Dose Response and Time Course of Activin Effects on lnh-а Expression
We first investigated the dose response and time course relationships between activin and Inh-a production. Figure 1A shows that treatment with graded doses of activin for 48 h stimulated the production of 18-kDa Inh-a protein in a dose-dependent fashion (ED50 = 48.3 ± 6.7 ng/ml). Results from the time course experiments are shown in Figure IB. Small amounts of 18-kDa Inh-a protein were detected in the conditioned media from control (untreated) GC throughout the 72-h culture period. Comparison of media conditioned by treated GC with matched controls showed that activin stimulated a significant increase (~3-fold) in Inh-ot production at each time point tested. These results provide further support for the concept that the stimulatory effects of activin on Inh-a production are dose- and time-dependent.
Effect of Tyrphostin A23 and A63
To determine whether protein tyrosine kinase (PTK) activity is involved in the activin response, we tested the effect of tyrphostin A23, a PTK inhibitor that has been shown to inhibit receptor PTK autophosphorylation, including IGF-I-dependent tyrosine phosphorylation. Figure 2 shows the results of this experiment. In the presence of increasing concentrations of A23, there was a dose-dependent decrease (IC50 = 6.3 ±1.6 jjlM) in activin stimulation of 18-kDa Inh-a production. At 30 |xM A23, there was a 100% inhibition of the activin responses (Fig. 2A). Treatment with the inactive isomer, tyrphostin A63 (negative control), had no significant effect (p > 0.05) on the stimulation of Inh-a by activin (Fig. 2A). Figure 2B shows that increasing concentrations of A23 caused an almost identical dose-dependent decrease (/C50 = 4.1 ± 0.7 (xM) in activin-stimulated Inh-a mRNA levels in these cells, with 30 |xM A23 inhibiting Inh-a by nearly 100%. No effect of A63 on activin-stimulated Inh-a mRNA was observed (data not shown). Buy Asthma Inhalers Online
To address the issue of toxicity, we counted the number of viable GC in the A23- and activin-treated cultures using trypan blue dye. The results show that this inhibition did not result from decreases in the number of viable GC (control = 100 ± 3.6%; 300 ng/ml activin = 113 ± 1% \p = 0.28]; 30 (xM A23 = 115 ± 6.1.% [p = 0.19]; activin + A23 = 117 ± 5.5% \p = 0.14]).
FIG. 1. Dose response and time course study of activin effects on Inh-a production. GC were cultured as indicated, and the amount of lnh-a in conditioned medium was analyzed by Western immunoblot-ting and densitometry. A) Dose response relationship between activin A on the levels of 18-kDa lnh-a in 48-h conditioned medium analyzed by densitometry. B)
Time course of activin stimulation of 18-kDa lnh-a analyzed by densitometry. Data are mean ± SEM of four separate experiments with one well per data point per experiment.
FIG. 2. Effects of the protein kinase inhibitor tyrphostin A23. GC were cultured for 48 h with 300 ng/ml activin A and/or increasing doses of either A23 or its inactive isomer, A63. After culture, the amount of Inh-a protein and mRNA were measured in conditioned medium and cells, respectively. A) Upper panel, typical Western immunoblot. Lower panel, 18-kDa Inh-a analyzed by densitometry. Data are mean ± SEM; n = 3 experiments with one well per treatment per experiment. B) Upper panel, typical Northern blot. Lower panel, the 1.5-kb Inh-a mRNA analyzed by densitometry after normalization to the cyclophilin mRNA levels. *p < 0.05 when compared to activin A treatment alone. Data are mean ± SEM; n = 3 experiments.
FIG. 3. Inhibitory effects of IGFBPs and anti-IGF-l antibody on Inh-a production. GC were cultured for 48 h as indicated in the Materials and Methods section, and the amount of Inh-a in conditioned medium was measured by Western immuno-blotting. A) Effects of graded doses of IGFBP-4 and -5. Upper panel, typical Western immunoblot. Lower panel, 18-kDa Inh-a analyzed by densitometry. Data are mean ± SEM of three separate experiments with one well per treatment per experiment. B) Effect of anti-IGF-l monoclonal antibody and control mouse ascites fluid. Upper panel, typical Western immunoblot. Lower panel, levels of 18-kDa Inha analyzed by densitometry. *p < 0.05 compared to activin A treatment alone. Data are mean ± SEM of three separate experiments with one well per treatment per experiment.