Active beta-catenin signaling is an inhibitory pathway for human immunodeficiency virus replication in peripheral blood mononuclear cells

The Wnt/β-catenin pathway is involved in cell functions governing development and disease. In modeling postentry restriction of human immunodeficiency virus (HIV) replication in astrocytes, we reported that part of this natural resistance to productive replication of HIV in astrocytes involved expression of proteins of the Wnt/β-catenin signaling pathway. We determined here whether induction of β-catenin signaling in peripheral blood mononuclear cells (PBMCs) can modulate HIV replication. Given that lithium is an inducer of β-catenin signaling, we used it as a tool to determine the impact of β-catenin signaling on HIV replication in PBMCs. We demonstrated that lithium inhibited the replication of T-tropic and primary isolates of HIV by >90% and did so in noncytotoxic/noncytostatic concentrations and in a β-catenin-dependent manner. Specifically, inhibiting β-catenin signaling by transfection of dominant-negative mutant constructs to either T-cell factor 4, the downstream effector of Wnt signaling, or β-catenin, the central mediator of this pathway, abrogated the ability of lithium to inhibit HIV replication. Moreover, when Wnt/β-catenin signaling was inhibited, the level of HIV replication was enhanced by fourfold. To confirm the in vivo relevance of the β-catenin pathway in repressing HIV replication, we evaluated HIV-positive antiretroviral therapy-naive patients who were on lithium therapy. These patients demonstrated a reduction in viral load, which increased as the dose of lithium was reduced. Collectively, these data indicate that β-catenin signaling is an intrinsic molecular pathway restricting HIV replication in PBMCs.     

IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability

Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.     

T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.     

Ca2+/calmodulin-stimulated PDE1 regulates the beta-catenin/TCF signaling through PP2A B56 gamma subunit in proliferating vascular smooth muscle cells

The phenotypic change of vascular smooth muscle cells (VSMCs), from a 'contractile' phenotype to a 'synthetic' phenotype, is crucial for pathogenic vascular remodeling in vascular diseases such as atherosclerosis and restenosis. Ca(2+)/calmodulin-stimulated phosphodiesterase 1 (PDE1) isozymes, including PDE1A and PDE1C, play integral roles in regulating the proliferation of synthetic VSMCs. However, the underlying molecular mechanism(s) remain unknown. In this study, we explore the role and mechanism of PDE1 isoforms in regulating β-catenin/T-cell factor (TCF) signaling in VSMCs, a pathway important for vascular remodeling through promoting VSMC growth and survival. We found that inhibition of PDE1 activity markedly attenuated β-catenin/TCF signaling by downregulating β-catenin protein. The effect of PDE1 inhibition on β-catenin protein reduction is exerted via promoting glycogen synthase kinase 3 (GSK3)β activation, β-catenin phosphorylation and subsequent β-catenin protein degradation. Moreover, PDE1 inhibition specifically upregulated phosphatase protein phosphatase 2A (PP2A) B56γ subunit gene expression, which is responsible for the effects of PDE1 inhibition on GSK3β and β-catenin/TCF signaling. Furthermore, the effect of PDE1 inhibition on β-catenin was specifically mediated by PDE1A but not PDE1C isozyme. Interestingly, in synthetic VSMCs, PP2A B56γ, phospho-GSK3β and phospho-β-catenin were all found in the nucleus, suggesting that PDE1A regulates nuclear β-catenin protein stability through the nuclear PP2A-GSK3β-β-catenin signaling axis. Taken together, these findings provide direct evidence for the first time that PP2A B56γ is a critical mediator for PDE1A in the regulation of β-catenin signaling in proliferating VSMCs.     

IFN-γ Induces Gastric Cancer Cell Proliferation and Metastasis Through Upregulation of Integrin β3-Mediated NF-κB Signaling

Interferon γ (IFN-γ), a multifunctional cytokine, was upregulated in the resected gastric cancer tissue. However, whether IFN-γ is involved in the regulation of gastric cancer has not been well elucidated. Herein, we aimed to investigate the effects and mechanism of IFN-γ on gastric cancer. In this study, we found a vital role of IFN-γ in enhancing proliferation, inhibiting apoptosis, and promoting cell migration and invasion in gastric cancer cells SGC-7901 and MGC-803. Additionally, IFN-γ activated nuclear factor κB (NF-κB) signaling pathway by upregulating the phosphorylation expression of p65 and IκBα, and induced the expression of integrin β3 in vitro. Therefore, to further investigate the relationship between IFN-γ and integrin β3, SGC-7901 cells were transfected with integrin β3 siRNA. And then cells expressed lower cell viability, migration, and invasion rates, while cell apoptosis was significantly enhanced. Meanwhile, expression of integrin β3, MMP-2, MMP-9, and NF-κB, including p65 and IκBα, and the nuclear translocation of NF-κB/p65 were dramatically repressed, whereas IFN-γ significantly improved the effects. Moreover, in vivo, the experiment of xenograft model and pulmonary metastasis model also retarded in integrin β3 siRNA group. And the expression of integrin β3, MMP-2, MMP-9, and NF-κB was repressed. However, the treatment with IFN-γ improved tumor volume, lung/total weight, tumor nodules, and the protein expression described above compared with integrin β3 siRNA group. Overall, the results indicated that IFN-γ induces gastric cancer cell proliferation and metastasis partially through the upregulation of integrin β3-mediated NF-κB signaling. Hence, the inhibition of IFN-γ or integrin β3 may be the key for the treatment of gastric cancer.     

Confluence induced threonine41/serine45 phospho-β-catenin dephosphorylation via ceramide-mediated activation of PP1cγ

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101–11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-β-catenin at threonine41/serine45. The effect of confluence on β-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of β-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented β-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cγ prevented dephosphorylation of β-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cγ to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced β-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes

Although elevation of the blood glucose level is a causal adverse effect of treatment with interferon (IFN), the precise underlying molecular mechanism is largely unknown. We examined the effects of type I and type II IFN (IFN-β and IFN-γ) on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. IFN-β suppressed insulin-induced tyrosine phosphorylation of IRS-1 without affecting its expression, whereas IFN-γ reduced both the protein level and tyrosine phosphorylation. Although both IFNs stimulated phosphorylation of STAT1 (at Tyr(701)) and STAT3 (at Tyr(705)) after treatment for 30 min, subsequent properties of induction of the SOCS isoform were different. IFN-β preferentially induced SOCS1 rather than SOCS3, whereas IFN-γ strongly induced SOCS3 expression alone. In addition, adenovirus-mediated overexpression of either SOCS1 or SOCS3 inhibited insulin-induced tyrosine phosphorylation of IRS-1, whereas the reduction of IRS-1 protein was observed only in SOCS3-expressed cells. Notably, IFN-β-induced SOCS1 expression and suppression of insulin-induced tyrosine phosphorylation of IRS-1 were attenuated by siRNA-mediated knockdown of STAT1. In contrast, adenovirus-mediated expression of a dominant-negative STAT3 (F-STAT3) attenuated IFN-γ-induced SOCS3 expression, reduction of IRS-1 protein, and suppression of insulin-induced glucose uptake but did not have any effect on the IFN-β-mediated SOCS1 expression and inhibition of insulin-induced glucose uptake. Interestingly, pretreatment of IFN-γ with IL-6 synergistically suppressed insulin signaling, even when IL-6 alone had no significant effect. These results indicate that type I and type II IFN induce insulin resistance by inducing distinct SOCS isoforms, and IL-6 synergistically augments IFN-γ-induced insulin resistance by potentiating STAT3-mediated SOCS3 induction in 3T3-L1 adipocytes.     

Induction of indoleamine 2,3-dioxygenase by interferon-gamma in human lens epithelial cells: Apoptosis through the formation of 3-hydroxykynurenine

Interferon-gamma (IFN-γ) is known to cause apoptosis of lens epithelial cells and cataract formation, but the molecular mechanisms underlying these effects are unknown. IFN-γ induces the expression of indoleamine 2,3-dioxygenase (IDO) and thereby enhances the production of kynurenines from l-tryptophan. The present study was designed to investigate the role of IDO and kynurenines in the IFN-γ-mediated apoptosis of lens epithelial cells and to determine the signaling pathways involved. IFN-γ stimulated the synthesis of IDO and activated the JAK–STAT1 signaling pathway in human lens epithelial cells (HLE-B3) in a dose-dependent manner. Meanwhile, fludarabine, an inhibitor of STAT1 activation, blocked IFN-γ-mediated IDO expression. N-Formylkynurenine, kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) were detected in cells, with 3OHKyn concentrations being higher than those of the other kynurenines. The intracellular production of kynurenines was completely blocked by 1-methyl-dl-tryptophan (MT), an inhibitor of IDO. Kyn- and 3OHKyn-modified proteins were detected in IFN-γ-treated cells. The induction of IDO by IFN-γ in HLE-B3 cells caused increases in intracellular ROS, cytosolic cytochrome c and caspase-3 activity, along with a decrease in protein-free thiol content. These changes were accompanied by apoptosis. At equimolar concentrations, 3OHKyn caused higher levels of apoptosis than the other kynurenines in HLE-B3 cells. MT and a kynurenine 3-hydroxylase inhibitor (Ro61-8048) effectively inhibited IFN-γ-mediated apoptosis in HLE-B3 cells. Our results show that the induction of IDO by IFN-γ is JAK–STAT1 pathway-dependent and that this induction causes 3OHKyn-mediated apoptosis in HLE-B3 cells. These data suggest that IDO-mediated kynurenine formation could play a role in cataract formation related to chronic inflammation.     

Cell-type-specific type I interferon antagonism influences organ tropism of murine coronavirus

Previous studies have demonstrated that mouse hepatitis virus (MHV) hepatotropism is determined largely by postentry events rather than by availability of the viral receptor. In addition, mutation of MHV nonstructural protein 2 (ns2) abrogates the ability of the virus to replicate in the liver and induce hepatitis but does not affect replication in the central nervous system (CNS). Here we show that replication of ns2 mutant viruses is attenuated in bone marrow-derived macrophages (BMM) generated from wild-type (wt) mice but not in L2 fibroblasts, primary astrocytes, or BMM generated from type I interferon receptor-deficient (IFNAR(-/-)) mice. In addition, ns2 mutants are more sensitive than wt virus to pretreatment of BMM, but not L2 fibroblasts or primary astrocytes, with alpha/beta interferon (IFN-α/β). The ns2 mutants induced similar levels of IFN-α/β in wt and IFNAR(-/-) BMM, indicating that ns2 expression has no effect on the induction of IFN but rather that it antagonizes a later step in IFN signaling. Consistent with these in vitro data, the virulence of ns2 mutants increased to near that of wt virus after depletion of macrophages in vivo. These data imply that the ability of MHV to replicate in macrophages is a prerequisite for replication in the liver and induction of hepatitis but not for replication or disease in the CNS, underscoring the importance of IFN signaling in macrophages in vivo for protection of the host from hepatitis. Our results further support the notion that viral tissue tropism is determined in part by postentry events, including the early type I interferon response.     

STAT1-independent control of a neurotropic measles virus challenge in primary neurons and infected mice

Neurons are chiefly nonrenewable; thus, cytolytic immune strategies to clear or control neurotropic viral infections could have lasting neurologic consequences. IFN-γ is a potent antiviral cytokine that is critical for noncytolytic clearance of multiple neurotropic viral infections, including measles virus (MV); however, the downstream pathways through which IFN-γ functions in neurons have not been defined. Unlike most cell types studied to date in which IFN-γ affects gene expression via rapid and robust activation of STAT1, basal STAT1 levels in primary hippocampal neurons are constitutively low, resulting in attenuated STAT1 activation and consequently slower kinetics of IFN-γ-driven STAT1-dependent gene expression. Given this altered expression and activation of STAT1 in neurons, we sought to determine whether STAT1 was required for IFN-γ-mediated protection from infection in neurons. To do so, we evaluated the consequences of MV challenge of STAT1-deficient mice and primary hippocampal neurons explanted from these mice. Surprisingly, the absence of STAT1 did not restrict the ability of IFN-γ to control viral infection either in vivo or ex vivo. Moreover, the canonical IFN-γ-triggered STAT1 gene expression profile was not induced in STAT1-deficient neurons, suggesting that IFN-γ regulates neuronal STAT1-independent pathways to control viral replication.     

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.     

TWEAK signals through JAK–STAT to induce tumor cell apoptosis

The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK–STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK–STAT dependent, as JAK inhibitors block IFNγ?dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK–STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNβ, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNβ can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK–STAT pathway to induce IFNβ, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.