Modulation of 72-Kilodalton Type IV Collagenase: RESULTS(2)
Equimolar amounts of glutathione did not affect the basal expression of MMP-2 (Fig. 1B). According to semiquantitative den-sitometric analysis and taking the vitamin C-free cell media as 100%, residual activity was approximately 60% in the 29 ^g/ml dose (range from 53% to 72%, n = 8) and 18% in the 200 ^g/ml vitamin C dose (range from 15% to 30%, n = 8). ANOVA on ranks showed differences between all treatments (p < 0.0001). Ascorbic acid did not have a direct effect on enzymatic activity as demonstrated by the gel-substrate assay of purified human proenzyme 72-kDa type IV collagenase in the presence of equivalent amounts of ascorbic acid (Fig. 1C). buy flovent inhaler
Western blotting of all the assayed media revealed the presence of a 71-kDa molecular mass band detected with the antibody directed against MMP-2. The relative intensity of this band decreased to 72% with 29 ^g/ml (range from 70% to 78%, n = 8) and to 55.6% with 200 ^g/ml of vitamin C (range from 45.0% to 65.1%, n = 5) compared to that for the cells incubated in absence of vitamin C (Fig. 2A). ANOVA on ranks revealed significant differences between all groups (p < 0.001).
MMP-2 mRNA showed a diminished expression when the concentration of vitamin C increased (Fig. 2B). Relative densitometric intensity decreased to 63% with 29 ^g/ml of vitamin C (range from 58% to 72%, n = 4) and to 8.7% with 200 ^g/ml (range from 6% to 11.0%, n = 4). Statistical differences reached p < 0.0001 between groups. In contrast, intensity of bands for type I collagen increased in the opposite way. The band of p-actin did not change in the presence of different concentrations of vitamin C (Fig. 2B).
Transcription rate for MMP-2, as measured by relative densitometric intensity in the run-on assays, decreased to 13% (range 11% to 17%, n = 3) in those nuclei obtained from cells stimulated with 200 ^g/ml of vitamin C as compared to the basal rate in the absence of the vitamin (Fig. 3).
FIG. 2. Western and Northern blot. A) Immunoreactive MMP-2 decreased in the media of vitamin C-stimulated WISH cells. Lanes 1-5 contained 0, 10, 29, 100, and 200 ^g/ml of vitamin C, respectively. B) Basal levels of MMP-2 mRNA (lane 1) decreased in the cells stimulated with 29, 100, and 200 ^g/ml of vitamin C (lanes 2-4). No effect was observed in p-actin mRNA, and the opposite effect was documented in type I collagen mRNA.
FIG. 3. Run-on assay. WISH cells were stimulated with 0, 10, 29, 50, 100, and 200 ^,g/ml of vitamin C (lanes 1-6), and the newly synthesized labeled RNA were hybridized against membrane-linked probes for p-actin and MMP-2. A progressive decrease in the transcriptional rate of the MMP-2 mRNA depending on the vitamin C dose was observed.