Article

Interleukin (IL)-15 and IL-2 reciprocally regulate expression of the chemokine receptor CX3CR1 through selective NFAT1- and NFAT2-dependent mechanisms

Molecular Signaling Section, Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2004; 279(47):48520-34. DOI: 10.1074/jbc.M406978200
Source: PubMed

ABSTRACT We have recently reported that interleukin (IL)-15 and IL-2, which signal through IL-2Rbetagamma, oppositely regulate expression of the proinflammatory chemokine receptor CX3CR1. Here we delineate molecular mechanisms responsible for this paradox. By using a luciferase reporter plasmid, we identified a 433-bp region spanning the major transcriptional start point of human CX3CR1 that, when expressed in human peripheral blood mononuclear cells (PBMCs), possessed strong constitutive promoter activity. IL-2 and IL-15 treatment increased and abolished this activity, respectively, mimicking their effects on endogenous CX3CR1. IL-2 and IL-15 have been reported to also have opposite effects on the immunoregulatory transcription factor NFAT (nuclear factor of activated T cells), and the 433-bp region contains a kappaB-like NFAT site. The effects of IL-15 and IL-2 on both CX3CR1 reporter activity and endogenous CX3CR1 transcription in PBMCs were abolished by the NFAT inhibitors cyclosporin A and VIVIT. Moreover, mutation of the kappaB-like NFAT sequence markedly attenuated IL-2 and IL-15 modulation of CX3CR1 promoter-reporter activity in PBMCs. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific recruitment of NFAT1 but not NFAT2 to the CX3CR1 promoter, whereas IL-2 had the converse effect. This appears to be relevant in vivo because mouse CX3CR1 mRNA was expressed in both PBMCs and splenocytes from NFAT1-/- mice injected with recombinant IL-15 but was undetectable in cells from IL-15-injected NFAT1+/+ BALB/c mice; as predicted, IL-2 up-regulated cx3cr1 in both mouse strains to a similar extent. Thus, by pharmacologic, genetic, and biochemical criteria in vitro and in vivo, our results suggest that IL-15 and IL-2 oppositely regulate CX3CR1 gene expression by differentially recruiting NFAT1 and NFAT2 to a kappaB-like NFAT site within the CX3CR1 promoter. We propose that expression of CX3CR1 and possibly other immunoregulatory genes may be determined in part by the balance of NFAT1 and NFAT2 activity in leukocytes.

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Available from: David H Mcdermott, Jul 27, 2015
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Results of this study demonstrated that mouse CX3CL1 is predominately shed by ADAM10-dependent mechanisms as shown by pharmacological inhibition and transcriptional silencing. As described for human CX3CL1, shedding of mouse CX3CL1 is enhanced by ionomycin treatment, which also involves ADAM10-activity. Deletion and replacement of the cleavage region of mouse CX3CL1 could not further abrogate its release suggesting that other domains of mouse CX3CL1 may influence this process. And indeed, release of mouse CX3CL1 was found to be further influenced by the chemokine domain and also the cytoplasmic tail. However, truncation of the cytoplasmic tail led also to impaired cellular trafficking. In fact, when wt mouse CX3CL1 and the truncated variant were sufficiently expressed on the cell surface, both were still shed. Additionally, adhesion and transmigration experiments with human PBMCs revealed that the truncation did not affect the function of the transmembrane mouse CX3CL1 as an adhesion and a transmigration molecule. This latter finding suggests that potential signalling events induced by the cytoplasmic tail of CX3CL1 are not involved in the recruitment of leukocytes to the inflamed tissue. This work indicates that multiple molecular determinants within mouse CX3CL1 influence its shedding via ADAM10, suggesting that exchange of several domains of mouse CX3CL1 cannot completely prevent its shedding. The cytoplasmic tail of mouse CX3CL1 appears to be relevant for correct trafficking of mouse CX3CL1 but neither for shedding, adhesion or transmigration. Nevertheless, shedding of CX3CL1 by ADAM10 and ADAM17 could still represent a crucial step within the leukocyte recruitment process in developing vascular diseases such as atherosclerosis. Leukozyten werden aus dem Blutstrom durch die Gefäßwand hin zum entzündeten Gewebe rekrutiert. Dieser Vorgang erfolgt in mehreren Schritten und wird zum Teil durch Chemokine reguliert. Innerhalb der großen Familie der Chemokine, die eine gerichtete Zellwanderung vermitteln, existiert ein ungewöhnliches Mitglied, das CX3CL1 (Fraktalkin), was als transmembranes und auch lösliches Molekül vorkommt. Dieses lösliche CX3CL1 Molekül wird im speziellen von der Metalloprotease ADAM10 durch limitierte Proteolyse (Shedding der Ektodomäne) gebildet. Durch die Bindung an den Rezeptor CX3CR1 vermittelt das transmembrane CX3CL1 außerdem Adhäsion und Transmigration von leukozytären Subpopulationen. Bis heute ist allerdings nicht klar, wie ADAM10 einen Beitrag zur Funktion des murines CX3CL1 leistet. Das Ziel dieser Arbeit war es nun, die molekularen Faktoren innerhalb des murinem CX3CL1, verantwortlich für die Spaltung und Funktion dieses Moleküls, zu charakterisieren. Ergebnisse dieser Studie haben mittels pharmakologischer Inhibition und transkriptionellen Silencing gezeigt, dass das murine CX3CL1 vorwiegend auch durch ADAM10 gespalten wird. Wie auch für das humane CX3CL1 beschrieben, wird das ADAM10 vermittelte Shedding von murinem CX3CL1 durch die Behandlung mit Ionomycin weiter verstärkt. Depletion und Ersatz der Spaltstellenregion von murinem CX3CL1 konnten die Freisetzung von löslichem CX3CL1 nicht weiter vermindern. Weiterhin konnte gezeigt werden, dass die Freisetzung von murinem CX3CL1 auch durch die Chemokindomäne und den zytoplasmatischen Teil beeinflusst wird. Allerdings führte die Trunkierung dieses zytoplasmatischen Teils zu einer Beeinträchtigung der zellulären Verteilung des Moleküls. Jedoch wurden das wt CX3CL1 und die verkürzte Variante nach gleichwertiger Oberflächenexpression immer noch in gleichem Maße gespalten. 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