A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFalpha; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFalpha vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFalpha trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.
"Transiently expressed Rab6a–GFP observed in the macrophage RAW 264.7 cell line shows typical labeling of the perinuclear Golgi complex and more diffuse staining in the cytoplasm (Figure 1B). LPS activates macrophages, initiating the synthesis of cytokines, chemokines and other secretory proteins . Immuno-staining of LPS-activated macrophages typically shows bright staining of the newly synthesized trans-membrane precursor of the TNF in the Golgi complex (Figure 1C). "
[Show abstract][Hide abstract] ABSTRACT: Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.
PLoS ONE 02/2013; 8(2):e57034. DOI:10.1371/journal.pone.0057034 · 3.23 Impact Factor
"The prototype mechanism may be the secretion of newly synthesized TNF by macrophages, which is reported to be organized in two steps (Stow et al., 2006, 2009; Lacy and Stow, 2011). First, the fusion of carriers originating from the Golgi complex with recycling endosomes is mediated by the Q-SNARE complex of STX6-STX7-Vti1b with the R-SNARE VAMP3, and second, the membrane fusion of the recycling endosome and the plasma membrane is mediated by the R-SNARE VAMP3 on the recycling endosome by pairing with the STX4–SNAP-23 Q-SNARE complex on the plasma membrane (Pagan et al., 2003; Murray et al., 2005a,b). On the other hand early studies performed in mast cells examining TNF secretion clearly showed the necessity for a stimulatory calcium signal or activation of PKC as addition of selective pharmacological blockers after some time completely arrested secretion despite the fact that TNF had already accumulated within the cell (Baumgartner et al., 1994). "
[Show abstract][Hide abstract] ABSTRACT: Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators - stored in granules - as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNARE) proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, secretory carrier membrane proteins, complexins, or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.
Frontiers in Immunology 06/2012; 3:143. DOI:10.3389/fimmu.2012.00143
"In activated macrophages, one of the major functions is to secrete pro-inflammatory cytokines such as TNFα (tumour necrosis factor α). Syntaxin-6 is up-regulated within a very short time period to meet the demand for TNFα trafficking and secretion. Since the up-regulation occurs rapidly, it is therefore likely that syntaxin-6 protein levels are regulated by post-translational or p53-mediated transcriptional mechanisms . These two SNAREs are present on intracellular membranes, isolated Golgi membranes and Golgi-derived TNFα vesicles, where they contribute to TNFα trafficking and secretion (Figure 2). "
[Show abstract][Hide abstract] ABSTRACT: Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions.
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