Robert H. Arch

Reactive oxygen species are downstream products of TRAF-mediated signal transduction

Members of the TNFR (tumor necrosis factor receptor) superfamily are involved in regulating activation and differentiation of cells as well as cell survival and programmed cell death/apoptosis. Multimerization of TNFRs can lead to recruitment of TRAFs (TNFR-associated factors) by the receptors resulting in activation of kinases and transcription factors, such as c-Jun N-terminal kinase and nuclear factor κB (NF-κB). Signal transduction triggered by TNF-α also induces an increase in intracellular reactive oxygen species (ROS). ROS have been suggested to play a role in NF-κB activation, which is thought to promote cell survival. However, oxidation of proteins and lipids by ROS can also result in apoptosis. The processes generating intracellular ROS and the mechanism(s) regulating the cellular redox status have not been fully elucidated. We investigated whether TRAFs play a role in controlling intracellular ROS levels. Our results indicate that recruitment of TRAFs to the plasma membrane of human embryonic kidney (HEK) 293 cells is crucial for activation of signaling pathways, which regulate ROS production in mitochondria. TRAF-mediated changes in ROS levels enhanced NF-κB activation but were not dependent on NF-κB-inducing kinase. Consistent with its anti-apoptotic function, Bcl-xL interfered with TRAF-mediated ROS generation but not NF-κB activation. Taken together, our results suggest a novel role of TRAFs in signal transduction pathways triggered by TNFR-related proteins, which balance cell survival and apoptosis by regulating the electron transport in mitochondria.

A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice

In lower organisms, such as Caenorhabditis elegans and Drosophila, many genes identified as key regulators of aging are involved in either detoxification of reactive oxygen species or the cellular response to oxidatively-damaged macromolecules. Transgenic mice have been generated to study these genes in mammalian aging, but have not in general exhibited the expected lifespan extension or beneficial behavioral effects, possibly reflecting compensatory changes during development. We administered a small-molecule synthetic enzyme superoxide dismutase (SOD) mimetic to wild-type (i.e. non-transgenic, non-senescence accelerated) mice starting at middle age. Chronic treatment not only reduced age-associated oxidative stress and mitochondrial radical production, but significantly extended lifespan. Treated mice also exhibited improved performance on the Morris water maze learning and memory task. This is to our knowledge the first demonstration that an administered antioxidant with mitochondrial activity and nervous system penetration not only increases lifespan, but rescues age-related cognitive impairment in mammals. SOD mimetics with such characteristics may provide unique complements to genetic strategies to study the contribution of oxidative processes to nervous system aging.

Glucocorticoid-Induced TNF Receptor Functions as a Costimulatory Receptor That Promotes Survival in Early Phases of T Cell Activation

Glucocorticoid-induced TNFR (GITR) is a member of the TNFR family that can inhibit the suppressive function of regulatory T cells and promote the survival and activation of T cells. However, little is known about the molecular mechanisms regulating T cell survival and activation downstream of GITR. To gain further insight into the cellular events and signaling pathways triggered by GITR, survival, proliferation, and cytokine production as well as activation of MAPKs and NF-κB were monitored after cross-linking of the receptor on naive and activated T cells. GITR cross-linking provided costimulation of naive and activated T cells and resulted in activation of MAPKs and NF-κB. Although GITR-induced signaling pathways augmented the survival of naive T cells, they were not sufficient to inhibit activation-induced cell death triggered by CD3 cross-linking of activated T cells. Differences in the contributions of GITR to cell survival between naive and activated T cells suggest that the receptor triggers specific pathways depending on the activation state of the T cell.

Glucocorticoid-Induced TNF Receptor, a Costimulatory Receptor on Naive and Activated T Cells, Uses TNF Receptor-Associated Factor 2 in a Novel Fashion as an Inhibitor of NF-κB Activation

Glucocorticoid-induced TNFR (GITR) has been implicated as an essential regulator of immune responses to self tissues and pathogens. We have recently shown that GITR-induced cellular events promote survival of naive T cells, but are insufficient to protect against activation-induced cell death. However, the molecular mechanisms of GITR-induced signal transduction that influence physiologic and pathologic immune responses are not well understood. TNFR-associated factors (TRAFs) are pivotal adapter proteins involved in signal transduction pathways of TNFR-related proteins. Yeast two-hybrid assays and studies in HEK293 cells and primary lymphocytes indicated interactions between TRAF2 and GITR mediated by acidic residues in the cytoplasmic domain of the receptor. GITR-induced activation of NF-κB is blocked by A20, an NF-κB-inducible protein that interacts with TRAFs and functions in a negative feedback mechanism downstream of other TNFRs. Interestingly, in contrast with its effects on signaling triggered by other TNFRs, our functional studies revealed that TRAF2 plays a novel inhibitory role in GITR-triggered NF-κB activation.

Tumor necrosis factor receptor (TNFR)-associated factor 5 is a critical intermediate of costimulatory signaling pathways triggered by glucocorticoid-induced TNFR in T cells.

Tumor necrosis factor receptor (TNFR) family members such as glucocorticoid-induced TNFR (GITR) control T cell activation, differentiation, and effector functions. Importantly, GITR functions as a pivotal regulator of physiologic and pathologic immune responses by abrogating the suppressive effects of T regulatory cells and costimulating T effector cells. However, the molecular mechanisms underlying GITR-triggered signal transduction pathways remain unclear. Interestingly, GITR-induced stimulation of TNFR-associated factor (TRAF) 5-deficient T cells resulted in decreased activation of nuclear factor κB as well as the mitogen-activated protein kinases p38 and extracellular signal-regulated protein kinase, whereas activation of c-Jun N-terminal kinase was less affected. Consistent with impaired signaling, costimulatory effects of GITR were diminished in TRAF5-/- T cells. In sum, our studies indicate that TRAF5 plays a crucial role in GITR-induced signaling pathways that augment T cell activation.

A Chemotactic Peptide from Laminin α5 Functions as a Regulator of Inflammatory Immune Responses via TNFα-mediated Signaling

Tissue injury triggers inflammatory responses that may result in release of degradation products or exposure of cryptic domains of extracellular matrix components. Previously, we have shown that a cryptic peptide (AQARSAASKVKVSMKF) in the α-chain of laminin-10 (α5β1γ1), a prominent basement membrane component, is chemotactic for both neutrophils (PMNs) and macrophages (Mφs) and induces matrix metalloproteinase-9 (MMP-9) production. To determine whether AQARSAASKVKVSMKF has additional effects on inflammatory cells, we performed microarray analysis of RNA from RAW264.7 Mφs stimulated with AQARSAASKVKVSMKF. Several cytokines and cytokine receptors were increased >3-fold in response to the laminin α5 peptide. Among these were TNF-α and one of its receptors, the p75 TNFR (TNFR-II), increasing 3.5- and 5.7-fold, respectively. However, the peptide had no effect on p55 TNFR (TNFR-I) expression. Corroborating the microarray data, the protein levels of TNF-α and TNFR-II were increased following stimulation of RAW264.7 cells with AQARSAASKVKVSMKF. In addition, we determined that the production of TNF-α and TNFR-II in response to AQARSAASKVKVSMKF preceded the production of MMP-9. Furthermore, using primary Mφs from mice deficient in TNFR-I, TNFR-II, or both TNF-α receptors (TNFRs), we determined that AQARSAASKVKVSMKF induces MMP-9 expression by Mφs through a pathway triggered by TNFR-II. However, TNF-α signaling is not required for AQARSAASKVKVSMKF-induced PMN release of MMP-9 or PMN emigration. These data suggest that interactions of inflammatory cells with basement membrane components may orchestrate immune responses by inducing expression of cytokines, recruitment of inflammatory cells, and release of proteinases.

Expression of CD30 and Ox40 on T lymphocyte subsets is controlled by distinct regulatory mechanisms

Members of the TNF receptor (TNFR) superfamily are cell‐surface proteins that can be found on most cell types including lymphocytes. Although some TNFR‐related molecules are constitutively expressed, others, such as CD30 and Ox40, are induced upon activation of lymphocytes. CD30 and Ox40 are predominantly expressed on activated T helper (Th)2 cells. Both receptors can activate c‐Jun N‐terminal kinase (JNK) and nuclear factor‐κB (NF‐κB) and have been suggested to play costimulatory roles in lymphocyte activation. To gain further insight into events triggered by both TNFR‐related molecules, a detailed analysis of their expression patterns has been prformed. We found that CD30 and Ox40 were coexpressed on Th2 cells. However, in contrast to CD30, Ox40 was also expressed on Th1 cells. Although expression of both receptors is augmented by interleukin‐4, only CD30 expression is dependent on signal transducer and activator of transcription (STAT)‐6‐mediated signaling. Differences in the regulatory pathways controlling expression of CD30 and Ox40 suggest distinct, functional effects triggered by the two TNFR‐related molecules during lymphocyte activation.

Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: Regulation at the interface between regulatory T cells and immune effector cells

Mammals and other higher vertebrates have developed an adaptive immune system to defy effectively countless pathogens and cancerous cells encountered during the lifetime of an individual. B and T lymphocytes, which are essential in orchestrating adaptive immune responses, express surface receptors specific for foreign and abnormal self-antigens. Genesis of this antigen receptor repertoire poses significant risks for autoimmunity caused by self-reactive lymphocytes. Therefore, organisms with adaptive immune systems have evolved central and peripheral tolerance mechanisms. In peripheral tissues, regulatory T (Treg) cells function in a dominant, cell-extrinsic manner to limit inflammatory responses and autoimmune disorders. To tap the potential clinical utility of these specialized lymphocytes, advances have been made in understanding how Treg cell-mediated suppression of immune effector cells is achieved and regulated. Importantly, signaling induced by a recently identified member of the tumor necrosis factor receptor (TNFR) family, termed glucocorticoid-induced TNFR family-related gene (GITR), abrogates the suppressive effects of Treg cells. GITR plays a pivotal role in controlling T cell-mediated responses in experimental models of organ-specific autoimmunity, chronic infection, and anti-tumor immunity. These findings highlight the importance of elucidating the molecular underpinnings of GITR-induced signaling. We propose that GITR employs adapter proteins, including TNFR-associated factors (TRAFs), to regulate diverse signaling pathways and transcriptional programs that control the interplay between Treg cells and immune effector cells.

Function of tumor necrosis factor receptor family members on regulatory T-cells.

Effector cells play a crucial role in the immune system of higher vertebrates in eliminating invading pathogens and transformed cells that could cause disease or death of the individual. To be effective and specific, immune responses have to distinguish between self and nonself. Mechanisms of central and peripheral tolerance have evolved to control effector cells that could respond to autoantigens. Regulatory T-cells (Treg cells) are critical modulators of effector cells in the periphery that suppress autoreactive T-cells but are also involved in modulating immune responses against invading pathogens. Identification of surface markers of Treg cells and the development of in vitro systems to study the suppressive function of Treg cells have revealed distinct phenotypic and functional subsets of Treg cells. Several tumor necrosis factor receptor (TNFR) family members have been shown to play a role in the development, homeostasis, and suppressor function of Treg cells. Recent findings suggest that TNFRs and other cell-surface molecules of Treg cells can be explored for therapeutic strategies targeting autoimmune disorders, cancer, and immune responses against pathogens.

Characterization of Binding Mode of Action of a Blocking Anti-Platelet-Derived Growth Factor (PDGF)-B Monoclonal Antibody, MOR8457, Reveals Conformational Flexibility and Avidity Needed for PDGF-BB To Bind PDGF Receptor-beta.

Platelet derived growth factor-BB (PDGF-BB) is an important mitogen and cell survival factor during development. PDGF-BB binds PDGF receptor-β (PDGFRβ) to trigger receptor dimerization and tyrosine kinase activation. We present the pharmacological and biophysical characterization of a blocking PDGF-BB monoclonal antibody, MOR8457, and contrast this to PDGFRβ. MOR8457 binds to PDGF-BB with high affinity and selectivity, and prevents PDGF-BB induced cell proliferation competitively and with high potency. The structural characterization of the MOR8457-PDGF-BB complex indicates that MOR8457 binds with a 2:1 stoichiometry, but that binding of a single MOR8457 moiety is sufficient to prevent binding to PDGFRβ. Comparison of the MOR8457-PDGF-BB structure with that of the PDGFRβ-PDGF-BB complex suggested the potential reason for this was a substantial bending and twisting of PDGF-BB in the MOR8457 structure, relative to the structures of PDGF-BB alone, bound to a PDGF-BB aptamer or PDGFRβ, which makes it nonpermissive for PDGFRβ binding. These biochemical and structural data offer insights into the permissive structure of PDGF-BB needed for agonism as well as strategies for developing specific PDGF ligand antagonists.