Molecular mechanisms of IL-18BP regulation in DLD-1 cells: pivotal direct action of the STAT1/GAS axis on the promoter level.

Interleukin (IL)-18, formerly known as interferon (IFN)-gamma-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-gamma, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-gamma. Mutational analysis revealed that a proximal gamma-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-gamma-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-gamma-stimulated DLD-1 colon carcinoma cells.

Interleukin-18 secretion and Th1-like cytokine responses in human peripheral blood mononuclear cells under the influence of the toll-like receptor-5 ligand flagellin.

Flagellin is the major protein component of the flagella from motile bacteria and was identified as the ligand for toll-like receptor (TLR)-5. Whereas its effects on epithelial cells have been studied in detail, activation of human peripheral blood mononuclear cells (PBMC) by flagellin is characterized only partially. By using the recombinant protein of Salmonella muenchen we confirm the proinflammatory nature of flagellin as detected by nuclear factor-kappaB activation and interleukin (IL)-8 production. Aim of the current study was to elucidate in PBMC effects of flagellin on IL-18 and Th1-like cytokine responses. We report that flagellin in pathophysiologically relevant concentrations augmented release of mature IL-18 by THP-1 monocytes, PBMC, and whole blood stimulated with nigericin or by ATP-mediated P2X7 purinergic receptor activation. Further key functions of the IL-18/IL-12/interferon-gamma (IFNgamma) pathway were upregulated by flagellin. Flagellin synergized with IL-12 for production of IFN-gamma and augmented secretion of interferon-inducible protein-10, a CXC-chemokine that is key to the generation of Th1-type responses. In contrast, neither IL-18-binding protein nor IL-4 was affected. Taken together, the present data demonstrate for the first time that flagellin at concentrations that are detectable in the blood compartment during sepsis efficiently enhances the IL-18/IL-12/IFNgamma pathway and thus Th1-like cytokine responses in PBMC.

The HIV protease inhibitor ritonavir synergizes with butyrate for induction of apoptotic cell death and mediates expression of heme oxygenase-1 in DLD-1 colon carcinoma cells.

The protease inhibitor ritonavir is an integral part of current antiretroviral therapy targeting human immunodeficiency virus. Recent studies demonstrate that ritonavir induces apoptotic cell death with high efficiency in lymphoblastoid cell lines. Moreover, ritonavir can suppress activation of the transcription factor nuclear factor-kappaB and is an inhibitor of interleukin-1beta and tumor necrosis factor-alpha production in peripheral blood mononuclear cells. Thus, ritonavir appears to have anti-inflammatory properties. In the present study, we investigated in DLD-1 colon carcinoma cell effects of ritonavir on apoptotic cell death and expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme that may be critically involved in the modulation of colonic inflammation. Compared to unstimulated control, ritonavir resulted in a moderate increase in the rate of apoptotic cell death as observed after 20 h of incubation. Notably, ritonavir potently synergized with the short-chain fatty acid butyrate for induction of caspase-3-dependent apoptosis in DLD-1 cells. Ritonavir enhanced mRNA and protein expression of HO-1 in DLD-1 cells. Ritonavir-induced HO-1 protein was suppressed by SB203580 or SB202190 and preceded by immediate upregulation of cellular c-Fos and c-Jun protein levels. This process was associated with induction of activator protein-1 as detected by electrophoretic mobility shift analysis. The present data suggest that ritonavir has the potential to curb colon carcinogenesis by reducing cell growth via mechanisms that include apoptosis and by simultaneously modulating colonic inflammation via induction of anti-inflammatory HO-1.

Nitric oxide differentially regulates pro- and anti-angiogenic markers in DLD-1 colon carcinoma cells.

Inducible nitric oxide (NO) synthase (iNOS) appears to be a marker of tumor progression in colon carcinogenesis. Here we investigated effects of NO on selected chemokines that differentially regulate angiogenesis, namely pro-angiogenic interleukin (IL)-8 as well as tumor-suppressive interferon-inducible protein-10 (IP-10) and monokine induced by interferon-gamma (MIG). These chemokines are expressed by DLD-1 colon carcinoma cells after stimulation with IL-1beta/interferon-gamma. Expression of IL-8 was markedly upregulated by NO. Moreover, NO enhanced expression of vascular endothelial growth factor (VEGF). In contrast, expression of IP-10 and MIG was suppressed by NO. The present data are consistent with previous observations that link NO to enhanced tumor angiogenesis and imply that NO-mediated upregulation of IL-8 and VEGF as well as downregulation of IP-10 and MIG may contribute to this phenomenon.

Biochemical characterization of the high affinity binding between the glycine receptor and gephyrin.

Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the beta-subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR beta-sub-unit (GlyRbeta-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496-654). By using isothermal titration calorimetry, a high affinity (K(d) = 0.2-0.4 microm) and low affinity (K(d) = 11-30 microm) binding site for the GlyRbeta-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyRbeta-loops per E domain in both cases. Binding of the GlyRbeta-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.