IFN-λ3 inhibits HIV infection of macrophages through the JAK-STAT pathway

Background

Interferon lambda 3 (IFN-λ3) is a newly identified cytokine with antiviral activity, and its single nucleotide polymorphisms are strongly associated with the treatment effectiveness and development of chronic hepatitis C virus infection. We thus examined the potential of IFN-λ3 to inhibit HIV replication and the possible mechanisms of the anti-HIV action by IFN-λ3 in human macrophages.

Principal Findings

Under different conditions (before, during, and after HIV infection), IFN-λ3 significantly inhibited viral replication in macrophages, which was associated with the induction of multiple antiviral cellular factors (ISG56, MxA, OAS-1, A3G/F and tetherin) and IFN regulatory factors (IRF-1, 3, 5, 7 and 9). This anti-HIV action of IFN-λ3 could be compromised by the JAK-STAT inhibitor. In addition, IFN-λ3 treatment of macrophages induced the expression of toll-like receptor 3 (TLR3) and two key adaptors (MyD88 and TRIF) in type I IFN pathway activation. However, HIV infection compromised IFN-λ3-mediated induction of the key elements in JAK-STAT signaling pathway.

Conclusions

These data indicate that IFN-λ3 exerts its anti-HIV function by activating JAK-STAT pathway-mediated innate immunity in macrophages. Future in vivo studies are necessary in order to explore the potential for developing IFN-λ3-based therapy for HIV disease.     

Regulation of low density lipoprotein receptor mRNA levels by estradiol 17β and chorionic gonadotropin in human placenta

Inhibition of synthesis of estradiol 17 by the addition of inhibitors of aromatase, a key enzyme in the biosynthesis of estradiol 17, or addition of tamoxifen - an estrogen receptor antagonist, to human placental minces resulted in an increase in the level of LDL-receptor mRNA. This increase could be blocked by the simultaneous addition of estradiol 17. A concentration dependent effect of estradiol 17 on the level of LDL-receptor mRNA was seen both in first trimester, and term placenta. Addition of human chorionic gonadotropin (hCG) to term placental minces also increased the LDL-receptor mRNA levels. When hCG and cycloheximide were added together, an additive effect was observed. The results obtained in this study suggest that the LDL-receptor mRNA levels in the human placenta are regulated by estradiol 17 and hCG.     

Regulation of the TGF-β pathway by deubiquitinases in cancer

The transforming growth factor-β (TGF-β) pathway regulates diverse cellular processes. It signals via serine/threonine kinase receptors and intracellular Smad and non-Smad effector proteins. In cancer cells, aberrant TGF-β signalling can lead to loss of growth inhibition and an increase in invasion, epithelial-to-mesenchymal transition (EMT) and metastasis. Therapeutic targeting of the pro-oncogenic TGF-β responses is currently being explored as a potential therapy against certain invasive and metastatic cancer types. The ubiquitin post-translational regulation system is emerging as a key regulatory mechanism for the control of TGF-β pathway components. In this review, we focus on the role of deubiquitinases (DUBs), which counteract the activity of E3 ubiquitin ligases. We will discuss the mechanisms by which specific DUBs control Smad and non-Smad TGF-β signalling routes, and how perturbation of the expression and function of DUBs contributes to misregulation of TGF-β signalling in cancer.     

Distribution of the α2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues

Summary The hepatic ₁-macroglobulin receptor (₂MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses ₂-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of ₂MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed ₂MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocytederived cells displayed marked ₂MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of ₂MR/LRP in certain cell types of most organs suggests two main routes for ₂MR/LRP ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.     

Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Aβ uptake and degradation by astrocytes

Accumulation of the amyloid β (Aβ) peptide within the brain is hypothesized to be one of the main causes underlying the pathogenic events that occur in Alzheimer disease (AD). Consequently, identifying pathways by which Aβ is cleared from the brain is crucial for better understanding of the disease pathogenesis and developing novel therapeutics. Cellular uptake and degradation by glial cells is one means by which Aβ may be cleared from the brain. In the current study, we demonstrate that modulating levels of the low-density lipoprotein receptor (LDLR), a cell surface receptor that regulates the amount of apolipoprotein E (apoE) in the brain, altered both the uptake and degradation of Aβ by astrocytes. Deletion of LDLR caused a decrease in Aβ uptake, whereas increasing LDLR levels significantly enhanced both the uptake and clearance of Aβ. Increasing LDLR levels also enhanced the cellular degradation of Aβ and facilitated the vesicular transport of Aβ to lysosomes. Despite the fact that LDLR regulated the uptake of apoE by astrocytes, we found that the effect of LDLR on Aβ uptake and clearance occurred in the absence of apoE. Finally, we provide evidence that Aβ can directly bind to LDLR, suggesting that an interaction between LDLR and Aβ could be responsible for LDLR-mediated Aβ uptake. Therefore, these results identify LDLR as a receptor that mediates Aβ uptake and clearance by astrocytes, and provide evidence that increasing glial LDLR levels may promote Aβ degradation within the brain.     

Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-β activation

Transforming growth factor-β (TGF-β) is a critical regulator of bone development and remodeling. TGF-β must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-β activation and TSP1 control of TGF-β activation is critical for regulation of TGF-β activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-β inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-β activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-β. Cultured MSCs express TSP1 and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. TSP1 and active TGF-β block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-β, since a peptide which blocks TSP1 TGF-β activation reduced TGF-β activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-β activity is a critical determinant of osteoblast differentiation.     

INHBA gene silencing inhibits gastric cancer cell migration and invasion by impeding activation of the TGF-β signaling pathway

Gastric cancer (GC) is the fourth largest cancer in the world, with a 5-year survival rate of <30%. Thus, this study intends to investigate the effects of inhibin βA (INHBA) gene silencing on the migration and invasion of GC cells via the transforming growth factor-β (TGF-β) signaling pathway. Initially, this study determined the expression of INHBA and the TGF-β signaling pathway-related genes in GC tissues. After that, to assess the effect of INHBA silencing on GC progression, GC cells were transfected with short hairpin RNAs that targeted INHBA in order to detect the expression of INHBA and the TGF-β signaling pathway-related genes, as well as cell migration, invasion, and proliferation abilities. Finally, a tumor xenograft model in nude mice was constructed to verify the effect that the silencing of INHBA had on tumor growth. Highly expressed INHBA and activated TGF-β signaling pathways were observed in GC tissues. In response to shINHBA-1 and shINHBA-2, the TGF-β signaling pathway was inhibited in GC cells, whereas the GC cell migration, invasion, proliferation, and tumor growth were significantly dampened. On the basis of the observations and findings of this study, INHBA gene silencing inhibited the progression of GC by inactivating the TGF-β signaling pathway, which provides a potential target in the treatment of GC.     

Oxidatively modified high density lipoprotein promotes inflammatory response in human monocytes–macrophages by enhanced production of ROS,TNF-α, MMP-9, and MMP-2

It has been proposed that high-density lipoprotein (HDL) loses its cardioprotective ability through oxidative modifications by reactive oxygen species (ROS) and promote atherogenesis. However, the pro-atherogenic pathways undergone by oxidized HDL remain poorly understood. Since monocytes play a crucial role in atherogenesis, this study was aimed to investigate the influence of both native and oxidized HDL (oxHDL) on monocytes-macrophages functions relevant to atherogenesis. HDL particles were isolated from human blood samples by ultracentrifugation and subjected to in vitro oxidation with CuSO(4). The extent of oxidation was quantitated by measurement of lipid peroxides. Human peripheral blood mononuclear cells were isolated and cultured under standard conditions. Cells were treated with native and oxHDL at varying concentrations for different time intervals and used for several analyses. Intracellular ROS production was assessed based on ROS-mediated DCFH fluorescence of the cells. The release of TNF-α and matrix metalloproteinases (MMPs) was quantitated using ELISA kit and gelatine zymography, respectively. Treatment of cells with oxidized HDL enhanced the production of ROS in a concentration-dependent way, while native HDL had no such effect. Further, the release of TNF-α, MMP-9, and MMP-2 was found to be remarkably higher in cells incubated with oxHDL than that of native HDL. Results demonstrate that oxidative modification of HDL induces pro-inflammatory response and oxidative stress in human monocytes-macrophages.     

Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family

Selenium is an essential micronutrient that function through selenoproteins. Selenium deficiency results in lower concentrations of selenium and selenoproteins. The brain maintains it's selenium better than other tissues under low-selenium conditions. Recently, the selenium-containing protein selenoprotein P (Sepp) has been identified as a possible transporter of selenium. The targeted disruption of the selenoprotein P gene (Sepp1) results in decreased brain selenium concentration and neurological dysfunction, unless selenium intake is excessive However, the effect of selenoprotein P deficiency on the processes of memory formation and synaptic plasticity is unknown. In the present studies Sepp1(-/-) mice and wild type littermate controls (Sepp1(+/+)) fed a high-selenium diet (1 mg Se/kg) were used to characterize activity, motor coordination, and anxiety as well as hippocampus-dependent learning and memory. Normal associative learning, but disrupted spatial learning was observed in Sepp1(-/-) mice. In addition, severe alterations were observed in synaptic transmission, short-term plasticity and long-term potentiation in hippocampus area CA1 synapses of Sepp1(-/-) mice on a 1 mg Se/kg diet and Sepp1(+/+) mice fed a selenium-deficient (0 mg Se/kg) diet. Taken together, these data suggest that selenoprotein P is required for normal synaptic function, either through presence of the protein or delivery of required selenium to the CNS.     

Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

Transforming growth factor-β (TGF-β) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context–dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-β signaling. However, the molecular mechanism(s) involved in the regulation of TGF-β signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-β receptor (TβRI) with TGF-β phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TβRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA–mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-β–induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-β in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TβRI in the membrane, balancing TGF-β signaling via the Smad and JNK pathways.     

Balancing acts: the role of TGF-β in the mucosal immune system

The gastrointestinal mucosal immune system faces unique challenges in dealing not only with fed antigens but also both commensal and pathogenic bacteria. It is tasked with digesting, transporting and using nutritional antigens while protecting the host from pathogenic organisms. As such, mechanisms that mediate effective immunity and immune tolerance are active within the gut environment. To accomplish this, the mucosal immune system has evolved sophisticated mechanisms that safeguard the integrity of the mucosal barrier. Transforming growth factor-β (TGF-β) emerges as a key mediator, balancing the tolerogenic and immunogenic forces at play in the gut. In this review, we discuss the role of TGF-β in the generation and functioning of gut lymphocyte populations. We highlight recent findings, summarize controversies, outline remaining questions and provide our personal perspectives.     

Induction and activation of the p53 pathway: a role for the protein kinase CK2?

Protein kinase CK2 has many established in vitro substrates, but it is only within the past few years that we have begun to ascertain which of these are its real physiological targets, how their phosphorylation may contribute towards regulating normal cell physiology, and how phosphorylation of these proteins might influence the development of diseases such as cancer. One of the well-characterised in vitro substrates for CK2 is the tumour suppressor protein, p53. However, the physiological nature of this interaction has never been fully established. In the present article, we summarise a recent study from our laboratory showing that phosphorylation of p53 at Ser392, the sole site modified by CK2 in vitro, is regulated by a novel mechanism where the stoichiometry of phosphorylation is governed by the rate of turnover of the p53 protein. Such a model is entirely consistent with phosphorylation by a constitutively active protein kinase such as CK2. In contrast to this, while there is overwhelming evidence that CK2 phosphorylates p53 in vitro and is the only detectable Ser392 protein kinase in cell extracts, our data raise uncertainty as to whether this interaction truly reflects events underpinning Ser392 phosphorylation in vivo. We consider the possible role of CK2 in regulating the p53 response in a wider context and suggest key issues that should be addressed experimentally to provide a more cohesive picture of the relationship between this important protein kinase and a pivotal anti-cancer surveillance system in cells.     

Downregulation of cPLA2γ expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway

Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA₂γ in chemotaxis of human breast cancer cells. Inhibition of cPLA₂γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA₂γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA₂γ regulated migration. Knockdown of cPLA₂γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA₂γ also impaired the phosphorylation of integrin β1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA₂γ plays an important role in cancer cell chemotaxis.     

Intercellular variation in signaling through the TGF-β pathway and its relation to cell density and cell cycle phase

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment.     

Substance P inhibits high urea-induced apoptosis through the AKT/GSK-3β pathway in human corneal epithelial cells

To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the relationship between SP and the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. An in vitro model of chronic renal failure (CRF)-related dry eye was used to study HCECs that were treated with high urea concentrations. Cell proliferation was assayed using a cell counting kit-8 test. Besides, cell apoptosis was evaluated by flow cytometry. Furthermore, the effects of SP and the AKT inhibitor perifosine on the urea-treated HCECs were examined using immunofluorescence, quantitative real time polymerase chain reaction (qRT-PCR), and Western blot analysis. SP markedly reduced the number of apoptotic HCECs and decreased the cleaved caspase-3 expression levels while contributing to increased cellular proliferation (P < 0.05). The Western blot analysis and qRT-PCR experiments revealed that SP significantly increased the expression of p-AKT and p-GSK-3β (P < 0.05); additionally, these increases were attenuated after the perifosine inhibition of the AKT signaling pathway (P < 0.05). These in vitro experiments demonstrated that SP may protect against the apoptotic damage of HCECs caused by the high urea condition. The underlying mechanism may be related to the activation of the AKT/GSK-3β signaling pathway.     

Evolution of the TGF-β signaling pathway and its potential role in the ctenophore, Mnemiopsis leidyi

The TGF-β signaling pathway is a metazoan-specific intercellular signaling pathway known to be important in many developmental and cellular processes in a wide variety of animals. We investigated the complexity and possible functions of this pathway in a member of one of the earliest branching metazoan phyla, the ctenophore Mnemiopsis leidyi. A search of the recently sequenced Mnemiopsis genome revealed an inventory of genes encoding ligands and the rest of the components of the TGF-β superfamily signaling pathway. The Mnemiopsis genome contains nine TGF-β ligands, two TGF-β-like family members, two BMP-like family members, and five gene products that were unable to be classified with certainty. We also identified four TGF-β receptors: three Type I and a single Type II receptor. There are five genes encoding Smad proteins (Smad2, Smad4, Smad6, and two Smad1s). While we have identified many of the other components of this pathway, including Tolloid, SMURF, and Nomo, notably absent are SARA and all of the known antagonists belonging to the Chordin, Follistatin, Noggin, and CAN families. This pathway likely evolved early in metazoan evolution as nearly all components of this pathway have yet to be identified in any non-metazoan. The complement of TGF-β signaling pathway components of ctenophores is more similar to that of the sponge, Amphimedon, than to cnidarians, Trichoplax, or bilaterians. The mRNA expression patterns of key genes revealed by in situ hybridization suggests that TGF-β signaling is not involved in ctenophore early axis specification. Four ligands are expressed during gastrulation in ectodermal micromeres along all three body axes, suggesting a role in transducing earlier maternal signals. Later expression patterns and experiments with the TGF-β inhibitor SB432542 suggest roles in pharyngeal morphogenesis and comb row organization.     

Prenatal diagnosis of familial hypercholesterolemia caused by the “Lebanese” mutation at the low density lipoprotein receptor locus

Summary Here, we report the prenatal diagnosis of familial hypercholesterolemia in a Christian-Arab family that carries the Lebanese mutation, a single base substitution that creates a HinfI restriction site, at the low density lipoprotein (LDL) receptor locus. Polymerase chain reaction amplification and restriction analysis were performed on genomic DNA extracted from a chorionic villus sample. In conjunction with karyotype analysis, the fetus was identified as a heterozygous female. Analysis of LDL receptor restriction fragment length polymorphisms confirmed the presence of a male parent marker and revealed that the fetus inherited the mutant gene from its mother. This technique offers a simple and rapid diagnostic tool that can be carried out at an early stage of gestation. It is recommended for families and population groups with molecularly defined LDL receptor mutations.     

Why does inflammation persist: a dominant role for the stromal microenvironment?

Inflammatory responses occur within tissue microenvironments, with functional contributions from both haematopoietic (lymphocytic) cells and stromal cells (including macrophages and fibroblasts). These environments are complex--a compound of many different cell types at different stages of activation and differentiation. Traditional models of inflammatory disease highlight the role of antigen-specific lymphocyte responses and attempt to identify causative agents. However, recent studies have indicated the importance of tissue microenvironments and the innate immune response in perpetuating the inflammatory process. The prominent role of stromal cells in the generation and maintenance of these environments has begun to challenge the primacy of the lymphocyte in regulating chronic inflammatory processes. Sensible enquiries into factors regulating the persistence of inflammatory disease necessitate an understanding of the mechanisms regulating tissue homeostasis and remodelling during inflammation. This article highlights recent insights into the factors regulating dynamic aspects of inflammation, focusing particularly on mononuclear cell infiltrates, their interactions with stromal cells in tissues and the relevance of these interactions to existing and possible future therapies. A key feature of current research has been a growing appreciation that disordered spatial and temporal interactions between infiltrating immune cells and resident stromal cells lie at the heart of disease persistence.     

Reversine increases the plasticity of lineage-committed preadipocytes to osteogenesis by inhibiting adipogenesis through induction of TGF-β pathway in vitro

Reversine has been reported to reverse differentiation of lineage-committed cells to mesenchymal stem cells (MSCs), which then enables them to be differentiated into other various lineages. Both adipocytes and osteoblasts are known to originate from common MSCs, and the balance between adipogenesis and osteogenesis in MSCs is reported to modulate the progression of various human diseases, such as obesity and osteoporosis. However, the role of reversine in modulating the adipogenic potential of lineage-committed preadipocytes and their plasticity to osteogenesis is unclear. Here we report that reversine has an anti-adipogenic function in 3T3-L1 preadipocytes in vitro and alters cell morphology and viability. The transforming growth factor-β (TGF-β) pathway appears to be required for the anti-adipogenic effect of reversine, due to reversine-induced expression of genes involved in TGF-β pathway and reversal of reversine-inhibited adipogenesis by inhibition of TGF-β pathway. We show that treatment with reversine transformed 3T3-L1 preadipocytes into MSC-like cells, as evidenced by the expression of MSCs marker genes. This, in turn, allowed differentiation of lineage-committed 3T3-L1 preadipocytes to osteoblasts under the osteogenic condition in vitro. Collectively, these findings reveal a new function of reversine in reversing lineage-committed preadipocytes to osteogenesis in vitro, and provide new insights into adipose tissue-based regeneration of osteoblasts.