Inducible NOS-induced chloride intracellular channel 4 (CLIC4) nuclear translocation regulates macrophage deactivation

Abstract

Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the proto anti-inflammatory transition of the cells because IL-1β and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1β remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor L-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1β in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.protein nitrosylation | IL-1beta | phagocytosis C hloride intracellular channel 4 (CLIC4) is the most wellcharacterized member of a family of channel proteins conserved from Caenorhabditis elegans to humans. Although the chloride-selective channel activity of various members has long been established by multiple groups (1-3), the signaling and adaptor functions of soluble CLIC4 and other members have only recently come to the fore. Soluble CLIC4, originally identified as a p53-and c-myc-responsive proapoptotic protein (4, 5), is important for PKCdependent keratinocyte differentiation (6) and a modulator of TGFβ signaling in multiple cell types (7-9). Many of these proapoptotic and differentiation functions of CLIC4 are dependent on its translocation to the nucleus (6,8,10). CLIC4 exhibits redox sensitivity in both its soluble and membrane-associated states (11,12). Indeed, a central mechanism for induction of nuclear CLIC4 is nitric oxide (NO)-dependent modification (S-nitrosylation) of the protein that induces a conformational change, enhancing its association with nuclear transporters and thus its nuclear levels (13).Recently, a role for CLIC4 has been implicated in innate immunity. CLIC4 is an IRF3-dependent early response gene in LPSstimulated macrophages, transrepressed by the anti-inflammatory activity of the glucocorticoid receptor (14). Moreover, CLIC4 knockout macrophages exhibit dysregulation of multiple inflammatory mediators during the early response to LPS, in part as a consequence of altered IRF3 activity (15). These studies suggest that CLIC4 is important in macrophage early functions in response to stimulation but do not address later aspects of macrophage biology related to deactivation and phagocytosis. Furthermore, information is not available to reveal if the CLIC4 channel or nuclear activity is involved in these functions. Therefore, we investigated the role of CLIC4 activity in infl...

Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer

“…Peritoneal macrophages were harvested from the peritoneal cavity of C57BL/6 mice and purified by adherence to tissue culture plastic. 56 Bone marrow cells were isolated from C57BL/6 mice and cultured with DMEM supplemented with 10% fetal bovine serum (Gibco, 10100147) and 20 ng/ml recombinant murine M-CSF (PeproTech, 315-02). Culture fluid was exchanged with fresh culture medium every 3 d. Under these conditions, an adherent macrophage monolayer was obtained at d 7.…”

The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1β transcription and activate the NLRP3 inflammasome

“…CLIC4 is an NF-kB and IRF-3 early response gene in LPS stimulated macrophages [31]. CLIC4 knockout macrophages exhibit dysregulation of multiple inflammatory mediators during the early response to LPS [32,33]. In response to LPS, CLIC4 is S-nytrosylated and translocates into the nucleus where functions to enhance TGF-ß signalling [33].…”

Emerging biological roles of Cl− intracellular channel proteins

“…This effect is transient and requires both the putative NLS of CLIC4 and the nuclear import machinery. Nuclear translocation of CLIC4 has also been observed in macrophages that are exposed to lipopolysaccharide (LPS) and interferon γ (IFNγ) (Malik et al, 2012). It was shown previously that transforming growth factor β (TGFβ) induces nuclear translocation of CLIC4 (Shukla et al, 2009).…”

“…In theory, CLIC translocation could be regulated by posttranslational modifications. For instance, LPS-and IFNγ-induced nuclear translocation of CLIC4 in macrophages depends on Snitrosylation, which can induce a conformational change that promotes the association of CLIC4 with the nuclear import machinery (Malik et al, 2012). Other post-translational modifications found or predicted for the CLIC proteins include phosphorylation (Suh et al, 2007), ubiquitylation (Wagner et al, 2012) and palmitoylation (Fang et al, 2016); but biochemical details and functional outcomes remain to be determined.…”

“…(9) TGFβ induces translocation of CLIC4 into the nucleus were it prevents the dephosphorylation of SMAD proteins to enhance transcription (Shukla et al, 2009). INFγ and LPS promote CLIC4 S-nitrosylation (N) and nuclear translocation (Malik et al, 2012). TGFβR, transforming growth factor β receptor, IFNR, interferon receptor; GPCR, G-protein-coupled receptor.…”