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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.018671

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TPL2-mediated activation of ERK1 and ERK2 regulates the processing of pre-TNF in LPS-stimulated macrophages

¹ MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
² Division of Immune Cell Biology, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK
³ Division of Immunoregulation, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK
⁴ Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

View larger version (35K): [in this window] [in a new window]   Fig. 1. Effect of protein kinase inhibitors on TLR-induced production of intracellular and extracellular TNF in murine macrophages. (A) RAW cells were pre-treated for 60 minutes with or without 2 µM PD 184352 (PD) and then either left untreated or were stimulated for 4 hours with 100 ng/ml LPS. The cell culture supernatants were collected and the levels of TNF analysed using the beadlyte system (histogram). The cell lysates (30 µg protein) were subjected to SDS-PAGE and, after transfer to nitrocellulose, the membrane was probed with a murine anti-TNF-specific antibody or an anti-actin antibody (lower panel). (B) As in A, except that BMDM were used instead of RAW cells. (C) As in A, except that primary human monocytes-macrophages were used instead of RAW cells and 10 µM U0126 (U0) was used as an additional MKK1 inhibitor. (D) As in A, except that cells were stimulated for 4 hours with 100 ng/ml FSL1. The results are shown as mean±s.e.m. for three (A,B) or two (C,D) separate experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 2. The TPL2 pathway regulates TNF production mainly at the post-translational level. (A) BMDM from wild-type (wt, black squares) and TPL2-deficient (–/–, white squares) mice were either left untreated or were stimulated with 10 ng/ml LPS for the times (hours) indicated. The levels of TNF in the cell culture supernatant were analysed by standard ELISA. Each square represents a determination using a different mouse. (B) BMDM were lysed in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 100 nM okadaic acid, 2 mM Na4P2O7, a mixture of protease inhibitors and 1% Nonidet-P 40. Lysates from wild-type and TPL2-deficient mice were either left untreated or were stimulated with 100 ng/ml LPS for the times (minutes) indicated, and were immunoblotted for pre-TNF as in Fig. 1 or for tubulin. (C) The experiment was carried out as in A except that total RNA was purified from the lysates, reverse transcribed and the amount determined by real-time quantitative PCR.

View larger version (76K): [in this window] [in a new window]   Fig. 3. LPS-induced pre-TNF intracellular distribution is not affected by preventing ERK1/2 activation. (A-D) RAW cells were pre-treated for 60 minutes with 10 µM of the TACE inhibitor TAPI-1, with or without 2 µM PD 184352, and then either left untreated or stimulated for 4 hours with 100 ng/ml LPS. The medium was removed, the cells fixed, and thawed cryosections prepared as described in Materials and Methods. The sections were labelled with antibodies against TNF followed by species-specific antibodies and protein-A gold. Micrographs in A-C are taken from the most intensely labelled cells to illustrate the structure of the labelled compartments detected by quantitative methods (D). (A) Labelling is located over lamellipodia-like (Lam) structures and endosome/lysosome profiles (End/Lys); (B) labelling is concentrated in putative peripheral endosomes with pleiomorphic tubulovesicular morphology (arrows); (C) labelling is found over a stack of Golgi cisternae (Golgi). (D) For RAW cells treated with LPS, or PD 184352 plus LPS, values are means of percentage gold counts from two EM grids ± range. PM, plasma membrane; PER, peripherally located tubulo-vesicular structures; DEEP, tubulo-vesicular structures located deep in the cell; MVB/LYS, multi-vesicular bodies; Lys, endolysosomes with heterogeneous content; ER, endoplasmic reticulum; NE, nuclear envelope; MIT, mitochondria. For unbiased counting methods and further details on compartment identification refer to Watt et al. (Watt et al., 2002). Scale bars: 100 nm.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Genetic ablation of TPL2 blocks pre-TNF expression at the cell surface. BMDMs were stimulated with LPS (1 µg/ml) for (A,B) 1.5 or (C,D) 3 hours in (A,C) the presence or (B,D) absence of Brefeldin A. Permeabilised (A,C) and intact (B,D) cells were stained with PE-conjugated anti-TNF and APC-conjugated anti-F4/80. Histograms are gated on F4/80+ cells. Grey histograms denote wild-type cells and solid lines denote TPL2-deficient cells. Unstimulated cells (solid lines) and isotype-control staining (grey histogram) are shown in the bottom panel.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Inhibition of ERK1/2 prevents translocation of pre-TNF to the cell membrane. RAW cells were plated on Labtek chambers and then pre-treated for 60 minutes with 10 µM of the TACE inhibitor TAPI-1 (A-F), with (C,F) or without (A,B,D,E) 2 µM PD 184352, and were then either left untreated (A,D) or were stimulated for 4 hours with 100 ng/ml LPS (B,C,E,F). Cells were either left unpermeabilised (A-C) or were permeabilised with 0.1% (w/v) saponin (D-F). TNF was visualised using an antibody coupled to Alexa-Fluor-488-conjugated anti-sheep IgG by confocal microscopy at 600-fold magnification. Similar results were obtained in three independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 6. TACE phosphorylation at Thr735 is mediated by the TPL2-MKK1/2-ERK1/2 pathway. (A) The C-terminal tail of TACE [TACE(693-827)] was expressed in bacteria as a GST-fusion, purified and phosphorylated with or without ERK2, as described (Morton et al., 2006). The indicated amounts (ng) of TACE(693-827) were spotted onto nitrocellulose and probed with an antibody that recognises TACE phosphorylated at Thr735 (pTACE Thr735). (B) RAW cells were pre-treated for 60 minutes without (–) or with (+) 10 µM U0126 (U0) or 2 µM PD 184352 (PD) and then either left untreated or were stimulated for 45 minutes with 100 ng/ml LPS. Immunoblotting was carried out as in Fig. 1 using an antibody that recognises ERK1/2 phosphorylated at the TEY motif (pERK), one that recognises TACE phosphorylated at Thr735 or an antibody that recognises the phosphorylated and unphosphorylated forms of TACE equally well (TACE). (C) BMDM from wild-type (WT) or TPL2-deficient (TPL2–/–) mice were either left untreated or were stimulated with 100 ng/ml LPS for the times (minutes) indicated. Immunoblotting was performed as in B.