Inhibition of Receptor Internalization by Monodansylcadaverine Selectively Blocks p55 Tumor Necrosis Factor Receptor Death Domain Signaling

Abstract

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.Tumor necrosis factor (TNF), 1 originally defined by its antitumoral activity, is now recognized as a pleiotropic cytokine exerting a wide variety of immunoregulatory activities (for review, see Refs. 1-3). TNF action is mediated by two types of cell surface receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular masses, respectively. Both receptors mediate distinct TNF responses (4 -6). The majority of activities of soluble TNF appears to be mediated by TR55 (7-11). Like other cytokine receptors, the cytoplasmic domain of both TNF receptors lacks intrinsic enzymatic activities. The activation of intracellular signaling enzyme systems is initiated by a selective interplay between the cytoplasmic domain of the TNF receptors and a number of recently identified TNF receptor-associated proteins (see Ref. Results from numerous studies have revealed that TNF signaling further involves activation of downstream enzyme systems at multiple subcellular compartments such as the plasmamembrane, endosomes, mitochondria, the cytosol, and the nucleus (for review, see Refs. 11, 23, and 24). Membrane-associated enzyme systems transmitting TR55 signals include plasmamembrane-bound phospholipases such as phosphatidylcholine-specific phospholipase C (25), which generates the lipid second messenger molecule 1,2-diacylglycerol and a N-SMase (9), producing ceramide by sphingomyelin hydrolysis. Ceramide generated at the plasmamembrane triggers activation of a 97-kDa ceramide-activated protein kinase (26), recently suggested to be identical with the "kinase suppressor of ras" (27). Ceramide-activated protein kinase belongs to a family of proline-directed protein kinases (PDPK) (9), including members of the mitogen-activated protein kinases (28). TNF signaling further involves intracellular membrane compartments like caveolae and endosomes harboring an acid SMase (A-SMase) (9,29,30). In mitochondria, TNF induces reactive oxygen species that are generated at the level of the oxidative phosphorylation complex III (31). T...

C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes

“…MDC is a pharmacological inhibitor of receptor-mediated endocytosis [18,21], while filipin, which binds cholesterol in the plasma membrane, impairs the invagination and subsequent internalisation of caveolae [23,24]. Under our experimental conditions, pretreatment of HAEC with MDC (43 mmol/l) for 30 min completely blocked the uptake of Alexa Fluor 488-labelled C-peptide (p<0.01 vs control) (Fig.…”

“…The dose and duration of treatment for each compound, none of which showed any toxic effect, was determined experimentally on the basis of previous reports [17][18][19][20]. After 30 min, fluorescent Cpeptide was added for 30 min and the cells were put back in the incubator.…”

Caspase-resistant Golgin-160 Disrupts Apoptosis Induced by Secretory Pathway Stress and Ligation of Death Receptors

“…Additionally, overexpression of caspase-resistant BAP31 disrupts Fas-induced apoptosis , implying cross-talk between the secretory pathway and death receptors. Recent reports also suggest that internalization of the TNF receptor I after its ligation is important for initiation of apoptosis (Schutze et al, 1999;SchneiderBrachert et al, 2004), indicating a potential function for membrane trafficking in TNF receptor signaling. Apoptosis also was impaired after treatment with TRAIL and ligation of Fas in cells expressing caspase-resistant golgin-160 (although impairment was less dramatic after Fas ligation than TNF-␣ or TRAIL treatment).…”

Differential activation of JNK1 isoforms by TRAIL receptors modulate apoptosis of colon cancer cell lines

“…Receptor internalisation occurs following treatment with rhTRAIL and agonistic DR4/5 antibodies On ligation by TRAIL, the TRAIL receptor-ligand complexes can be internalised and surface bound vs internalised TNF receptors have been shown to induce different signal transduction pathways resulting different cellular responses (Schutze et al, 1999;Schneider-Brachert et al, 2004;Varfolomeev et al, 2005;Kohlhaas et al, 2007). We hypothesised that unlike rhTRAIL, agonistic DR4/ 5 antibodies do not trigger receptor internalisation resulting in JNK activation in a different cellular compartment.…”