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Regulation of transforming growth factor beta 1 action by multiple transducing pathways: evidence for both G
P H Howe1, C C Bascom, M R Cunningham
1Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232.
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.
Insights
Cholera toxin (CT) inhibits transforming growth factor beta 1 (TGF-β1)-stimulated protooncogene expression and GTP binding in AKR-2B cells. However, CT did not affect TGF-β1-induced anchorage-independent growth, suggesting distinct intracellular pathways mediate TGF-β1 effects.
Area of Science:
- Cell biology
- Molecular biology
- Signal transduction
Background:
- Transforming growth factor beta 1 (TGF-β1) is a key regulator of cell growth and differentiation.
- Cholera toxin (CT) is known to modulate cellular signaling pathways through ADP-ribosylation.
- Understanding the intracellular mechanisms of TGF-β1 action is crucial for deciphering its diverse biological roles.
Purpose of the Study:
- To investigate the effects of CT on TGF-β1-stimulated cellular responses in AKR-2B fibroblasts.
- To elucidate the role of CT in modulating TGF-β1 signaling pathways, including protooncogene expression, GTP binding, and GTPase activity.
- To determine whether CT influences TGF-β1-mediated cell growth under anchorage-dependent and -independent conditions.
Main Methods:
- AKR-2B fibroblast cells were treated with CT and TGF-β1.
- Protooncogene mRNA expression (c-sis, c-myc) was assessed using decay and nuclear runon experiments.
- GTP binding and GTPase activity were measured in cell membranes.
- TGF-β1 binding to its receptor was analyzed using radioreceptor assays and affinity labeling.
- Cellular growth under anchorage-independent (soft agar) and -dependent conditions was evaluated.
- The effects of pertussis toxin were also examined for comparison.
Main Results:
- CT inhibited TGF-β1-stimulated c-sis and c-myc mRNA expression via decreased transcriptional activation, not increased message decay.
- CT did not affect TGF-β1 binding to its receptor.
- CT ADP-ribosylated a Mr 45,000 protein in AKR-2B cell membranes, inhibiting TGF-β1-stimulated [gamma-35S]GTP binding and GTPase activity.
- Unexpectedly, CT did not inhibit TGF-β1-stimulated anchorage-independent growth; pertussis toxin did.
- CT inhibited TGF-β1-stimulated monolayer growth, while pertussis toxin had no effect.
Conclusions:
- TGF-β1 utilizes multiple intracellular signaling pathways with distinct toxin sensitivities.
- CT interferes with specific TGF-β1-mediated signaling events, likely involving G proteins, but not anchorage-independent growth.
- Pertussis toxin selectively inhibits TGF-β1-induced anchorage-independent growth, highlighting pathway divergence.