Single binding sites for transcription factors NF-IL6 and NF-iB are present in the promoter of the interleukin (IL) 6 gene. Previous studies of internally deleted promoter mutants demonstrated that these two sites are important for the transcriptional regulation of this gene. In this report, we describe the synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-KB. Cotransfection of NF-IL6 with the NF-cB p65 subunit resulted in strong synergistic activation of an IL-6 promoter-reporter construct. Both the NF-IL6 and NF-cB binding sites in the IL-6 promoter were required for synergistic activation. Similar synergistic activation was observed in the IL-8 promoter, which also contains both NF-IL6 and NF-cB binding sites. Furthermore, we demonstrated that NF-IL6 and the NF-KB p65 subunit directly associated via the basic leucine-zipper domain of NF-IL6 and the Rel homology domain of p65. Since the promoters of many other genes involved in the inflammatory and acute-phase responses also contain binding sites for NF-IL6 and NF-cB, the cooperation between these two factors may have an important role in these responses. We also discuss the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production.

In animal societies, chemical communication plays an important role in conflict and cooperation. For ants, cuticular hydrocarbon (CHC) blends produced by non-nestmates elicit overt aggression. We describe a sensory sensillum on the antennae of the carpenter ant Camponotus japonicus that functions in nestmate discrimination. This sensillum is multiporous and responds only to non-nestmate CHC blends. This suggests a role for a peripheral recognition mechanism in detecting colony-specific chemical signals.

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late “transitional” stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)–induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho–guanine nucleotide exchange factors.

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/ Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes. INTRODUCTIONNeurite outgrowth is of prime importance in the formation of neuronal networks. Rho-family GTPases (RhoA, Rac1, and Cdc42), which regulate actin dynamics in a diversity of cellular functions (Van Aelst and D'Souza-Schorey, 1997;Hall, 1998), also play central roles in neuronal morphogenesis during the development of neuronal networks (Mueller, 1999;Luo, 2000). Rac1 and Cdc42, implicated in the formation of lamellipodia and filopodia in nonneuronal cells, respectively (Hall, 1998), have been accepted as positive regulators of neurite outgrowth (Luo, 2000). Our recent study using fluorescence resonance energy transfer (FRET)-based probes has shown the localized and intermittent activation of Rac1 and Cdc42 within the neurite tips of PC12 cells stimulated with nerve growth factor (NGF) (Aoki et al., 2004). This activation is largely dependent on phosphatidylinositol 3-kinase (PI3-kinase) activity, in agreement with the results of biochemical studies (Yasui et al., 2001;Nusser et al., 2002).Activation of PI3-kinase has been shown to promote neurite outgrowth in NGF-stimulated PC12 cells and sympathetic neurons (Kobayashi et al., 1997;Kuruvilla et al., 2000) and also to support cell survival (Bibel and Barde, 2000;Kaplan and Miller, 2000). Although PI3-kinase is a multifunctional signaling molecule having various effectors (Cantley, 2002), a close linkage between Rac1/Cdc42 and PI3-kinase has been found in a wide range of morphological responses to external stimuli Merlot and Firtel, 2003). Furthermore, a local positive feedback loop comprised of Rac1/Cdc42 and PI3-kinase has been hypothesized to be responsible for the symmetry breaking and persistent activation required for morphogenesis (Merlot a...

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Natural killer (NK) cells express multiple activating receptors that initiate signaling cascades through DAP10-or immunoreceptor tyrosine-based activation motif-containing adapters, including DAP12 and FcR ␥ . Among downstream signaling mediators, the guanine nucleotide exchange factor Vav1 carries out a key role in activation. However, whether Vav1 regulates only some or all NK cell-activating pathways is matter of debate. It is also possible that two other Vav family molecules, Vav2 and Vav3, are involved in NK cell activation. Here, we examine the relative contribution of each of these exchange factors to NK cell-mediated cytotoxicity using mice lacking one, two, or all three Vav proteins. We found that Vav1 deficiency is sufficient to disrupt DAP10-mediated cytotoxicity, whereas lack of Vav2 and Vav3 profoundly impairs FcR ␥ -and DAP12-mediated cytotoxicity. Our results provide evidence that these three Vav proteins function specifically in distinct pathways that trigger NK cell cytotoxicity.