Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals.
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Approximately 15 different alpha, or CXC, chemokines have thus far been isolated from 11 species of mammals. Among the best studied chemokines are the 12 human proteins that are encoded by 11 paralogous genes. In order to better understand the evolution and function of this group of genes, we isolated and characterized six novel GRO and GRO-related cDNA sequences from the cow (Bos taurus), the sheep (Ovis aries), the rabbit (Oryctolagus cuniculus), and the guinea pig (Cavia porcellus). The amino acid sequence of the diverged guinea pig GRO or KC gene is only 50%-60% similar to presumed orthologs from other species, while the sheep and cow GRO proteins are 90%-99% similar to each other. The presence of multiple GRO genes in the cow, the rabbit, and the sheep is consistent with what has been observed for humans. Phylogenetic analyses of amino acid sequences from 44 proteins indicate that genes orthologous to many of the 11 known from humans exist in other species. One such gene, interleukin 8, or IL8, has been isolated from nine species, including the rodent guinea pig; however, this gene is absent in the rat and the mouse, indicating a unique gene loss event in the rat/mouse (muroid rodent) lineage. The KC (or MIP2) gene of rodents appears to be orthologous to the GRO gene found in other taxonomic orders. Combined evidence from different sources suggests that IP10 and MIG share sister taxon relationships on the evolutionary tree, while the remaining paralogous genes represent independent lineages, with limited evidence for kinship between them. This observation indicates that these genes originated nearly contemporaneously via a series of gene duplication events. Relative-rate tests for synonymous and nonsynonymous nucleotide substitutions in the KC and IL8 genes did not detect rate heterogeneity; however, there are several notable features regarding the IL8 genes. For example, the IL8 proteins from two Old World monkeys are as similar to one another as they are to the IL8 protein from humans, and all observed nucleotide differences between the IL8 genes of the two monkeys cause amino acid changes; in other words, there are no synonymous differences between them. (+info)
Cutting edge: clustered AU-rich elements are the target of IL-10-mediated mRNA destabilization in mouse macrophages.
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In the present study we show that IL-10-mediated inhibition of inflammatory gene expression can be mediated by an AU-rich element (ARE) cluster present in the 3' untranslated region (3'UTR) of sensitive genes. A series of chloramphenicol acetyl transferase (CAT) reporter gene constructs were prepared in which different fragments from the IL-10-sensitive KC mRNA 3'UTR were placed downstream of the coding region of the reporter gene CAT. CAT mRNA containing the KC 3'UTR was markedly destabilized as compared with the control CAT mRNA, and the decay rate was further increased in cells stimulated with IL-10. The KC 3'UTR contains an ARE cluster and three isolated ARE motifs. The ARE cluster spanning nucleotides 378-399 appeared to be both necessary and sufficient to mediate sensitivity to IL-10 because a 116-nucleotide fragment that contains the cluster conferred sensitivity, while mutation of the sequence between positions 378 and 399 eliminated sensitivity. The destabilizing effect of IL-10 was relatively selective, as the stability of chimeric CAT mRNAs was not modulated in cells treated with IFN-gamma or IL-4. (+info)
Isolation of the CXC chemokines ENA-78, GRO alpha and GRO gamma from tumor cells and leukocytes reveals NH2-terminal heterogeneity. Functional comparison of different natural isoforms.
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Chemokines are a family of chemotactic peptides affecting leukocyte migration during the inflammatory response. Post-translational modification of chemokines has been shown to affect their biological potency. Here, the isolation and identification of natural isoforms of the neutrophil chemoattractants GRO alpha and GRO gamma and the epithelial-cell-derived neutrophil attractant-78 (ENA-78), is reported. Cultured tumor cells produced predominantly intact chemokine forms, whereas peripheral blood monocytes secreted mainly NH2-terminally truncated forms. The order of neutrophil chemotactic potency of these CXC chemokines was GRO alpha > GRO gamma > ENA-78 both for intact and truncated forms. However, truncated GRO alpha (4,5,6-73), GRO gamma (5-73) and ENA-78(8,9-78) were 30-fold, fivefold and threefold more active than the corresponding intact chemokine. As a consequence, truncated GRO alpha (4,5,6-73) was 300-fold more potent than intact ENA-78 indicating that both the type of chemokine and its mode of processing determine the chemotactic potency. Similar observations were made when intact and truncated GRO alpha, GRO gamma and ENA-78 were compared for their capacity to induce an increase in the intracellular calcium concentration in neutrophilic granulocytes, and to desensitize the calcium response towards the CXC chemokine granulocyte chemotactic protein-2 (GCP-2). It must be concluded that physiological proteolytic cleavage of CXC chemokines in general enhances the inflammatory response, whereas for CC chemokines NH2-terminal processing mostly results in reduced chemotactic potency. (+info)
Elevated constitutive IkappaB kinase activity and IkappaB-alpha phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-alpha transcription.
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The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-alpha, is up-regulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF)-kappaB consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducible mechanisms for transcription of this gene are fairly well delineated, the mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood. Recently, we demonstrated an increased rate of IkappaB-alpha degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kappaB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkappaB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkappaB-alpha phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kappaB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkappaB-alpha in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkappaB that is phosphorylated at Ser-32 reacted with IkappaB-alpha in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-alpha or IKK-beta are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappaB, the IkappaB-alpha wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-alpha transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kappaB. (+info)
Role of clathrin-mediated endocytosis in CXCR2 sequestration, resensitization, and signal transduction.
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CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines interleukin-8, melanoma growth-stimulatory activity/growth-regulatory protein, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we explored the mechanism responsible for ligand-induced CXCR2 endocytosis. Here, we demonstrate that dynamin, a component of clathrin-mediated endocytosis, is essential for CXCR2 endocytosis and resensitization. In HEK293 cells, dynamin I K44A, a dominant-negative mutant of dynamin that inhibits the clathrin-mediated endocytosis, blocks the ligand-stimulated CXCR2 sequestration. Furthermore, co-expression of dynamin I K44A significantly delays dephosphorylation of CXCR2 after ligand stimulation, suggesting that clathrin-mediated endocytosis plays an important role in receptor dephosphorylation and resensitization. In addition, ligand-mediated receptor down-regulation is attenuated when receptor internalization is inhibited by dynamin I K44A. Interestingly, inhibition of receptor endocytosis by dynamin I K44A does not affect the CXCR2-mediated stimulation of mitogen-activated protein kinase. Most significantly, our data indicate that the ligand-stimulated receptor endocytosis is required for CXCR2-mediated chemotaxis in HEK293 cells. Taken together, our findings suggest that clathrin-mediated CXCR2 internalization is crucial for receptor endocytosis, resensitization, and chemotaxis. (+info)
Mast cell migratory response to interleukin-8 is mediated through interaction with chemokine receptor CXCR2/Interleukin-8RB.
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To explore the role of chemokines in mast cell chemotaxis and accumulation at sites of inflammation, we first investigated the response of human mast cells to 18 different chemokines by induction of intracellular calcium mobilization in the human mast cell line, HMC-1. Only a subgroup of CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR) tripeptide motif, which included interleukin-8 (IL-8), growth-regulated oncogene alpha (GROalpha), neutrophil-activating peptide-2 (NAP-2), and epithelial cell-derived neutrophil activating peptide-78 (ENA-78), induced calcium flux in the cells. These observations suggested that the receptor CXCR2 (IL-8RB) should be expressed on the surface of these cells. Using the RNAse protection assay, CXCR2 mRNA, but not CXCR1 (IL-8RA) mRNA expression was detected in HMC-1 cells. Flow cytometry analysis documented the surface expression of CXCR2. A binding analysis performed with 125I-IL-8 determined that there were approximately 3,600 high affinity IL-8 binding sites per HMC-1 cell, with a calculated kd of 1.2 to 2 nmol/L. The activity of this receptor was further explored using IL-8, which was found to induce dose-dependent chemotactic and haptotactic responses in both HMC-1 cells and in vitro cultured human cord blood-derived mast cells. These results show the expression of functional CXCR2 receptors on the surface of human mast cells, which may play an important role in mast cell recruitment during the genesis of an inflammatory response. (+info)
Stimulation of peripheral cannabinoid receptor CB2 induces MCP-1 and IL-8 gene expression in human promyelocytic cell line HL60.
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Using the recently developed methodology of nucleic acid microarrays spotted with specific cDNAs probes belonging to different gene families, we showed for the first time that nanomolar concentrations of the cannabinoid ligand CP-55940 upregulated the expression of two different members of the chemokine gene family: the alpha-chemokine interleukin-8 (IL-8) and the beta-chemokine monocyte chemotactic protein-1 (MCP-1), in the promyelocytic cell line HL60 transfected with peripheral cannabinoid receptors (CB2). These genomic modulations observed on large-scale cDNA arrays were first confirmed by Northern blot studies. Furthermore, ELISA evaluations in culture supernatants indicated that the cannabinoid-induced activation of these two chemokine genes was followed by enhanced expression and secretion of the corresponding proteins. These upregulations initially observed in transfected HL60 cells overexpressing CB2 receptors, also occurred in normal non-transfected HL60 cells. The enhancement of IL-8 and MCP-1 gene transcription and protein production was shown to be pertussis toxin sensitive attesting that this phenomenon was a Gi protein-coupled receptor-mediated process as expected for cannabinoid receptors. More specifically, the abolition of the cannabinoid-induced effect by the specific CB2 antagonist SR 144528 indicated a strict peripheral cannabinoid-mediated process. Altogether, our data highlight a possible new function of peripheral cannabinoid receptors in the modulation of immune and inflammatory responses. (+info)
NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells.
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IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells. (+info)