Please click here to obtain permission to reproduce this image.
Click on image to view larger version.
Figure 4 The effect of pravastatin (Prava) and mevalonate (MEV) on the production and messenger ribonucleic acid (mRNA) levels of transforming growth factor-beta1 (TGF-beta1). (A) Bioassay of TGF-beta1 activity in a conditioned medium of monocyte cultures incubated with pravastatin (1–10 µM) or pravastatin (5 µM) plus mevalonate (100 µM). A conditioned medium from the cultures was collected after 24 h and acidified for mink-lung epithelial cell bioassay. Mink-lung cells respond to TGF-beta1 released in the culture medium, with a dose-dependent decrease in deoxyribonucleic acid (DNA) synthesis, as evaluated by 3H-thymidine incorporation. A comparison with the standard curve and multiplication by the dilution factor (1:10) yielded a dose-dependent increase in the TGF-beta1 concentration (ng/ml) in the conditioned medium of monocyte cultures. Mevalonate reversed the TGF-beta1–dependent effect on mink-lung cells. The results are expressed as the mean value ± SD of three independent experiments performed in triplicate. Comparisons among treatment conditions were carried out by analysis of variance, followed by Dunnett’s test. *p < 0.05, **p < 0.01 and p = NS versus untreated (control) cells. (B) Representative Northern blot analysis of TGF-beta1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in human peripheral blood monocytes after incubation with pravastatin (5 µM) alone or mevalonate (100 µM) together with pravastatin (5 µM). Mononuclear cells were cultured 24 h in serum-free conditions. Total RNA (5 µg per lane) was electrophoresed on 1.5% formaldehyde agarose gels, transferred to nitrocellulose and hybridized with 32P random primer-labeled TGF-beta1 complementary DNA. The sizes of the transcripts were determined by comparison with 28S and 18S ribosomal RNA. Molecular size markers are shown on the left. Lane 1 = untreated cells; lane 2 = pravastatin-treated cells; and lane 3 = cell treated with pravastatin plus mevalonate. Quantitative data obtained from imaging are expressed in arbitrary units.
|
