Multiple kinases in the interferon-gamma response.

Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are essential for responses to interferons (IFNs), most cytokines, and some growth factors. JAK/STAT signaling is not, however, sufficient for a full IFN-gamma response. Here, a convenient, robust, and quantitative flow cytometry-based kinome-wide siRNA screen has identified nine additional kinases as required for the IFN-gamma class II HLA response, seven for an antiviral response, and two for the cytopathic response to encephalomyocarditis virus (EMCV). As one example, inhibition of the IFN-gamma response by siRNA to ataxia telangiectasia-mutated (ATM) differentially affects a spectrum of IFN-gamma-stimulated mRNAs, with inhibitions being seen as early as 1 h after IFN-gamma stimulation. The implication of ATM, with its previously recognized function in chromatin decondensation, in the control of transcription early in the IFN-gamma response highlights both a role for ATM in cytokine responses and a possible correlation with the chromatin decondensation recently observed in response to IFN-gamma in mammalian cells. This work has, therefore, revealed the simplicity, power, and convenience of quantitative flow cytometry-based siRNA screens, a requirement for ATM and multiple additional kinases in the IFN-gamma response and a possible requirement for two of these kinases in the cytopathic response to EMCV.

Genetic characterization of the chemokine receptor CXCR4 gene in lagomorphs: comparison between the families Ochotonidae and Leporidae.

Chemokines receptors are transmembrane proteins that bind chemokines. Chemokines and their receptors are known to play a crucial role in the immune system and in pathogen entry. There is evidence that myxoma virus, the causative agent of myxomatosis, can use the chemokine receptor CXCR4 to infect cells. This virus causes a benign disease in its natural host, Sylvilagus, but in the European rabbit (Oryctolagus cuniculus) it causes a highly fatal and infectious disease known as myxomatosis. We have characterized the chemokine receptor CXCR4 gene in five genera of the order Lagomorpha, Ochotona (Ochotonidae), and Oryctolagus, Lepus, Bunolagus and Sylvilagus (Leporidae). In lagomorphs, the CXCR4 is highly conserved, with most of the protein diversity found at surface regions. Five amino acid replacements were observed, two in the intracellular loops, one in the transmembrane domain and two in the extracellular loops. Oryctolagus features unique amino acid changes at the intracellular domains, putting this genus apart of all other lagomorphs. Furthermore, in the 37 European rabbits analysed, which included healthy rabbits and rabbits with clinical symptoms of myxomatosis, 14 nucleotide substitutions were obtained but no amino acid differences were observed.

Control of gp130 expression by the mitogen-activated protein kinase ERK2.

Interleukin (IL)-6-type cytokines such as IL-6, oncostatin M (OSM) and leukaemia inhibitory factor (LIF) signal through receptor complexes that are critically dependent on gp130. The latter is the common signal-transducing molecule that couples these cytokines to their downstream effectors, Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). IL-6-type cytokine signalling additionally involves the recruitment and activation of extracellular signal-regulated kinase (ERK) 1 and ERK2. Both STATs and ERKs regulate responses mediated by members of the IL-6 family. Here, we show that ERK2, but not ERK1, also controls the expression and function of gp130 per se, as silencing ERK2 in human osteosarcoma U2OS cells inhibits the expression of gp130. This does not simply reflect quantitative differences between ERK1 and ERK2, and the effects are not restricted to osteosarcoma cells, as they can be extended to several other cancer cell types analysed to date (such as breast, prostate, lung and cervical cancer cells). Importantly, ERK2 binds to the GP130 promoter, where it perhaps interacts with the transcriptional machinery. Indeed, its role in the transcriptional regulation of the GP130 gene was corroborated using luciferase reporter assays and messenger RNA stability experiments. Considering the pivotal role that gp130 has in cancer and inflammation these data thus identify novel non-overlapping functions for ERK1 and ERK2 that are biologically relevant.

DAPK2 is a novel modulator of TRAIL-induced apoptosis.

Targeting molecules involved in TRAIL-mediated signalling has been hailed by many as a potential magic bullet to kill cancer cells efficiently, with little side effects on normal cells. Indeed, initial clinical trials showed that antibodies against TRAIL receptors, death receptor (DR)4 and DR5, are well tolerated by cancer patients. Despite efficacy issues in the clinical setting, novel approaches to trigger TRAIL-mediated apoptosis are being developed and its clinical potential is being reappraised. Unfortunately, as observed with other cancer therapies, many patients develop resistance to TRAIL-induced apoptosis and there is thus impetuous for identifying additional resistance mechanisms that may be targetable and usable in combination therapies. Here, we show that the death-associated protein kinase 2 (DAPK2) is a modulator of TRAIL signalling. Genetic ablation of DAPK2 using RNA interference causes phosphorylation of NF-κB and its transcriptional activity in several cancer cell lines. This then leads to the induction of a variety of NF-κB target genes, which include proapoptotic DR4 and DR5. DR4 and DR5 protein expression is correspondingly increased on the cell surface and this leads to the sensitisation of resistant cells to TRAIL-induced killing, in a p53-independent manner. As DAPK2 is a kinase, it is imminently druggable, and our data thus offer a novel avenue to overcome TRAIL resistance in the clinic.

Genetic variation at chemokine receptor CCR5 in leporids: alteration at the 2nd extracellular domain by gene conversion with CCR2 in Oryctolagus, but not in Sylvilagus and Lepus species.

Whereas in its natural host (Sylvilagus sps.) the effects of myxoma virus infections are benign, in European rabbit (Oryctolagus cuniculus), it causes a highly infectious disease with very high mortality rate, known as myxomatosis. There is evidence that, as with HIV-1 virus in human, myxoma virus may use chemokine receptors such as CCR5 of the host target cell for entry and activation of pathways of immune avoidance. We have characterized and compared CCR5 genes of leporid species with different susceptibility levels to myxomatosis. The CCR5 protein of O. cuniculus differs markedly from all those known from other species. The most striking was the replacement of a specific peptide motif of the second extracellular loop (ECL2) by a motif, which in other species characterizes the CCR2 molecules. While absent in Sylvilagus and Lepus species, this CCR2 imposed CCR5-ECL2 alteration was observed in all genomes of 25 European rabbits, representing the subspecies O. cuniculus algirus and O. cuniculus cuniculus. Allelic variation at the rabbit CCR5 locus confirmed that the gene conversion predates the subspecies split (1-2 Ma).