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TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator
1Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany.
Abstract:
We investigated intracellular signaling events involved in fibronectin-accelerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresis and western blotting. Proteins were sequenced with electrospray ionization mass spectrometry. Apoptosis was quantitated by flow cytometry. We detected a cluster of acidic, high molecular-weight proteins that were only tyrosine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleavage, yielding a 23-kD fragment. At 8 h, intact Ly-GDI was decreased to 33% on fibronectin, compared with 69% on PolyHema (P<0.05). Inhibition of tyrosine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated apoptosis in TNF-alpha-treated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activation and that caspase-3 inhibition decreased apoptosis. We conclude that tyrosine phosphorylation of Ly-GDI, followed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alpha-mediated apoptosis on fibronectin.
Insights
Fibronectin accelerates tumor necrosis factor-alpha (TNF-alpha)-mediated programmed cell death (apoptosis) in neutrophils. This process involves tyrosine phosphorylation and caspase-3-mediated cleavage of Ly-GDI, a key signaling regulator.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Neutrophil apoptosis is crucial for resolving inflammation.
- Fibronectin and TNF-alpha are key mediators in inflammatory processes.
- Understanding the signaling pathways regulating neutrophil apoptosis is vital.
Purpose of the Study:
- To elucidate the intracellular signaling events underlying fibronectin-accelerated TNF-alpha-mediated neutrophil apoptosis.
- To identify key proteins and molecular mechanisms involved in this accelerated apoptotic process.
Main Methods:
- Two-dimensional gel electrophoresis and western blotting were employed to analyze protein expression and phosphorylation.
- Proteins of interest were sequenced using electrospray ionization mass spectrometry.
- Apoptosis was quantified using flow cytometry.
- Specific inhibitors were used to probe signaling pathways, including tyrosine phosphorylation and caspase-3 activation.
Main Results:
- A cluster of proteins, including Ly-GDI (leukocyte-specific guanine nucleotide-dissociation inhibitor), were identified as tyrosine phosphorylated specifically upon TNF-alpha stimulation in the presence of fibronectin.
- Fibronectin significantly increased TNF-alpha-induced Ly-GDI cleavage, resulting in a 23-kD fragment and reduced intact Ly-GDI levels.
- Inhibition of tyrosine phosphorylation abrogated Ly-GDI phosphorylation, cleavage, and the accelerated apoptotic response.
- Ly-GDI cleavage was found to be dependent on caspase-3 activation, and its inhibition reduced apoptosis.
Conclusions:
- Tyrosine phosphorylation of Ly-GDI is a critical early event in fibronectin-accelerated TNF-alpha-mediated neutrophil apoptosis.
- Subsequent caspase-3-mediated cleavage of phosphorylated Ly-GDI is a key downstream signaling event driving this accelerated apoptosis.
- This study reveals a novel signaling pathway linking fibronectin interaction to enhanced neutrophil apoptosis via Ly-GDI regulation.