KRAS, HRAS and EGFR Mutations in Sporadic Sebaceous Gland Hyperplasia.

Sporadic sebaceous gland hyperplasia (SGH) is a benign skin lesion, with a high prevalence in the general population. Although SGH has been attributed to both extrinsic and intrinsic factors, the underlying genetic changes have not yet been characterized. Recently, HRAS and KRAS mutations have been identified in sebaceous naevus, a hamartoma sharing histological characteristics with SGH. Therefore we screened 43 SGH for activating mutations in RAS genes and other oncogenes. We identified a wide spectrum of mutually exclusive activating HRAS (8/43), KRAS (11/43) and EGFR mutations (7/31) in altogether 60% of the lesions investigated. A RAS and EGFR wildtype status was found in 15 normal sebaceous glands in the head and neck area. Our findings indicate that activating HRAS, KRAS and EGFR mutations play a major role in the pathogenesis of sporadic SGH. These results support the concept that SGH is a true benign neoplasm rather than a reactive hyperplasia.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

Membrane trafficking of death receptors: implications on signalling.

Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.

Lymphotoxin-beta receptor signalling regulates cytokine expression via TRIM30α in a TRAF3-dependent manner.

Our earlier studies indicated that activation of the lymphotoxin beta receptor (LTßR) by T cell derived LTα(1)ß(2) regulates inflammatory cytokine expression. While characterizing the cellular and molecular mechanisms responsible for the down regulation of the inflammatory reaction after LTßR stimulation we were able to identify the specific induction of TRIM30α expression as a result of LTßR signalling in mouse macrophages. Furthermore, we could demonstrate that LTßR activation in these cells results in the down regulation of pro-inflammatory cytokine (e.g. TNF and IL-6) and mediator expression upon TLR4 and TLR9 re-stimulation, demonstrating that LTßR activation on mouse macrophages dampens pro-inflammatory cytokine and mediator expression. Thus, LTßR signalling renders macrophages hypo-responsive to subsequent stimulation with TLR ligands. The observation of an LTßR-mediated TLR-tolerance in the human monocyte cell line THP-1 suggests that similar signalling mechanisms seem to exist in human cells. Signalling pathway analysis clearly demonstrated that LTßR-induced TRIM30α expression is mediated by an IκBα-dependent signalling pathway. Furthermore, the LTßR-induced TRIM30α expression seems to be TRAF3 dependent. Our data suggest that LTßR activation on mouse macrophages is involved in the control of pro-inflammatory cytokine and mediator expression by activation of a signalling pathway that controls exacerbating inflammatory cytokine production.

E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin.

Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.