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Tumor necrosis factor-mediated biological activities involve a G-protein-dependent mechanism
C Q Earl1, J M Stadel, M A Anzano
1Department of Clinical Investigation, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406-0939.
Abstract:
The guanine nucleotide binding protein (G-protein) dependency of several of the activities of tumor necrosis factor (TNF), including cytotoxicity, inhibition of lipoprotein lipase activity, blockade of 3T3-L1 differentiation, and receptor binding were examined. TNF induced killing of the TNF-sensitive cell line L929S (ED50 = 30 pM), but had little to no effect on the TNF-resistant cell line L929R (ED50 = 5,300 pM). TNF-induced cytotoxicity in L929S was antagonized in a dose-dependent manner by pertussis toxin (sevenfold increase in ED50). However, TNF-induced cytotoxicity in L929R cells was only minimally affected by pretreatment with a high dose (50 ng/ml) of pertussis toxin (1.5-fold increase in ED50). Parallel biochemical investigations revealed that inhibition was accompanied by toxin-induced ADP ribosylation of a Gi alpha-like subunit in L929 and 3T3-L1 cell membranes. Pertussis toxin also significantly reduced TNF-induced inhibition of lipoprotein lipase activity in 3T3-L1 adipocytes and TNF blockade of 3T3-L1 preadipocyte differentiation. However, pertussis toxin pretreatment of L929S, L929R, and 3T3-L1 cell cultures had little to no effect on TNF receptor binding. These data indicate that several TNF-induced biological activities in the L929 and 3T3-L1 cell lines are partially dependent upon a pertussis toxin-sensitive G-protein.
Insights
Tumor necrosis factor (TNF) activities like cell killing and differentiation blockade depend on G-proteins. Pertussis toxin, which targets G-proteins, significantly reduced these TNF effects, indicating G-protein involvement in TNF signaling.
Area of Science:
- Cellular biology
- Molecular signaling
- Immunology
Background:
- Tumor necrosis factor (TNF) is a key cytokine involved in inflammation and immunity.
- The precise molecular mechanisms underlying TNF's diverse cellular activities are not fully elucidated.
- Guanine nucleotide-binding proteins (G-proteins) are known regulators of various cellular processes.
Purpose of the Study:
- To investigate the dependency of TNF's biological activities on G-proteins.
- To determine if pertussis toxin-sensitive G-proteins mediate TNF-induced cytotoxicity, lipoprotein lipase inhibition, and cell differentiation blockade.
Main Methods:
- Utilized TNF-sensitive (L929S) and TNF-resistant (L929R) cell lines for cytotoxicity assays.
- Administered pertussis toxin to cells to assess its effect on TNF-induced responses.
- Performed biochemical analysis, including ADP ribosylation, to identify G-protein involvement.
- Examined TNF receptor binding in the presence of pertussis toxin.
Main Results:
- Pertussis toxin dose-dependently antagonized TNF-induced cytotoxicity in L929S cells.
- TNF-induced cytotoxicity in L929R cells was minimally affected by pertussis toxin.
- Pertussis toxin reduced TNF-induced inhibition of lipoprotein lipase activity and blockade of 3T3-L1 differentiation.
- Pertussis toxin did not significantly affect TNF receptor binding.
Conclusions:
- Several TNF-induced biological activities, including cytotoxicity and differentiation blockade, are partially dependent on a pertussis toxin-sensitive G-protein.
- G-protein signaling pathways are implicated in mediating specific TNF cellular responses.
- The findings provide insights into the molecular mechanisms of TNF action.