Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2^−/− mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.     

Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.     

Trihydrophobin 1 is a new negative regulator of A-Raf kinase

Our previous work indicated that instead of binding to B-Raf or C-Raf, trihydrophobin 1 (TH1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1-372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase-dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G(0)/G(1) phase-arrested. Taken together, our data provide a clue to understanding the cellular function of TH1 on Raf isoform-specific regulation.     

MICAL-1 is a negative regulator of MST-NDR kinase signaling and apoptosis

MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.     

Casein kinase 1 is a novel negative regulator of E-cadherin-based cell-cell contacts

Cadherins are the most crucial membrane proteins for the formation of tight and compact cell-cell contacts. Cadherin-based cell-cell adhesions are dynamically established and/or disrupted during various physiological and pathological processes. However, the molecular mechanisms that regulate cell-cell contacts are not fully understood. In this paper, we report a novel functional role of casein kinase 1 (CK1) in the regulation of cell-cell contacts. Firstly, we observed that IC261, a specific inhibitor of CK1, stabilizes cadherin-based cell-cell contacts, whereas the overexpression of CK1 disrupts them. CK1 colocalizes with E-cadherin and phosphorylates the cytoplasmic domain of E-cadherin in vitro and in a cell culture system. We show that the major CK1 phosphorylation site of E-cadherin is serine 846, a highly conserved residue between classical cadherins. Constitutively phosphorylated E-cadherin (S846D) is unable to localize at cell-cell contacts and has decreased adhesive activity. Furthermore, phosphorylated E-cadherin (S846D) has weaker interactions with beta-catenin and is internalized more efficiently than wild-type E-cadherin. These data indicate that CK1 is a novel negative regulator of cadherin-based cell-cell contacts.     

CD4+ cell memory: the enigma of Th1 cells

Despite a wealth of information pertaining to functional and phenotypic attributes of memory CD4(+) cells, the mechanisms that underlie the generation and persistence of memory in this subset are largely unknown. Recent work suggests that the development of memory might be differently regulated in T-helper-1 and T-helper-2 cells, owing to differences in their susceptibility to cell death. These studies support a new paradigm, in which memory T cells are heterogeneous in terms of their stage of maturation and function as well as mechanisms of homeostatic control.     

CD25+CD4+ cells contribute to Th2 polarization during helminth infection by suppressing Th1 response development

Mice infected with Schistosoma mansoni develop polarized Th2 responses in which Th1 responses are prevented by IL-10-mediated suppression of IL-12 production. We show that dendritic cells from infected mice are primed to make IL-12 in response to CD40 ligation, and that IL-10 acts by inhibiting this process. In infected mice, two subpopulations of CD4(+) cells, separable by their expression of CD25, make IL-10. CD25(+)CD4(+) cells expressed forkhead box P3, inhibited proliferation of CD4(+) T cells, and made IL-10, but little IL-5. In contrast, CD25(-)CD4(+) cells failed to express forkhead box P3 or to inhibit proliferation and accounted for all the IL-5, IL-6, and IL-13 produced by unseparated splenic populations. Thus, CD25(+) and CD25(-) subpopulations could be characterized as regulatory T cells (Treg cells) and Th2 cells, respectively. Consistent with their ability to make IL-10, both CD25(+) and CD25(-)CD4(+) T cells from infected mice were able, when stimulated with egg Ag, to suppress IL-12 production by CD40 agonist-stimulated dendritic cells. Additionally, in adoptive transfer experiments, both CD4(+) subpopulations of cells were able to partially inhibit the development of Th1 responses in egg-immunized IL-10(-/-) mice. The relationship of Treg cells in infected mice to natural Treg cells was strongly suggested by the ability of CD25(+)CD4(+) cells from naive mice to inhibit Th1 response development when transferred into egg-immunized or infected IL-10(-/-) mice. The data suggest that natural Treg cells and, to a lesser extent, Th2 cells play roles in suppressing Th1 responses and ensuring Th2 polarization during schistosomiasis.     

Protein kinase Czeta is a negative regulator of protein kinase B activity

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.     

Sphingosine 1-phosphate as a novel immune regulator of dendritic cells

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling. Recently, the role of S1P on the immune system has evoked great interest. In particular, several aspects of the effects on antigen-presenting cells (APCs) as dendritic cells (DC) in mice and humans have been reported. In this review, we focus on the role played by S1P on the DC system and its effects in immune-related pathological states.     

p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells.

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.     

Sphingosine 1-phosphate as a novel immune regulator of dendritic cells

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling. Recently, the role of S1P on the immune system has evoked great interest. In particular, several aspects of the effects on antigen-presenting cells (APCs) as dendritic cells (DC) in mice and humans have been reported. In this review, we focus on the role played by S1P on the DC system and its effects in immune-related pathological states.     

Ufd1-Npl4 is a negative regulator of cholera toxin retrotranslocation

The A1 chain of the cholera toxin (CT) undergoes retrotranslocation to the cytosol across the endoplasmic reticulum (ER) membrane by hijacking ER-associated degradation (ERAD). In the cytosol the CT A1 chain stimulates adenylyl cyclase. The VCP(Ufd1-Npl4) complex mediates retrotranslocation of emerging ER proteins. While one group reported that VCP is required for CT retrotranslocation, another group concluded the opposite. We show that VCP is dispensable for CT retrotranslocation, however RNAi of either Ufd1 or Npl4 induces an increase in adenylyl cyclase activity induced by CT. RNAi of VCP, Ufd1 or Npl4 did not affect adenylyl cyclase activity induced by forskolin. These findings are coherent with our previous report showing that depletion of Ufd1-Npl4 accelerates ERAD of reporter substrates. To integrate contradictory results we propose a new model, where Ufd1-Npl4 is a negative regulator of retrotranslocation, delaying the retrotranslocation of ERAD substrates independently of its association with VCP.     

Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.     

Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1

3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.     

Protein phosphatase 4 is a positive regulator of hematopoietic progenitor kinase 1

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic specific mammalian Ste20-like protein kinase and has been implicated in many cellular signaling pathways including T cell receptor (TCR) signaling. However, little is known about the in vivo regulation of HPK1. We present evidence that HPK1 is positively regulated by protein phosphatase 4 (PP4; also called PPX and PPP4), a serine/threonine phosphatase. We found that PP4 interacted with HPK1 and that the proline-rich region of HPK1 was necessary and sufficient for this interaction. We also found that PP4 had phosphatase activity toward HPK1 in vivo and that co-transfection of PP4 with HPK1 resulted in specific kinase activation of HPK1. Moreover, we found that the PP4-induced HPK1 kinase activation was accompanied by an increase in protein expression of HPK1. Pulse-chase analysis showed that PP4 increased the half-life of HPK1. Further studies showed that HPK1 was subject to regulation by ubiquitination and ubiquitin-targeted degradation and that PP4 inhibited HPK1 ubiquitination. In addition, we found that TCR stimulation enhanced the PP4-HPK1 interaction and that wild-type PP4 enhanced, whereas a phosphatase-dead PP4 mutant inhibited, TCR-induced activation of HPK1 in Jurkat T cells. Combined with the observation that PP4 enhanced HPK1-induced JNK activation, our studies identify PP4 as a positive regulator for HPK1 and the HPK1-JNK signaling pathway.     

Role of T‐bet,the master regulator of Th1 cells,in the cytotoxicity of murine CD4+ T cells

Although CD4⁺ T cells are generally regarded as helper T cells, some activated CD4⁺ T cells have cytotoxic properties. Given that CD4⁺ cytotoxic T lymphocytes (CTLs) often secrete IFN‐γ, CTL activity among CD4⁺ T cells may be attributable to Th1 cells, where a T‐box family molecule, T‐bet serves as the “master regulator”. However, although the essential contribution of T‐bet to expression of IFN‐γ has been well‐documented, it remains unclear whether T‐bet is involved in CD4⁺ T cell‐mediated cytotoxicity. In this study, to investigate the ability of T‐bet to confer cytolytic activity on CD4⁺ T cells, the T‐bet gene (Tbx21) was introduced into non‐cytocidal CD4⁺ T cell lines and their cytolytic function analyzed. Up‐regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4⁺ T cells but not in untransfected parental cells. In one cell line, T‐bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas^? target cells. Although T‐bet was found to repress up‐regulation of CD40L (CD154), which controls FasL‐mediated cytolysis, the extent of CD40L up‐regulation on in vitro‐differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T‐bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.