Bruton's tyrosine kinase together with PI 3-kinase are part of Toll-like receptor 2 multiprotein complex and mediate LTA induced Toll-like receptor 2 responses in macrophages

Lipoteichoic acid (LTA) of Gram-positive bacteria initiates innate immune responses via Toll-like receptor-2 (TLR2), resulting in the activation of intracellular signaling and production of inflammatory cytokines in macrophages. Although Bruton's tyrosine kinase (Btk) is biologically important molecule implicated in immune regulation and recently in TLR signaling its importance for LTA-TLR2 mediated responses has not been evaluated. In this study, we detected Btk in the LTA signaling complex with TLR2 and PI 3-kinase (PI3K). The constitutive interaction of these proteins was mediated via PI3K Src homology (SH3) -domain. Both Btk and PI3K were activated by LTA stimulation and the LTA induced cytokine expression was differentially modulated by these kinases. LTA induced the activation of nuclear factor kappaB (NFkappaB), however, only Btk inhibition affected the LTA induced Ser536 phosphorylation and DNA-binding of NFkappaB. In conclusion, our results demonstrate that Btk and PI3K occupy important roles in TLR2-induced activation of macrophages, resulting in selective regulation of cytokines.     

Phosphoinositide turnover in Toll-like receptor signaling and trafficking

Lipid components in biological membranes are essential for maintaining cellular function. Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PI), regulate many critical cell processes involving membrane signaling, trafficking, and reorganization. Multiple metabolic pathways including phosphoinositide kinases and phosphatases and phospholipases tightly control spatio-temporal concentration of membrane phosphoinositides. Metabolizing enzymes responsible for PI 4,5-bisphosphate (PI(4,5)P2) production or degradation play a regulatory role in Toll-like receptor (TLR) signaling and trafficking. These enzymes include PI 4-phosphate 5-kinase, phosphatase and tensin homolog, PI 3-kinase, and phospholipase C. PI(4,5)P2 mediates the interaction with target cytosolic proteins to induce their membrane translocation, regulate vesicular trafficking, and serve as a precursor for other signaling lipids. TLR activation is important for the innate immune response and is implicated in diverse pathophysiological disorders. TLR signaling is controlled by specific interactions with distinct signaling and sorting adaptors. Importantly, TLR signaling machinery is differentially formed depending on a specific membrane compartment during signaling cascades. Although detailed mechanisms remain to be fully clarified, phosphoinositide metabolism is promising for a better understanding of such spatio-temporal regulation of TLR signaling and trafficking. [BMB Reports 2014; 47(7): 361-368]     

The receptor for urokinase regulates TLR2 mediated inflammatory responses in neutrophils

The urokinase-type plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol (GPI) anchored membrane protein, regulates urokinase (uPA) protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR) stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT) neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role.     

Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2

Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen that causes significant morbidity and mortality in immunocompromised people. An understanding of how CMV induces and circumvents host immunity is of critical importance in efforts to design effective therapeutics. It was recently discovered that mere cell contact by CMV particles leads to profound modulation of cellular gene expression, including induction of inflammatory cytokines and interferon-stimulated genes characteristic of innate immune detection. These findings suggest that a membrane receptor recognizes a CMV envelope protein(s), leading to innate immune activation. Here, we show that the pattern recognition receptors Toll-like receptor 2 (TLR2) and CD14 recognize CMV virions and trigger inflammatory cytokine production. Induction of inflammatory cytokines is mediated via TLR2-dependent activation of NF-kappa B. Since many of the pathological processes associated with CMV disease are facilitated or directly mediated by inflammatory cytokines, identification of the host membrane detection machinery may ultimately lead to improved therapeutics.     

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.     

Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling

MUC1 is a membrane-tethered mucin-like glycoprotein expressed on the surface of various mucosal epithelial cells as well as hematopoietic cells. Recently, we showed that MUC1 suppresses flagellin-induced Toll-like receptor (TLR) 5 signaling both in vivo and in vitro through cross talk with TLR5. In this study, we determined whether phosphoinositide 3-kinase (PI3K), a negative regulator of TLR5 signaling, is involved in the cross talk between MUC1 and TLR5 using various genetically modified epithelial cell lines. Our results showed 1) activation of MUC1 induced recruitment of the PI3K regulatory subunit p85 to the MUC1 cytoplasmic tail (CT) as well as Akt phosphorylation, 2) MUC1-induced Akt phosphorylation required the presence of Tyr(20) within the PI3K binding motif of the MUC1 CT, and 3) mutation of Tyr(20) or pharmacological inhibition of PI3K activation failed to block MUC1-induced suppression of TLR5 signaling. We conclude that whereas PI3K is downstream of MUC1 activation and negatively regulates TLR5 signaling, it is not responsible for MUC1-induced suppression of TLR5 signaling.     

Phosphoinositide 3-kinase, Src, and Akt modulate acute ventilation-induced vascular permeability increases in mouse lungs

To determine the role of phosphoinositide 3-OH kinase (PI3K) pathways in the acute vascular permeability increase associated with ventilator-induced lung injury, we ventilated isolated perfused lungs and intact C57BL/6 mice with low and high peak inflation pressures (PIP). In isolated lungs, filtration coefficients (K(f)) increased significantly after ventilation at 30 cmH(2)O (high PIP) for successive periods of 15, 30 (4.1-fold), and 50 (5.4-fold) min. Pretreatment with 50 microM of the PI3K inhibitor, LY-294002, or 20 microM PP2, a Src kinase inhibitor, significantly attenuated the increase in K(f), whereas 10 microM Akt inhibitor IV significantly augmented the increased K(f). There were no significant differences in K(f) or lung wet-to-dry weight (W/D) ratios between groups ventilated with 9 cmH(2)O PIP (low PIP), with or without inhibitor treatment. Total lung beta-catenin was unchanged in any low PIP isolated lung group, but Akt inhibition during high PIP ventilation significantly decreased total beta-catenin by 86%. Ventilation of intact mice with 55 cmH(2)O PIP for up to 60 min also increased lung vascular permeability, indicated by increases in lung lavage albumin concentration and lung W/D ratios. In these lungs, tyrosine phosphorylation of beta-catenin and serine/threonine phosphorylation of Akt, glycogen synthase kinase 3beta (GSK3beta), and ERK1/2 increased significantly with peak effects at 60 min. Thus mechanical stress activation of PI3K and Src may increase lung vascular permeability through tyrosine phosphorylation, but simultaneous activation of the PI3K-Akt-GSK3beta pathway tends to limit this permeability response, possibly by preserving cellular beta-catenin.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Crosstalk between Phosphoinositide 3-kinase/Akt signaling pathway with DNA damage response and oxidative stress in cancer

The phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway is one of the well-characterized and most important signaling pathways activated in response to DNA damage. This review discusses the most recent discoveries on the involvement of PI3K/Akt signaling pathway in cancer development, as well as stimulation of some important signaling networks involved in the maintenance of cellular homeostasis upon DNA damage, with an exploration of how PI3K/Akt signaling pathway contributes to the regulation of modulators and effectors underlying DNA damage response, the intricate, protein-based signal transduction network, which decides between cell cycle arrest, DNA repair, and apoptosis, the elimination of irreparably damaged cells to maintain homeostasis. The review continues by looking at the interplay between cell cycle checkpoints, checking the repair of damage inflicted to the DNA before entering DNA replication to facilitate DNA synthesis, and PI3K/Akt signaling pathway. We then investigate the challenges the cells overcome to ameliorate damages induced by oxidative activities, for example, the recruitment of many pathways and factors to maintain integrity and hemostasis. Finally, the review provides a discussion of how cells use the PI3K/Akt signaling pathway to regulate the balance between these networks.     

Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor

Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer's role in pIgR-pIgA transcytosis.     

Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation

Melanophores are pigmented cells capable of quick colour changes through coordinated transport of their intracellular pigment granules. We demonstrate the involvement of phosphoinositide 3-kinase (PI3-K) in Xenopus and Labrus aggregation by the use of the PI3-K inhibitor, LY-294002. In Xenopus, wortmannin-insensitive PI3-K was found to be essential for the aggregation, mitogen-activated protein kinase (MAPK) activation and tyrosine phosphorylation of a 280-kDa protein, and for the maintenance of low cyclic adenosine 3′:5′-monophosphate (cAMP) during the aggregated state. Pre-aggregated cells disperse completely to LY-294002 at 50–100 μM, involving a transient elevation in cAMP due to adenylate cyclase (AC) stimulation or to inhibition of cyclic nucleotide phosphodiesterase (PDE). The inactive analogue LY-303511 did not induce dispersion at the same concentrations. PDE4 and/or PDE2 was found to be involved in melanosome aggregation. The similar kinetics of LY-294002 and various PDE inhibitors indicates that the elevation of cAMP might be due to inhibition of PDE. In Labrus melanophores, LY-294002 had a less dramatic effect, probably due to less dependence on PDE in regulation of cAMP levels. In Xenopus aggregation, we suggest that melatonin stimulation of the Mel1c receptor via G_βγ activates PI3-K that, directly or indirectly via MAPK, activates PDE.     

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.     

Multiple roles of Toll-like receptor signaling in atherosclerosis

PURPOSE OF REVIEW: Toll-like receptors are key regulators of both innate and adaptive immune responses. This review outlines the recently emerged multiple roles of Toll-like receptor signaling in atherosclerosis. RECENT FINDINGS: Mice deficient in TLR4, TLR2 and MyD88 all have reduced atherosclerosis which establishes that Toll-like receptor-dependent pathways contribute to disease development. Although it is likely that total "infectious burden" contributes to atherosclerosis progression, endogenous ligands may also initiate and modulate Toll-like receptor signaling pathways. CD36, with established roles in recognition of endogenous ligands and atherosclerotic disease, facilitates TLR2 signaling and might therefore represent a bridge between endogenous lipid ligands and Toll-like receptor pathways. Furthermore, lipoprotein oxidation generates ligands that activate Toll-like receptor pathways. At the same time, Toll-like receptor activation may be inhibited by accumulating oxidized phospholipids, which could result in reduced dendritic cell maturation and impaired immunological priming. SUMMARY: Activation of Toll-like receptor signaling can promote atherosclerosis by multiple mechanisms, while some beneficial Toll-like receptor pathways may be inhibited by lipid accumulation. Due to their central role in the disease process, Toll-like receptor signaling pathways represent a target of immunomodulatory therapy with the goal of tipping the balance from excessive chronic inflammation towards resolution of inflammation, while not compromising host defense or atheroprotective immune functions.     

Phosphoinositide 3-kinase in disease: timing, location, and scaffolding

When PI3Ks are deregulated by aberrant surface receptors or modulators, accumulation of PtdIns(3,4,5)P3 leads to increased cell growth, proliferation and contact-independent survival. The PI3K/PKB/TOR axis controls protein synthesis and growth, while PtdIns(3,4,5)P3-mediated activation of Rho GTPases directs cell motility. PI3K activity has been linked to the formation of tumors, metastasis, chronic inflammation, allergy and cardiovascular disease. Although increased PtdIns(3,4,5)P3 is a well-established cause of disease, it is seldom known which PI3K isoform is implied. Recent work has demonstrated that PI3Kgamma contributes to the control of cAMP levels in the cardiac system, where the protein acts as a scaffold, but not as a lipid kinase.     

Phosphoinositide 3-kinase controls early and late events in mammalian cell division

Phosphoinositide 3-kinase (PI3K) plays a crucial role in triggering cell division. To initiate this process, PI3K induces two distinct routes, of which one promotes cell growth and the other regulates cyclin-dependent kinases. Fine-tuned PI3K regulation is also required for later cell cycle phases. Here, we review the multiple points at which PI3K controls cell division and discuss its impact on human cancer.