TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages

doi:10.1016/j.cellsig.2013.12.009

Cellular Signalling

Volume 26, Issue 4, April 2014, Pages 683-690

https://doi.org/10.1016/j.cellsig.2013.12.009 Get rights and content

Highlights

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TNFR2 sensitises macrophages for TNF-induced pro-inflammatory signalling
•
TNFR2 enhances activation of the p38 MAPK and NF-κB pathways at low [TNF]
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TNFR1 is essential for activation of the p38 MAPK and NF-κB pathways
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TNF induces TRAF2 protein degradation via TNFR2 in macrophages
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TNF does not appear to induce TRAF2 degradation in human monocytes

Abstract

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2^−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1^−/− macrophages. In contrast, although TNFR2^−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.

Introduction

There are two separate TNF receptors: the type I receptor (TNFR1, p55, or p60) and the type II receptor (TNFR2, p75, or p80). Both receptors are widely expressed but TNFR1 is thought to be the major receptor required by a wide variety of cells for activation of the pro-inflammatory NF-κB and MAPK signalling pathways which in turn induce the expression of proteins of the inflammatory response (for reviews see [13], [17], [31]).

In contrast, TNFR2 displays cell-type specific expression and is the major TNFR expressed on activated T cells in which it signals the induction of apoptosis [9], [35] in a process involving sensitisation to TNF [9]. TNFR2 is also constitutively expressed in regulatory T cells and plays a role in their activation, proliferative expansion, and survival [7]. Endothelial cells express more equal levels of both receptors [20], but in these cells, TNFR1 is thought to be the major receptor involved in the activation of the NF-κB and MAPK pathways and subsequent induction of inflammatory response proteins [4], [21], [24], [36].

Immortalised macrophages are reported to express both receptors with TNFR1 being expressed weakly [23]. Both TNFR1- and TNFR2-deficient macrophage cell lines display reduced activation of the NF-κB and MAPK pathways in response to TNF compared to immortalised wild-type macrophages [23]. Experiments employing artificial ligands for the different receptors, also called muteins, have shown that TNFR2 cannot significantly activate pro-inflammatory signalling pathways independently of TNFR1 [6], [8].

Physiological signalling functions of TNFR2 that are distinct from TNFR1 have proved elusive to identify. In cell lines, TNFR2 has been reported to induce TRAF2 [18], [33] and ASK1 [34] protein degradation, and to regulate protein kinase B expression [16]. Recently, it has been shown in primary T cells and in tumour cell lines to activate the alternative NF-κB pathway [26]. Membrane-bound TNF has been shown to be a more potent activator of TNFR2 than the soluble form of the ligand [12], [26]. In addition, TNFR2 shedding in response to TNF serves to neutralise TNF and inhibit signalling [25].

TNFR2 has been reported to regulate apoptosis via a mechanism of ‘ligand-passing’, shown to involve TNFR2-mediated facilitation of the activation of TNFR1 [29]. In this mechanism, TNF binds much more rapidly to TNFR2 than TNFR1. When both receptors are in close proximity, the presence of TNFR2 increases the association rate of TNF with TNFR1 thereby sensitising the cell to TNFR1-mediated cytotoxicity [29]. However, TNFR2 is able to drive apoptosis independently of TNFR1, and cooperation between TNFR1 and TNFR2 in activating the NF-κB pathway has also been found to be additive rather than synergistic [32].

Since the TNFR1 receptor has been more strongly implicated in the activation of pro-inflammatory signalling pathways than TNFR2, blockade of TNFR1 appears a logical choice for therapy of chronic inflammatory diseases. Indeed, TNFR1 expressed on mesenchymal cells has been shown to play an important role in arthritis in mice [1]. TNFR2 has also been suggested to be involved in diverse processes that may be of beneficial function and thus its blockade could have deleterious implications. These include diverse neuro-, cardio-, and osteo-protective effects of TNFR2 suggested from experiments in TNFR^−/− mice. Activation of TNFR2 by TNF inhibits seizures [2] and attenuates cognitive dysfunction following brain injury [19]. TNFR2 appears not to affect myocardial infarct size, but does promote survival following myocardial infarction in mice [22]. Similarly, TNFR2 has been suggested to protect against myocardial ischaemia/reperfusion injury [10], and to reduce remodelling and hypertrophy following heart failure [14]. Furthermore, in experimental arthritis, TNFR2 has also been shown to protect against joint inflammation and erosive bone destruction through regulation of osteoclastogenesis [27].

Given the wide range of protective functions of TNFR2, and the lack of a clear consensus on the signalling function of this receptor, we sought to identify unique physiological TNFR2-dependent signalling processes that are distinct from TNFR1. We have focused on TRAF2 protein degradation, the TNFR2-dependent signalling event that is most strongly implicated from previous studies in cell lines. To see if TRAF2 degradation occurs in primary cells we have investigated the ability of TNFR2 to induce this process in primary wild-type and TNFR1- or TNFR2-deficient macrophages. LPS-treated macrophages strongly express TNF, and this allowed us to test the activation of the alternative NF-κB pathway by endogenous membrane-bound TNF. Since these cells express both TNF receptors we also investigated if, according to the ‘ligand-passing’ model, TNFR2 sensitises primary macrophages for activation of classical pro-inflammatory signalling pathways.

Section snippets

Materials

Lipopolysaccharide (LPS) (TLR-grade) was purchased from Alexis Biochemicals (Exeter, UK). Macrophage colony stimulating factor (MCSF) and TNF (human cells were treated with human TNF; murine cells were treated with murine TNF) and human granulocyte/macrophage colony stimulating factor (GM-CSF) were from PeproTech (London, UK). Antagonistic anti-murine TNFR1 (55R-170) and anti-murine TNFR2 (TR75-32.4) monoclonal antibodies were from BioLegend (Cambridge, UK). Antibodies for western blotting

Function of the TNFR2 in TNF-induced TRAF2 protein degradation: time and concentration dependence

Signalling via TNFR2 induced by high concentrations of soluble TNF has previously been shown to cause TRAF2 protein degradation [18], [33], but this has not been reported in macrophages. To determine if TRAF2 degradation occurs in murine macrophages, and to identify which receptor is responsible for this with the use of receptor-deficient macrophages, it was firstly necessary to determine the time and concentration dependence of the process in BMDM. For this cells were treated with a high dose

Discussion

TNFR1 is known to activate the NF-κB and MAPK pathways in response to TNF in a wide range of cell types. In this study, induction by TNF of p38 MAPK phosphorylation and IκBα degradation was completely blocked in TNFR1^−/− macrophages at all concentrations of TNF used, underscoring a dominant role for this receptor and showing that TNFR2 cannot significantly activate these pathways independently of TNFR1. The literature suggests that the role of TNFR2 in signalling is more complex. In

Conclusions

In macrophages high concentrations of soluble TNF induce TRAF2 protein degradation via TNFR2. TRAF2 degradation does not appear to be required for TNF-induced tolerance of p38 MAPK activation and TNF does not appear to induce TRAF2 degradation in human monocytes. However, in addition to its previously reported anti-inflammatory functions in mice, in macrophages TNFR2 may also act as an auxiliary receptor to TNFR1 to enable cells to activate pro-inflammatory signalling pathways in response to

Acknowledgements

We are grateful to GSK, Arthritis Research UK, and the Kennedy Institute of Rheumatology Trust for funding and members of the Joint Research Committee for useful discussions throughout the course of this project. We thank Simon Arthur for reading the manuscript.

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The tumor necrosis factor receptor superfamily (TNFRSF) and their ligands mediate lymphoid tissue development and homeostasis in addition to key aspects of innate and adaptive immune responses. T cells of the adaptive immune system express a number of TNFRSF members that are used to receive signals at different instructive stages and produce several tumor necrosis factor superfamily (TNFSF) members as effector molecules. There is also one example of a TNFRSF member serving as a ligand for negative regulatory checkpoint receptors. In most cases, the ligands in afferent and efferent phases are membrane proteins and thus the interaction with TNFRSF members must take place in immunological synapses and other modes of cell–cell interaction. A particular feature of the TNFRSF-mediated signaling is the prominent use of linear ubiquitin chains as scaffolds for signaling complexes that activate nuclear factor κ-B and Fos/Jun transcriptional regulators. This review will focus on the signaling mechanisms triggered by TNFRSF members in their role as costimulators of early and late phases of T cell instruction and the delivery mechanism of TNFSF members through the immunological synapses of helper and cytotoxic effector cells.
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Previously, it has been proposed that TNFR2 is required for TRAF2 degradation, leading to activation of the alternative NFκB pathway and contributing to MAPK and classical NFκB signalling. Ruspi et al. studied the signalling functions of TNFR2 in primary macrophages [74]. TNF-α induced TRAF2 degradation which was blocked in TNFR2-/- macrophages.
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As in these experiments TNF can activate TNFR1 as well as TNFR2, one could hypothesize a role for TNFR1-induced TRAF2 degradation. However, TNF-induced TRAF2 degradation was still observed in TNFR1-deficient BMDM, while it was impaired in TNFR2-deficient cells, illustrating that TRAF2 degradation is specific for TNFR2 signaling [37,47]. Of note, TNF-induced TRAF2 degradation was only observed in BMDM expressing similar levels of TNFR1 and TNFR2, while TRAF2 degradation did not occur in monocytes which express more TNFR1 than TNFR2 [47].
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^☆: The authors claim no conflict of interest. This study was funded by GlaxoSmithKline, Arthritis Research UK and the Kennedy Institute of Rheumatology Trust. MF was a consultant for GlaxoSmithKline.

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TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages☆

Highlights

Abstract

Introduction

Section snippets

Materials

Function of the TNFR2 in TNF-induced TRAF2 protein degradation: time and concentration dependence

Discussion

Conclusions

Acknowledgements

Trends Cell Biol.

FEBS Lett.

J. Mol. Cell. Cardiol.

Cell

FEBS Lett.

J. Biol. Chem.

Blood

J. Biol. Chem.

J. Biol. Chem.

Am. J. Pathol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell. Signal.

J. Exp. Med.

Ann. Neurol.

Am. J. Physiol. Lung Cell. Mol. Physiol.

J. Immunol.