Bruce D. Freedman

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter‐organelle crosstalk

We have investigated the mechanism of mitochondrial–nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m‐chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψm) and electron transport‐coupled ATP synthesis, which was also accompanied by an elevated steady‐state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor‐1 (RyR‐1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+‐responsive factors calineurin, calcineurin‐dependent NFATc (cytosolic counterpart of activated T‐cell‐specific nuclear factor) and c‐Jun N‐terminal kinase (JNK)‐dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF‐κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady‐state [Ca2+]i were normalized in genetically reverted cells which contain near‐normal mtDNA levels. We propose that the mitochondria‐to‐nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

BACKGROUND The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. RESULTS By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. CONCLUSIONS These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Macrophage activation through CCR5‐ and CXCR4‐mediated gp120‐elicited signaling pathways

Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV‐1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV‐1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell‐surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV‐1 entry, CCR5 and CXCR4 are G protein‐coupled receptors that can activate multiple intracellular signaling pathways. HIV‐1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl–, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion‐related tyrosine kinase Pyk2, mitogen‐activated protein kinases (MAPK), and phosphoinositol‐3 kinase. Activation of the MAPK leads to gp120‐induced expression of chemokines such as monocyte chemoattractant protein‐1 and macrophage‐inflammatory protein‐1β and the proinflammatory cytokine tumor necrosis factor α. These responses establish a complex cytokine network, which may enhance or suppress HIV‐1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.

F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

Actomyosin dynamics and T cell receptor signaling are tightly coupled to ensure proper dynamics and function of signaling microclusters within the immunological synapse.

Differential requirement for SLP-76 domains in T cell development and function.

The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.

Ezrin and Moesin Function Together to Promote T Cell Activation

The highly homologous proteins ezrin, radixin, and moesin link proteins to the actin cytoskeleton. The two family members expressed in T cells, ezrin and moesin, are implicated in promoting T cell activation and polarity. To elucidate the contributions of ezrin and moesin, we conducted a systematic analysis of their function during T cell activation. In response to TCR engagement, ezrin and moesin were phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localized to the distal pole complex (DPC). However, ezrin exhibited unique behaviors, including tyrosine phosphorylation and transient localization to the immunological synapse before movement to the DPC. To ask whether these differences reflect unique requirements for ezrin vs moesin in T cell signaling, we generated mice with conditional deletion of ezrin in mature T cells. Ezrin−/− T cells exhibited normal immunological synapse organization based upon localization of protein kinase C-θ, talin, and phospho-ZAP70. DPC localization of CD43 and RhoGDP dissociation inhibitor, as well as the novel DPC protein Src homology region 2 domain-containing phosphatase-1, was also unaffected. However, recruitment of three novel DPC proteins, ezrin binding protein of 50 kDa, Csk binding protein, and the p85 subunit of PI3K was partially perturbed. Biochemical analysis of ezrin−/− T cells or T cells suppressed for moesin using small interfering RNA showed intact early TCR signaling, but diminished levels of IL-2. The defects in IL-2 production were more pronounced in T cells deficient for both ezrin and moesin. These cells also exhibited diminished phospholipase C-γ1 phosphorylation and calcium flux. We conclude that despite their unique movement and phosphorylation patterns, ezrin and moesin function together to promote T cell activation.

WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse

ABSTRACT T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

HIV‐1 gp120‐induced TNF‐α production by primary human macrophages is mediated by phosphatidylinositol‐3 (PI‐3) kinase and mitogen‐activated protein (MAP) kinase pathways

Human immunodeficiency virus type 1 (HIV‐1) infection is initiated by binding of the viral envelope glycoprotein gp120 to CD4 followed by a chemokine receptor, but these interactions may also take place independently from infection. gp120 stimulation of primary human macrophages is known to trigger production of cytokines implicated in pathogenesis, particularly tumor necrosis factor α (TNF‐α), but the mechanisms have not been determined. We sought to define the pathways responsible for TNF‐α secretion by monocyte‐derived macrophages (MDM) following HIV‐1 gp120 stimulation. MDM exposure to recombinant macrophage‐tropic (R5) gp120 led to dose‐ and donor‐dependent release of TNF‐α, which was cyclohexamide‐sensitive and associated with up‐regulated message. Pretreatment with specific inhibitors of the mitogen‐activated protein kinases (MAPK) extracellular signal‐regulated kinase 1/2 (ERK‐1/2; PD98059, U0126) and p38 (SB202190, PD169316) inhibited the secretion of TNF‐α. gp120‐elicited TNF‐α production was also blocked by phosphatidylinositol‐3 kinase (PI‐3K) inhibitors (wortmannin, LY294002). Moreover, PI‐3K inhibition ablated gp120‐induced phosphorylation of p38 and ERK‐1/2. The response was inhibited by a CC chemokine receptor 5 (CCR5)‐specific antagonist, indicating that CCR5 was in large part responsible. These results indicate that gp120‐elicited TNF‐α production by macrophages involves chemokine receptor‐mediated PI‐3K and MAPK activation, that PI‐3K is an upstream regulator of MAPK in this pathway, and that p38 and ERK‐1/2 independently regulate TNF‐α production. These gp120‐triggered signaling pathways may be responsible for inappropriate production of proinflammatory cytokines by macrophages, which are believed to play a role in immunopathogenesis and in neurological sequelae of AIDS.

Initiation and termination of NF-κB signaling by the intracellular protozoan parasite Toxoplasma gondii

Signaling via the NF-κB cascade is critical for innate recognition of microbial products and immunity to infection. As a consequence, this pathway represents a strong selective pressure on infectious agents and many parasitic, bacterial and viral pathogens have evolved ways to subvert NF-κB signaling to promote their survival. Although the mechanisms utilized by microorganisms to modulate NF-κB signaling are diverse, a common theme is targeting of the steps that lead to IκB degradation, a major regulatory checkpoint of this pathway. The data presented here demonstrate that infection of mammalian cells with Toxoplasma gondii results in the activation of IKK and degradation of IκB. However, despite initiation of these hallmarks of NF-κB signaling, neither nuclear accumulation of NF-κB nor NF-κB-driven gene expression is observed in infected cells. However, this defect was not due to a parasite-mediated block in nuclear import, as general nuclear import and constitutive nuclear-cytoplasmic shuttling of NF-κB remain intact in infected cells. Rather, in T. gondii-infected cells, the termination of NF-κB signaling is associated with reduced phosphorylation of p65/RelA, an event involved in the ability of NF-κB to translocate to the nucleus and bind DNA. Thus, these studies demonstrate for the first time that the phosphorylation of p65/RelA represents an event downstream of IκB degradation that may be targeted by pathogens to subvert NF-κB signaling.

Chemokine receptor utilization and macrophage signaling by human immunodeficiency virus type 1 gp120: Implications for neuropathogenesis

Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 for entry. Macrophages and microglia (M/M) are the principal productively infected brain cells in HIV encephalopathy (HIVE), and neuronal injury is believed to result both from direct effects of viral proteins and indirect effects mediated by macrophage activation and secretion of neurotoxic products. In vitro, direct injury by the viral envelope glycoprotein gp120 can be mediated by neuronal CXCR4, but most HIV-1 isolates from the central nervous system (CNS) studied to date use CCR5 (R5 strains) rather than CXCR4 (X4 or R5X4 strains). Additionally, it remains unknown how HIV induces M/M activation and neurotoxin secretion. To address these issues, the authors analyzed a CNS-derived primary isolate, TYBE, and showed that it uses CXCR4 only and replicates efficiently in macrophages through CXCR4-mediated entry. The authors also showed that both R5 and X4 gp120 activate intracellular signals in macrophages through CCR5 and CXCR4, including calcium elevations; K+, Cl−and nonselective cation channel activation; phosphorylation of the nonreceptor tyrosine kinase Pyk2; and activation of p38 and SAPK/JNK mitogen-activated protein kinases (MAPKs). Finally, the authors showed that macrophages stimulated with gp120 produce soluble factors through MAPK-dependent pathways, including β-chemokines implicated in HIVE pathogenesis. The findings emphasize that both X4 and R5 HIV-1 isolates may contribute to HIVE pathogenesis, and that gp120/chemokine receptor interactions in M/M trigger specific signal transduction pathways that may affect M/M function and provide a mechanism underlying CNS injury.