Protein Phosphatase 1? and Cofilin Regulate Nuclear Translocation of NF-?B and Promote Expression of the Anti-Inflammatory Cytokine Interleukin-10 by T Cells.
ABSTRACT: While several protein serine/threonine kinases control cytokine production by T cells, the roles of serine/threonine phosphatases are largely unexplored. Here, we analyzed the involvement of protein phosphatase 1? (PP1?) in cytokine synthesis following costimulation of primary human T cells. Small interfering RNA (siRNA)-mediated knockdown of PP1? (PP1KD) or expression of a dominant negative PP1? (D95N-PP1) drastically diminished interleukin-10 (IL-10) production. Focusing on a key transcriptional activator of human IL-10, we demonstrate that nuclear translocation of NF-?B was significantly inhibited in PP1KD or D95N-PP1 cells. Interestingly, knockdown of cofilin, a known substrate of PP1 containing a nuclear localization signal, also prevented nuclear accumulation of NF-?B. Expression of a constitutively active nonphosphorylatable S3A-cofilin in D95N-PP1 cells restored nuclear translocation of NF-?B and IL-10 expression. Subpopulation analysis revealed that defective nuclear translocation of NF-?B was most prominent in CD4+ CD45RA- CXCR3- T cells that included IL-10-producing TH2 cells. Together these findings reveal novel functions for PP1? and its substrate cofilin in T cells namely the regulation of the nuclear translocation of NF-?B and promotion of IL-10 production. These data suggest that stimulation of PP1? could limit the overwhelming immune responses seen in chronic inflammatory diseases.
Project description:Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.
Project description:<h4>Background</h4>Cofilin is a member of the actin depolymerizing factor (ADF)/cofilin family, which regulates actin dynamics. Increasing evidence suggests that mitochondrial translocation of cofilin appears necessary for the regulation of apoptosis.<h4>Results</h4>We report that allyl isothiocyanate (AITC) potently induces mitochondria injury and apoptosis. These events were accompanied by a loss of polymerized filamentous actin (F-actin) and increase in unpolymerized globular actin (G-actin). AITC also induces dephosphorylation of cofilin through activation of PP1 and PP2A. Only dephosphorylated cofilin binds to G-actin and translocates to mitochondria during AITC-mediated apoptosis. Mechanistic study revealed that interruption of ROCK1/PTEN/PI3K signaling pathway plays a critical role in AITC-mediated dephosphorylation and mitochondrial translocation of cofilin and apoptosis. Our in vivo study also showed that AITC-mediated inhibition of tumor growth of mouse leukemia xenograft model is in association with dephosphorylation of cofilin.<h4>Conclusions</h4>These findings support a model in which induction of apoptosis by AITC stems primarily from activation of ROCK1 and PTEN, and inactivation of PI3K, leading in turn to activation of PP1 and PP2A, resulting in dephosphorylation of cofilin, which binds to G-actin and translocates to mitochondria, culminating in the dysfunction of mitochondria, release of cytochrome c and apoptosis.
Project description:<h4>Background</h4>Recent studies have revealed that cyclooxygenase-2 (COX-2) expression is down-regulated in aspirin-induced asthma (AIA). Various signal pathways (MAPKs, NF-?B and C/EBP) are involved in COX-2 regulation.<h4>Objective</h4>To investigate the regulation of COX-2 expression through MAP-kinase pathway activation and nuclear factor translocation in aspirin-induced asthma (AIA).<h4>Methods</h4>Fibroblasts were isolated from specimens of nasal mucosa (NM, N?=?5) and nasal polyps (NP, N?=?5). After IL-1? (1 ng/ml) incubation, COX-2 and phosphorylated forms of ERK, JNK and p38 MAPK were measured by Western blot. MAPK's role in IL-1?-induced COX-2 expression was assessed by treating cells with ERK (PD98059), JNK (SP600125) and p38 MAPK (SB203580) inhibitors (0.1-10 µM) prior to IL-1? exposure. NF-?B and C/EBP nuclear translocation was measured by Western blot and TransAM® after IL-1? (10 ng/ml) exposure.<h4>Results</h4>No differences were observed in the MAPK phosphorylation time-course between NM and NP-AIA fibroblasts. The p38 MAPK inhibitor at 10 µM significantly reduced IL-1?-induced COX-2 expression in NM fibroblasts (85%). In NP-AIA fibroblasts the COX-2 inhibition (65%) at 1 and 10 µM was not statistically significant compared to non-treated cells. ERK and JNK inhibitors had no significant effect in either the NM or NP-AIA cultures. The effect of IL-1? on NF-?B and C/EBP subunits' nuclear translocation was similar between NM and NP-AIA fibroblasts.<h4>Conclusions</h4>These results suggest that p38 MAPK is the only MAPK involved in IL-1?-induced COX-2 expression. NM and NP-AIA fibroblasts have similar MAPK phosphorylation dynamics and nuclear factor translocation (NF-?B and C/EBP). COX-2 downregulation observed in AIA patients appears not to be caused by differences in MAPK dynamics or transcription factor translocation.
Project description:The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKI (casein kinase I ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKI-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.
Project description:We have analysed activation of nuclear factor-kappaB (NF-kappaB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-kappaB1 and inhibitor of kappaB (I-kappaB) alpha using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-kappaBalpha degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-kappaB1. During pathway activation, relA was transported up a concentration gradient, resulting in a 3-4-fold increase in nuclear levels, but without any significant decrease in cytoplasmic concentration. IL-1 stimulation caused translocation of only 20% of the relA, but resulted in degradation of up to 70% of the cytoplasmic I-kappaBalpha. RelA nuclear translocation in fibroblasts correlated with DNA-binding activity measured by electrophoretic mobility shift assay (EMSA), both with respect to kinetics and IL-1 concentration-dependence. Clonal populations of cells demonstrated a marked degree of heterogeneity in the response to IL-1. The single-cell assay revealed the presence of responder and non-responder subpopulations, with an enhanced proportion of responder cells, and prolonged responses at higher concentrations of IL-1. Comparing different cell types demonstrated that whereas HepG2 cells, as fibroblasts, showed good correlation between nuclear translocation of relA and activation of DNA binding by relA-containing dimers, EL4 thymoma cells showed no effect on relA localization, even during induction of significant levels NF-kappaB activity, as measured by EMSA. The analysis shows that stimulation by IL-1 results in transient perturbation of the NF-kappaB system, which cycles between the resting and active states with net redistribution of a minor proportion of its DNA-binding component. In addition, it demonstrates significant cell-to-cell variations, as well as cell-type-specific differences in net relA nuclear transport in response to stimuli. The data are consistent with NF-kappaB constituting a dynamic and versatile system, regulated to a significant degree by binary events involving bidirectional trafficking between the cytoplasmic and nuclear compartments during pathway activation.
Project description:Dynorphin 1-17, (DYN 1-17) opioid peptide produces antinociception following binding to the kappa-opioid peptide (KOP) receptor. Upon synthesis and release in inflamed tissues by immune cells, DYN 1-17 undergoes rapid biotransformation and yields a unique set of opioid and non-opioid fragments. Some of these major fragments possess a role in immunomodulation, suggesting that opioid-targeted therapeutics may be effective in diminishing the severity of inflammatory disorders. This study aimed to examine the immunomodulatory effects of DYN 1-17 and major N-terminal fragments found in the inflammatory environment on nuclear factor-kappaB/p65 (NF-?B/p65) nuclear translocation and the release of interleukin-1beta (IL-1?) and tumor necrosis factor-alpha (TNF-?) from lipopolysaccharide (LPS)-stimulated, differentiated THP-1 cells. The results demonstrate that NF-?B/p65 nuclear translocation was significantly attenuated following treatment with DYN 1-17 and a specific range of fragments, with the greatest reduction observed with DYN 1-7 at a low concentration (10 nM). Antagonism with a selective KOP receptor antagonist, ML-190, significantly reversed the inhibitory effects of DYN 1-17, DYN 1-6, DYN 1-7 and DYN 1-9, but not other DYN 1-17 N-terminal fragments (DYN 1-10 and 1-11) on NF-?B/p65 nuclear translocation. DYN 1-17 and selected fragments demonstrated differential modulation on the release of IL-1? and TNF-? with significant inhibition observed with DYN 1-7 at low concentrations (1 nM and 10 pM). These effects were blocked by ML-190, suggesting a KOP receptor-mediated pathway. The results demonstrate that DYN 1-17 and certain N-terminal fragments, produced in an inflamed environment, play an anti-inflammatory role by inhibiting NF-?B/p65 translocation and the subsequent cytokine release through KOP receptor-dependent and independent pathways.
Project description:Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.
Project description:Interleukin (IL)-15 and its receptors are induced by tumor necrosis factor ? (TNF) in the cerebral endothelial cells composing the blood-brain barrier, but it is not yet clear how IL-15 modulates endothelial function. Contrary to the known induction of JAK/STAT3 signaling, here we found that nuclear factor (NF)- ?B is mainly responsible for IL-15 actions on primary brain microvessel endothelial cells and cerebral endothelial cell lines. IL-15-induced transactivation of an NF?B luciferase reporter resulted in phosphorylation and degradation of the inhibitory subunit I?B that was followed by phosphorylation and nuclear translocation of the p65 subunit of NF?B. An I?B kinase inhibitor Bay 11-7082 only partially inhibited IL-15-induced NF?B luciferase activity. The effect of IL-15 was mediated by its specific receptor IL-15R?, since endothelia from IL-15R? knockout mice showed delayed nuclear translocation of p65, whereas those from knockout mice lacking a co-receptor IL-2R? did not show such changes. At the mRNA level, IL-15 and TNF showed similar effects in decreasing the tight junction protein claudin-2 and increasing the p65 subunit of NF?B but exerted different regulation on caveolin-1 and vimentin. Taken together, NF?B is a major signal transducer by which IL-15 affects cellular permeability, endocytosis, and intracellular trafficking at the level of the blood-brain barrier.
Project description:DNA damage-induced NF-?B activation and the secretion of inflammatory cytokines play crucial roles in carcinogenesis and cellular senescence. However, the underlying mechanisms, especially the initial sensors and transducers connecting the nuclear DNA damage signal with cytoplasmic NF-?B activation remain incompletely understood. Here, we report that TRAF-interacting protein with forkhead-associated domain (TIFA), an established NF-?B activator in the cytosol, unexpectedly exhibited nuclear translocation and accumulation on damaged chromatin following genotoxic stress. Accordingly, we also found that DNA damage-induced transcriptional activation and the resulting secretion of classic NF-?B targets, including interleukin (IL)-6 and IL-8, was greatly enhanced in TIFA-overexpressing cells compared with control cells. Mechanistically, DNA damage-induced TIFA phosphorylation at threonine 9 (pThr-9), and this phosphorylation event, involving the pThr-binding forkhead-associated domain, was crucial for its enrichment on damaged chromatin and subsequent NF-?B activation. Moreover, in conjunction with its partner protein, the E3 ligase TNF receptor-associated factor 2 (TRAF2), TIFA relayed the DNA damage signals by stimulating ubiquitination of NF-?B essential modulator (NEMO), whose sumoylation, phosphorylation, and ubiquitination were critical for NF-?B's response to DNA damage. Consistently, TRAF2 knockdown suppressed TIFA overexpression-enhanced NEMO ubiquitination under genotoxic stress, and a unphosphorylatable Thr-9-mutated TIFA variant had only minor effects on NEMO poly-ubiquitination. Finally, in agreement with the model of DNA damage-associated secretory senescence barrier against carcinogenesis, ectopic TIFA expression limited proliferation of multiple myeloma cancer cells. In conclusion our results indicate that TIFA functions as a key transducer in DNA damage-induced NF-?B activation.
Project description:The polymorphic membrane protein D (PmpD) is a highly conserved outer membrane protein which plays an important role in pathogenesis during Chlamydia psittaci infection. In this study, we evaluated the ability of the N-terminus of PmpD (PmpD-N) to modulate the functions of chicken macrophages and the signaling pathway(s) involved in PmpD-N-induced Toll-like receptors (TLRs), as well as interleukin (IL)-6 and IL-10 cytokine secretions. Thus, HD11 macrophages were treated with exogenous and intracellular PmpD-N of C. psittaci. The chlamydial growth was evaluated by enumeration of chlamydial loads in the infected macrophages. The phagocytic function of macrophages following PmpD-N treatment was detected by fluorescein-labeled Escherichia coli (E. coli). The concentration of nitric oxide (NO) secreted by HD11 macrophages was measured by the amount of NO2- in the culture supernatant using the Griess method. The cytokine secretions were assessed using multiplex cytokine ELISA kits. Expression levels of TLRs, myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-?B) were analyzed by a Western blotting assay, as well as a luciferase assay, while NF-?B p65 nuclear translocation was assessed by confocal microscopy. The nuclear translocation of the transcription factor NF-?B was confirmed by evaluating its ability to combine with the corresponding promoter using the electrophoretic mobility shift assay (EMSA). After treatment with exogenous or endogenous PmpD-N, chlamydial loads and phagocytic functions were reduced significantly compared with those of the plasmid vector group, while NO secretions were reduced significantly compared with those of the lipopolysaccharide (LPS) treatment. Stimulation of HD11 cells with PmpD-N provoked the secretion of the Th2 cytokines, IL-6, and IL-10 and upregulated the expression of TLR2, TLR4, MyD88, and NF-?B. Furthermore, inhibition of TLR2, MyD88, and NF-?B in HD11 cells significantly decreased IL-6 and IL-10 cytokine levels, while NO production and phagocytosis increased significantly, strongly suggesting their involvement in PmpD-N-induced Th2 cytokine secretion and macrophage dysfunction. Our data indicate that C. psittaci PmpD-N inhibited macrophage functions by activating the Th2 immune response and the TLR2/MyD88/NF-?B signaling pathway.