Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

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.

Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1–Induced Apoptosis by Modifying Mucin O-Glycan Synthesis

“…Rab6a regulates the intra-Golgi localization of NMIIA and facilitates the action of protein kinases (37, 39). KD of Rab6a significantly changes the Golgi morphology from a classical perinuclear compact shape to fragmented, collapsed or expanded structures (40, 41). We detected Rab6a-giantin complex prominently in LNCaP, but not PC-3 and DU145 cells (Fig.…”

How Rab Proteins Determine Golgi Structure

“…Based on these findings, we suggest that Rab6 and Rab33b are both examples of Class 2 Golgi-associated Rab proteins (Table 2). As observed by electron microscopy, depletion of Rab6 results in increased cisternal continuity of the trans Golgi/TGN in endothelial cells (Ferraro et al, 2014), HeLa cells (Storrie et al, 2012), and macrophages (Micaroni et al, 2013). In addition, in HeLa cells at least, there is increase in cisternal number, a pronounced accumulation of both COPI- and clathrin-coated vesicles and coated membrane fission/fusion figures, and a dilation of the trans cisternae/TGN when Rab6 is depleted (Storrie et al, 2012).…”

“…In addition, in HeLa cells at least, there is increase in cisternal number, a pronounced accumulation of both COPI- and clathrin-coated vesicles and coated membrane fission/fusion figures, and a dilation of the trans cisternae/TGN when Rab6 is depleted (Storrie et al, 2012). In macrophages, cisternal dilatation and vesicle accumulation is less evident (Micaroni et al, 2013). These detailed findings indicate that Rab6 plays a key role in the maintenance of Golgi ribbon organization and vesicle trafficking that is only apparent by observation by electron microscopy.…”

Effect of Ramadan Fasting On Classically Activated, Oxidative Stress and Inflammation of Macrophage

“…Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease [59]. Aberrant TNF-alpha production and TNF receptor signaling have been associated with the pathogenesis of several diseases [31].…”