A trans-Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo

Abstract

The transmembrane precursor of tumor necrosis factor-alpha (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNA-vector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery.

Fig. 1.

TNF trafficking is inhibited by silencing p230 in HeLa cells. (a–c) HeLa cells transfected with control siRNA or p230 siRNA for 48 h and then transfected a second time with YFP-TNF (a and b) and GFP-Ecad (c) for a further 24 h. In b, myc-tagged full length p230 (myc-p230) was cotransfected with YFP-TNF. Monolayers were then incubated with TACE inhibitor for 2 h, fixed in paraformaldehyde and cell surface TNF stained with rabbit anti-TNF antibodies followed by Alexa647-conjugated anti-rabbit IgG. Monolayers were then permeabilized and stained with human anti-p230 antibodies followed by Alexa594-conjugated goat anti-human IgG (a and c) or monoclonal anti-myc antibodies followed by Alexa568-conjugated anti-mouse IgG (b). (d) HeLa cells were either mock transfected (Con) or transfected with p230 siRNA for 72 h, as indicated, and lysed in reducing buffer and extracts subjected to SDS/PAGE on a 4–12% gradient polyacrylamide gel. Proteins were transferred to a PVDF membrane and probed with affinity-purified rabbit anti-p230 antibodies by using chemiluminescence detection. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-golgin −97 antibodies. (Scale bars, 10 μm.)

Fig. 2.

TNF in p230 labeled tubules leaving the TGN in live macrophages. (a) RAW macrophages were transfected with YFP-p230-GRIP and then fixed in 4% paraformaldehyde. Fixed macrophages were permeabilized and stained with rabbit anti-TNF antibodies. Images show YFP-p230-GRIP (green) labeled tubule (yellow arrow) containing a bolus (white arrow) of TNF (red) extending from a region of the TGN. (b) Quantification of events observed in live RAW macrophages transfected with either GFP-TNF, YFP-p230-GRIP or YFP-golgin-97-GRIP. In LPS stimulated macrophages, a 3-fold increase in GFP-TNF carrier budding from the Golgi is observed. LPS stimulation also results in a nearly 3-fold increase in YFP-p230-GRIP tubulations and carrier budding, while having no effect on the activity of YFP-golgin-97-GRIP. The number of cells analyzed for each condition ranged from 20 to 40. Data are expressed as mean ± SEM. ***, P < 0.0001; **, P < 0.01 by unpaired two-tailed t test. (Scale bar, 10 μm.)

Fig. 3.

Post-Golgi TNF trafficking is inhibited by silencing p230 in stimulated RAW macrophages. RAW macrophages were transfected with control miRNA or p230 miRNA-1, as indicated, for 96 h and treated with 100 ng/ml of LPS in serum-free RPMI medium 1640 at 37°C for 2 h in the presence of the TACE inhibitor. Stimulated macrophages were then fixed in 4% paraformaldehyde. (a) Fixed macrophages were permeabilised and stained with human anti-p230 antibodies followed by Alexa647-conjugate goat anti-human IgG and with rabbit anti-TNF antibodies followed by Alexa568-conjugated anti-rabbit IgG. (b and c) Fixed macrophages were analyzed for cell surface TNF by staining with rabbit anti-TNF antibodies followed by Alexa568-conjugated anti-rabbit IgG. Representative flow cytometry plots of GFP+ cells shown in c. (Scale bars, 10 μm.)

Fig. 4.

Peritoneal macrophages from transgenic mice expressing p230 miRNA are depleted in p230 and impaired in TNF secretion. Peritoneal macrophages obtained from either empty miRNA vector (control) or p230 miRNA retrogenic mice were fixed in 4% paraformaldehyde and stained with anti-human p230 antibodies, followed by goat Alexa594 conjugated anti-human IgG (a) or rabbit anti-human GCC88 antibodies or rabbit anti-human GCC185 antibodies, followed by goat Alexa568 conjugated anti-rabbit IgG (b). (c and d) Peritoneal macrophages from control and p230 miRNA transgenic mice were activated with 100 ng/ml of LPS in the presence of the TACE inhibitor for 2 h. Macrophages were fixed in 4% paraformaldehyde and cell surface TNF detected in nonpermeabilized cells by using rabbit anti-mouse TNF antibodies, followed by goat Alexa568 conjugated anti-rabbit IgG. (d) For intracellular TNF detection, macrophages were fixed and permeabilized, and stained with rabbit anti-mouse TNF antibodies, followed by goat anti-rabbit Alexa 568 conjugated antibodies. (Scale bars, 10 μm.)