Phenotypic maturation, cytokine secretion, and migration are distinct functional characteristics of dendritic cells (DCs). These functions are independently regulated by a number of extracellular variables, such as type, strength, and persistence of an array of soluble and membrane-bound mediators. Since the exact composition of these variables in response to infection may differ between individuals, the intracellular signaling pathways activated by these extracellular networks may more closely correlate with DC function and predict the course of adaptive immunity. We found that activation of p38 kinase (p38K), extracellular signal-related kinase 1/2 (ERK1/2), and phosphatidylcholine-specific phospholipase C (PC-PLC) enhanced cytokine secretion, whereas p38K, cyclic adenosine monophosphate (cAMP), and PC-PLC enhanced migration. In contrast, phosphatidylinositol 3-kinase (PI3K)/Akt-1 and cAMP inhibited cytokine secretion while ERK1/2 inhibited migration. Migration and cytokine secretion further differed in their sensitivity to inhibition over time. However, although DCs could be manipulated to express migration, cytokine secretion, or both, the level of activation or persistence of intracellular pathway signaling was not predictive. Our results suggest a modular organization of function. We hypothesize that the expression of specific DC functions integrates a large variety of activating and inhibitory variables, and is represented by the formation of a functional unit of molecular networks-the signal response module (SRM). The combined activities of these modules define the functional outcome of DC activation.
TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-␣-and Fas-induced apoptosis in vivo.160 LEVD 163 was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)-and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1-and TRAF2-mediated nuclear factor-B activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF-and Fasinduced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.
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