TNF is a cytokine with important functions in inflammation and immunity. Binding to its
receptor TNF-Receptor-1 (TNFR1) induces the formation of a signalling platform which leads
to TNF-target gene transcription and consequently the induction of cellular immune
responses. To understand and possibly influence the effects observed upon TNF stimulation,
it is important to define the molecular machinery responsible for signal transduction. In order
to resolve the composition of the TNF-Receptor signalling complex (TNF-RSC) our group
developed a highly sensitive modified tandem affinity purification method (moTAP). Using
this technique, we recently reported on the recruitment of the two In-Between-RING (IBR)
family members HOIL-1 (heme-oxidized IRP2 Ub ligase-1) and HOIP (HOIL-1 interacting
protein) to the TNF-Receptor signalling complex (TNF-RSC) (reviewed in Haas et al, 2009).
Together, HOIL-1 and HOIP form an E3 ligase complex that is capable of generating linear
ubiquitin chains and therefore referred to as “linear ubiquitin chain assembly complex”
(LUBAC) (Kirisako et al, 2006).
In addition to HOIL-1 and HOIP, we also identified Sharpin (SHANK-associated RH domain-interacting protein) as a further component that is recruited to the TNF-RSC in a stimulation-dependent manner. In this study I show that Sharpin forms an endogenous, stimulation-independent
complex with HOIL-1 and HOIP. Although the mRNA levels of HOIL-1 and
HOIP are unchanged in the absence of Sharpin, their respective protein levels are strongly
diminished. These data indicate that Sharpin, HOIL-1 and HOIP form a tripartite complex. As
previously shown for HOIL-1 and HOIP, overexpression of Sharpin together with HOIP also
stabilises the TNF-RSC and leads to activation of NF-kB. Therefore, LUBAC does not only
consist of HOIL-1 and HOIP, but also includes Sharpin.
A spontaneous point deletion in the Sharpin gene ablating its expression leads to the
development of chronic proliferative dermatitis (cpdm) in mice (HogenEsch et al, 1993;
HogenEsch et al, 1999; Seymour et al, 2007). Cpdm mice develop a multi-organ
inflammatory disease with prominent skin lesions and display severe abnormalities in
lymphoid architecture, including absence of Peyer’s patches, marginal zones, germinal
centres, and follicular dendritic cells. Mouse embryonic fibroblasts (MEFs) and primary
keratinocytes generated from these mice show impaired TNF-induced NF-kB and MAPK
signalling. Although activation of these pathways is not completely dependent on LUBAC
activity, its presence is required for full activation as demonstrated by proper induction of
TNF-target genes. In line with these TNF-induced signalling defects, cpdm-derived cells are
prone to TNF-mediated cell death, which is partially apoptotic and partially necroptotic. Thus,
loss of Sharpin results in a cell death favouring dysregulation of TNF-induced signalling
which is responsible for the inflammatory phenotype observed in cpdm mice.
Following the identification of a role for LUBAC in TNF signalling I determined whether it
also played a role in signalling by the TNF related ligands TRAIL and CD95 ligand
(CD95L/FasL). As compared to TNF, absence of a LUBAC component also sensitised cells to
TRAIL- and CD95L- induced cell death, indicating that LUBAC plays a crucial role in cell
survival. Accordingly, non-apoptotic TRAIL- and CD95L-induced signalling was also
affected in the absence of LUBAC as determined by altered NF-κB and MAPK responses,
respectively.
Taken together, the results obtained in this thesis determine LUBAC as a novel regulator of
various death receptor-driven signalling pathways implicated in gene activation, cell death
induction and, as a consequence, innate immunity and inflammation.