Reversibility of IGFBP-4 and -5 Inhibition
To address the question of reversibility, we tested the ability of exogenously added IGF-I to counteract the inhibitory effects of IGFBP-4 and -5 on Inh-a production. As shown in Figure 4, cotreatment with increasing concentrations of IGF-I reversed the inhibitory effects of both IGFBP-4 and -5. At high IGF-I concentrations (> 30 nM), there was a complete reversal of the inhibitory effect of 30 nM IGFBP-4 or -5. At the 100 nM dose of IGF-I, the levels of Inh-a in the conditioned medium were greater (p < 0.05) than that observed with activin alone (Fig. 4). This suggests that interactions between IGF-I and activin might contribute to greater effects on Inh-a production.
Effects of Activin and IGF-I on lnh-а Expression
To further explore this interaction, GC were treated for 48 h with 300 ng/ml activin or 30 nM IGF-I, and the levels of Inh-a protein and mRNA were measured in conditioned medium and cells, respectively. Control (untreated) cells produced a small amount of 18-kDa Inh-a spontaneously, and the amount was stimulated —300% and —180% by activin and IGF-I, respectively (Fig. 5A). Cotreatment with activin and IGF-I produced a further increase in 18-kDa Inh-a (-130%; p < 0.05) when compared to that produced by activin alone. As shown in Figure 5B, activin and IGF-I also increased the levels of 1.5-kb Inh-a mRNA, and the relative increases were of the same magnitude as those seen for the Inh-a protein. These results show that activin and IGF-I stimulation leads to significant parallel increases in Inh-a protein and mRNA levels in GC cultured in serum-free medium. buy ortho tri-cyclen online
In addition, we incubated the cells with 300 ng/ml activin in the presence of increasing concentrations of IGF-I. The results show that IGF-I enhanced activin-stimulated 18-kDa Inh-a production in a dose-dependent manner (ED5 = 4.3 ± 0.9 nM). IGF-I also produced similar elevations in activin-stimulated 1.5-kb Inh-a mRNA levels (data not shown).
FIG. 4. Reversibility of the IGFBP-4 and -5 inhibitory effects on lnh-a production. GC were cultured for 48 h with 300 ng/ml activin A and 30 nM IGFBP-4 or -5, either alone or together with increasing doses of IGF-I. After culture, the amount of lnh-a protein was measured in conditioned medium. Upper panel, typical Western immunoblot. Lower panel, the 18-kDa lnh-a after densitometry. *p < 0.05 compared to activin A treatment alone. Data are mean ± SEM of three experiments with one well per treatment per experiment.
FIG. 5. Effect of activin and IGF-I on Inh-a expression. GC were cultured for 48 h in the absence (control) and presence of activin A (300 ng/ml) and/or IGF-I (30 nM). After culture, the amounts of Inh-a protein and mRNA were measured in the conditioned medium and cells, respectively. A) Upper panel, typical Western immunoblot. Lower panel, intensity of 18-kDa Inh-a analyzed by densitometry. Data are mean ± SEM of four separate experiments with one well per treatment per experiment. B) Upper panel, typical Northern blot of 1.5-kb Inh-a mRNA and 1.0-kb cyclophilin mRNA. Lower panel, intensity of 1.5-kb Inh-a mRNA analyzed by