IntroductionReceptor activator of NF-B (nuclear factor-B) ligand (RANKL; also called TRANCE [tumor necrosis factor (TNF) activationinduced cytokine], ODF [osteoclast differentiation factor], and OPGL [osteoprotegerin ligand]) 1-4 is a key factor stimulating the differentiation and activation of osteoclasts and, therefore, is essential for bone remodeling. 5 The binding of RANKL to its receptor RANK leads to recruitment of TNF receptor-associated factor 6 (TRAF6) to the cytoplasmic domain of RANK, thereby resulting in the activation of distinct signaling cascades mediated by mitogen-activated protein (MAP) kinases, including c-Jun N-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinase (ERK). 6 It has been shown that JNK1-activated c-Jun signaling in cooperation with nuclear factor of activated T cells (NFAT) is key to RANKL-regulated osteoclast differentiation. 7 In addition, stimulation of p38 results in the downstream activation of the mi/Mitf (microphthalmia/microphthalmia transcription factor), which controls the expression of the genes encoding tartrate-resistant acid phosphatase (TRAP) and cathepsin K, indicating the importance of p38 signaling cascades. 6 Although our understanding of signaling pathways associated with osteoclast differentiation has advanced considerably recently, the mechanism of RANKL-mediated osteoclastogenesis, specifically the molecular linkage between TRAF6 and MAP kinases, is still unknown.At high concentrations, reactive oxygen species (ROSs) cause oxidative stress that has been viewed as deleterious phenomena, including inflammatory response, apoptosis, or ischemia. 8 Recent studies, however, indicate that small nontoxic amounts of ROS may play a role as a second messenger in the various receptor signaling pathways. [9][10][11][12][13] Osteoclasts have shown to be activated by ROSs to enhance bone resorption, 14 but little attention has been given to the role of ROSs in differentiation of macrophages and monocytes into osteoclasts. Signaling molecules such as JNK and p38, which are known to be essential for osteoclast differentiation, 6,7 are sensitive to activation by ROSs. 11,12 Thus, we hypothesized that signaling cascade(s) can be modulated by ROSs in bone marrow monocyte-macrophage lineage (BMM) cells.Here, we show that RANKL generates ROSs in BMM cells. Examination of the mechanism by which RANKL generates ROSs revealed the involvement of TRAF6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1). These data suggest that RANKL-mediated ROS production serves to regulate RANKL signaling pathways, including JNK and p38 activation required for osteoclast differentiation. Materials and methods Reagents and plasmids2Ј,7Ј-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Leiden, The Netherlands); all other chemicals and FLAG (five NH2-terminally deleted epitope-tagged) epitope (M2) were from Expression constructs encoding FLAG-tagged wild-type RANK, HAtagged TRAF6 (amino acids [aa] 289-530), and...

A novel member of the tumor necrosis factor (TNF) cytokine family, designated TRANCE, was cloned during a search for apoptosis-regulatory genes using a somatic cell genetic approach in T cell hybridomas. The

TRANCE (tumor necrosis factor [TNF]–related activation-induced cytokine) is a new member of the TNF family that is induced upon T cell receptor engagement and activates c-Jun N-terminal kinase (JNK) after interaction with its putative receptor (TRANCE-R). In addition, TRANCE expression is restricted to lymphoid organs and T cells. Here, we show that high levels of TRANCE-R are detected on mature dendritic cells (DCs) but not on freshly isolated B cells, T cells, or macrophages. Signaling by TRANCE-R appears to be dependent on TNF receptor–associated factor 2 (TRAF2), since JNK induction is impaired in cells from transgenic mice overexpressing a dominant negative TRAF2 protein. TRANCE inhibits apoptosis of mouse bone marrow–derived DCs and human monocyte-derived DCs in vitro. The resulting increase in DC survival is accompanied by a proportional increase in DC-mediated T cell proliferation in a mixed leukocyte reaction. TRANCE upregulates Bcl-xL expression, suggesting a potential mechanism for enhanced DC survival. TRANCE does not induce the proliferation of or increase the survival of T or B cells. Therefore, TRANCE is a new DC-restricted survival factor that mediates T cell–DC communication and may provide a tool to selectively enhance DC activity.

Studies of bone and the immune system have converged in recent years under the banner of osteoimmunology. The immune system is spawned in the bone marrow reservoir, and investigators now recognize that important niches also exist there for memory lymphocytes. At the same time, various factors produced during immune responses are capable of profoundly affecting regulation of bone. Mechanisms have evolved to prevent excessive interference by the immune system with bone homeostasis, yet pathologic bone loss is a common sequela associated with autoimmunity and cancer. There are also developmental links, or parallels, between bone and the immune system. Cells that regulate bone turnover share a common precursor with inflammatory immune cells and may restrict themselves anatomically, in part by utilizing a signaling network analogous to lymphocyte costimulation. Efforts are currently under way to further characterize how these two organ systems overlap and to develop therapeutic strategies that benefit from this understanding.

AMP-activated protein kinase (AMPK, also known as SNF1A) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Here, using the Drosophila model system, we generated the first animal model with no AMPK activity and discovered physiological functions of the kinase. Surprisingly, AMPK-null mutants were lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restored many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC; also known as MLC2) was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, our results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures.

TRAF2 is believed to mediate the activation of NF-kappaB and JNK induced by the tumor necrosis factor receptor (TNFR) superfamily, which elicits pleiotropic responses in lymphocytes. We have investigated the physiological roles of TRAF2 in these processes by expressing a lymphocyte-specific dominant negative form of TRAF2, thereby blocking this protein's effector function. We find that the TNFR superfamily signals require TRAF2 for activation of JNK but not NF-kappaB. In addition, we show that TRAF2 induces NF-kappaB-independent antiapoptotic pathways during TNF-induced apoptosis. Inhibition of TRAF2 leads to splenomegaly, lymphadenopathy, and an increased number of B cells. These findings indicate that TRAF2 is involved in the regulation of lymphocyte function and growth in vivo.

Background: Peanut allergy affects 0.6% of the US population. At the present time, allergen avoidance is the only therapeutic option. Animal models of food-induced anaphylaxis would facilitate attempts to design novel immunotherapeutic strategies for the treatment of peanut allergy. Objective: The purpose of this study was to develop a murine model of IgE-mediated peanut hypersensitivity that closely mimics human peanut allergy. Methods: C3H/HeJ mice sensitized orally with freshly ground whole peanut and cholera toxin as adjuvant were challenged orally 3 and 5 weeks later with crude peanut extract. Anaphylactic reactions were determined. T-and B-cell responses to Ara h 1 and Ara h 2, the major peanut allergens, were characterized by evaluating splenocyte proliferative responses and IgE antibody concentrations. Furthermore, IgE antibodies in the sera of patients with peanut allergy and mice were compared for antibody binding to Ara h 2 isoforms and allergenic epitopes. Results: Peanut-specific IgE was induced by oral peanut sensitization, and hypersensitivity reactions were provoked by feeding peanut to sensitized mice. The symptoms were similar to those seen in human subjects. Ara h 1-and Ara h 2-specific antibodies were present in the sera of mice with peanut allergy. Furthermore, these Ara h 2-specific IgE antibodies bound the same Ara h 2 isoforms and major allergenic epitopes as antibodies in the sera of human subjects with peanut allergy.

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE),