The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

The KB-motif is an important regulatory element both for constitutive lymphoid-specific as well as ubiquitous inducible transcriptional activity. We

Steroid refractory inflammation is an unmet medical need in the management of inflammatory diseases. Thus, mechanisms, improving steroid sensitivity and simultaneously decreasing inflammation have potential therapeutic utility. The FK506‐binding protein 51 (FKBP51) is reported to influence steroid sensitivity in mental disorders. Moreover, biochemical data highlight a connection between FKBP51 and the IKK complex. The aim of this study was to elucidate whether FKBP51 inhibition had utility in modulating steroid resistant inflammation by increasing the sensitivity of the glucocorticoid receptor (GR) signalling and simultaneously inhibiting NFκB‐driven inflammation. We have demonstrated that FKBP51 silencing in a bronchial epithelial cell line resulted in a 10‐fold increased potency for dexamethasone towards IL1beta‐induced IL6 and IL8, whilst FKBP51 over‐expression of FKBP51 reduced significantly the prednisolone sensitivity in a murine HDM‐driven pulmonary inflammation model. Immunoprecipitation experiments with anti‐FKBP51 antibodies, confirmed the presence of FKBP51 in a complex comprising Hsp90, GR and members of the IKK family. FKBP51 silencing reduced NFκB (p50/p65) nucleus translocation, resulting in reduced ICAM expression, cytokine and chemokine secretion. In conclusion, we demonstrate that FKBP51 has the potential to control inflammation in steroid insensitive patients in a steroid‐dependent and independent manner and thus may be worthy of further study as a drug target.

NF-B and activator protein 1 (AP-1) are dimeric transcription factors involved in transcriptional regulation in many cells, including neurons. We have examined their activity during mouse cerebellum development, a postnatal process starting just after birth and completed by the fourth postnatal (PN) week. The activity of these factors was analyzed by binding of nuclear extracts to a synthetic oligonucleotide representing the B site of human immunodeficiency virus or the AP-1 site of the urokinase promoter. NF-B activity was observed from 7 PN, was restricted to the developing cerebellum, and was not observed in the early postnatal neocortex and hippocampus. On the other hand, AP-1 activity was not found in cerebellum but was present in both neocortex and hippocampus. Moreover, a B-driven transgene was found to be increasingly expressed in the cerebellum from 5 PN to 10 PN but not in the adult. The regulation of NF-B activation in mouse cerebellum was analyzed by intraperitoneal injection of glutamate receptor antagonists to 9 PN mice, which abolished NF-B-binding activity, suggesting an endogenous loop of glutamate receptor activation. Glutamate receptor agonists, on the other hand, induced NF-B nuclear translocation in the cerebellum of 5 PN mice, which is a stage in which NF-B is not yet endogenously activated. This effect was specific for NF-B and not observed for AP-1. In adult mice, NF-B activity was absent in the cerebellum and was not induced by intraperitoneal injection of glutamate receptor agonists. These data show that NF-B is specifically activated during cerebellum development and indicate an important role of glutamate receptors in this process.

Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-alpha or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde. Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at approximately 45 min after Gro-alpha stimulation. This multiplexing demonstrated that Gro-alpha-induced CXCR2 internalization was tightly correlated with Gro-alpha-induced NFAT translocation, also on the single cell level. The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities.