Activation of liver X receptor suppresses the production of the IL-12 family of cytokines by blocking nuclear translocation of NF-κBp50.

There is now convincing evidence that liver X receptor (LXR) is an important modulator of the inflammatory response; however, its mechanism of action remains unclear. This study aimed to examine the effect of LXR on the IL-12 family of cytokines and examined the mechanism by which LXR exerted this effect. We first demonstrated that activation of murine-derived dendritic cells (DC) with a specific agonist to LXR enhanced expression of LXR following activation with LPS, suggesting a role in inflammation. Furthermore, we showed LXR expression to be increased in vivo in dextrane sulphate sodium-induced colitis. LXR activation also suppressed production of IL-12p40, IL-12p70, IL-27 and IL-23 in murine-derived DC following stimulation with LPS, and specifically targeted the p35, p40 and EBI3 subunits of the IL-12 cytokine family, which are under the control of the NF-κB subunit p50 (NF-κBp50). Finally, we demonstrated that LXR can associate with NF-κBp50 in DC and that LXR activation prevents translocation of the p50 subunit into the nucleus. In summary, our study indicates that LXR can specifically suppress the IL-12 family of cytokines though its association with NF-κBp50 and highlights its potential as a therapeutic target for chronic inflammatory diseases.

Syntaxin-4 is essential for IgE secretion by plasma cells.

The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

2D-DIGE: comparative proteomics of cellular signalling pathways.

Two-dimensional (2-D) gel electrophoresis concerted with protein identification by mass spectrometry (MS) is an extremely powerful method for comparative expression profiling of complex protein samples such as cell lysates. The highly resolutive 2-D electrophoresis allows the separation of heterogeneous protein samples on the basis of isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance ((1) J Biol Chem 250: 4007-4021, 1975; (2) Electrophoresis 14: 1067-1073, 1993). Consequently, it provides a comprehensive view of a proteome state ((3) Electrophoresis 21: 1037-1053, 2000), where variations in protein expression levels, isoforms, or post-translational modifications (e.g. phosphorylation) can be highlighted and investigated ((4) Electrophoresis 21: 2196-2208, 2000). Furthermore, this allows the identification of biological markers that characterize a specific physiological or pathological background of a cell or a tissue ((5) Proteomics 1: 397-408, 2001; (6) J Bacteriol 179: 7595-7599, 1997). In this way one can compare the effects of a stimulus or drug on cells or tissue, or more importantly, analyse the effects of disease on the expression level of proteins. Relatively recently, conventional 2-D gel electrophoresis has been combined with protein labelling strategies using up to three different fluorescent dyes to allow comparative analysis of different protein samples within a single 2-D gel platform. In this technique, termed differential in-gel electrophoresis (DIGE), samples are labelled separately then combined and run on the same 2D gel minimizing experimental variation and greatly facilitating spot matching. When three CyDyes (Cy2, Cy3, and Cy5) have been used, three images of the gel are captured then superposed to localize the differentially regulated spots on the 2-D gel using image analysis software. This is an extremely powerful tool in comparative proteomics as these dyes provide a linear response to protein concentration up to five orders of magnitude and great sensitivity with detection down to 125 pg of a single protein, which is less than needed for MS identification. In this chapter, we describe the basic methods for protein labelling, optimization of the isoelectrofocusing parameters for the first dimension (where proteins are separated according to their isoelectric point (pI)), sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation for the second dimension (based on molecular weight (MW)), and different post-staining protocols of the 2-D gel and protein preparation for mass spectrometry identification.

Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.