REGULATION OF TRANSFORMING GROWTH FACTOR-BETA-1 ACTION BY MULTIPLE TRANSDUCING PATHWAYS - EVIDENCE FOR BOTH G-PROTEIN-DEPENDENT AND G-PROTEIN-INDEPENDENT SIGNALING

Abstract

The effects of cholera toxin (CT) on transforming growth factor .beta.1-stimulated protooncogene expression, [.gamma.-35S]GTP binding, GTPase activity and growth under anchorage-independent and -dependent conditions were studied in AKR-2B fibroblast cells. CT was shown to inhibit TGF.beta.1-stimulated c-sis and c-myc mRNA expression. Actinomycin D decay and nuclear runon experiments demonstrated that this inhibition was not due to an increased decay of protooncogene message, but to a decreased transcriptional activation. These inhibitory effects were not secondary to changes in the ability of TGF.beta.1 to bind to its receptor(s) since radioreceptor assays and affinity labeling studies demonstrated that CT had no effect on TGF.beta.1 binding. ADP ribosylation of AKR-2B plasma membranes with [.alpha.-32P]NAD+ revealed a Mr 45,000 protein as the major CT substrate. The labeling of this Mr 45,000 protein in membranes could be inhibited by prior pretreatment of the cells with increasing concentrations of CT. Treatment of membranes with nanogram concentrations of CT abolished the increase in [.gamma.-35S]GTP binding following addition of TGF.beta.1 as well as decreased basal binding. Similarly, CT pretreatment of membranes inhibited TGF.beta.1-stimulated GTPase activity. Unexpectedly however, the stimulatory effects of TGF.beta.1 on anchorage-independent growth in soft agar were unaffected by CT. Only pertussis toxin was able to inhibit TGF.beta.1-induced colony formation in soft agar in a dose-dependent manner. Furthermore, differential effects of both CT and pertussis toxin were observed on TGF.beta.1-stimulated monolayer growth; CT was inhibitory, whereas pertussis toxin was without effect. These results suggest that the diverse biological effects of TGF.beta.1 are mediated through multiple intracellular pathways distinguishable by their toxin sensitivities.