markedly attenuated in PKC-downregulated cells. Secretion of the specific matrix proteins laminin and fibronectin by mesangial cells stimulated with either TGF-beta or Ang II was also diminished in PKC-downregulated cells and in cells pre-incubated with the specific PKC inhibitor, chelerythrine. There was no evidence of generalized cell toxicity or decreased non-specific protein synthesis caused by these PKC inhibitors.Conclusions, PKC is a key intermediary in the process by which TGF-beta and Ang II cause DNA synthesis and mesangial cell matrix protein production. Thus, PKC inhibitors deserve further study as potential therapeutic agents for a variety of glomerular diseases.