Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-γ, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-γ, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.

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Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-jB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-a and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-jB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-jB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IjB kinase a/b complex and the expression of some NF-jB family members. Treatment of macrophages with the c-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-jB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-a and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-jB signaling.Key words: Macrophages . NF-kB . Notch IntroductionMacrophages are essential cells for the innate immune response. They discriminate between pathogens and self through signals triggered by TLR, which recognize different pathogens' components, such as LPS, lipoproteins, or dsRNA, among others [1]. Activation of most TLR on the macrophage surface triggers a complex signaling pathway, which involves NF-kB activation (reviewed in [2]). In the classical NF-kB pathway, a ternary IkB kinase (IKK) complex, formed by IKK-a, IKK-b, and NF-kB essential modulator, is responsible for inducing IkB phosphorylation, allowing the release of sequestered cytoplasmic NF-kB from IkB and its translocation to the nucleus. Once in the nucleus, NF-kB controls the expression of multiple genes implicated in the inflammatory response, including cytokines, effector enzymes such as iNOS and COX-2, and adhesion molecules [2].Notch proteins encompass a family of transmembrane receptors composed of an extracellular subunit linked to a transmembrane and intracellular subunit via heterodimerization domains [3]. Ligand binding induces proteolytic cleavage of the transmembrane and intracellular receptor subunit by several proteases, including g-secretase [4], allowing the release of the intracellular domain of Notch (NIC), which then translocates to the nucleus and converts the CBF1 factor from a repressor to a transcriptional activator. Some NIC target genes have been characterized, including basic-helix-loop-helix transcription factors belonging to 2556the hairy/enhancer of split (HES) gene family [3]. Although some CBF1-independent Notch signaling can occur, its mechanism of action is not well characterized yet [5].Notch signaling is an evolutionarily conserved pathway that controls different aspects of tissue development and homeostasis [6]. In cells o...

Notch, a transmembrane receptor member of the homeotic epidermal growth factor-like family of proteins, participates in cell-to-cell signaling to control cell fate during development. Activated Notch-1 constructs lacking the extracellular region prevent differentiation of several mammalian cells in vitro. This effect, however, bypasses the normal mechanisms of cell-to-cell interactions in which Notch-1 participates. We investigated the role of Notch-1 in the hormone-induced adipocyte differentiation of 3T3-L1 fibroblasts, a paradigmatic model of adipogenesis that requires cell-to-cell contact. Unlike other differentiation models, Notch-1 expression and function were necessary conditions for adipogenesis. Impaired Notch-1 expression by antisense Notch-1 constructs prevented adipocyte differentiation. Strategies aimed at blocking putative Notch/ligand interactions also blocked adipogenesis, implicating Notch as a critical molecule in cell-to-cell signaling necessary for differentiation. Inhibition of Notch-1 expression or function decreased the expression of peroxisomal proliferatoractivated receptors ␦ and ␥, transcription factors that control adipocyte differentiation and that are up-regulated at cell confluence. These results implicate Notch in the commitment of 3T3-L1 cells to undergo adipogenesis by controlling the expression of the principal regulators of this process.

We describe the clinical features of 28 patients with juvenile dermatomyositis (JDM) and 1 patient with adult-onset dermatomyositis (DM), all of whom developed lipodystrophy (LD) that could be categorized into 1 of 3 phenotypes, generalized, partial, or focal, based on the pattern of fat loss distribution. LD onset was often delayed, beginning a median of 4.6 years after diagnosis of DM. Calcinosis, muscle atrophy, joint contractures, and facial rash were DM disease features found to be associated with LD. Panniculitis was associated with focal lipoatrophy while the anti-p155 autoantibody, a newly described myositis-associated autoantibody, was more associated with generalized LD. Specific LD features such as acanthosis nigricans, hirsutism, fat redistribution, and steatosis/nonalcoholic steatohepatitis were frequent in patients with LD, in a gradient of frequency and severity among the 3 sub-phenotypes. Metabolic studies frequently revealed insulin resistance and hypertriglyceridemia in patients with generalized and partial LD. Regional fat loss from the thighs, with relative sparing of fat loss from the medial thighs, was more frequent in generalized than in partial LD and absent from DM patients without LD. Cytokine polymorphisms, the C3 nephritic factor, insulin receptor antibodies, and lamin mutations did not appear to play a pathogenic role in the development of LD in our patients. LD is an under-recognized sequela of JDM, and certain DM patients with a severe, prolonged clinical course and a high frequency of calcinosis appear to be at greater risk for the development of this complication. High-risk JDM patients should be screened for Address reprint requests to: Lisa G. Rider, MD, Environmental Autoimmunity Group, National Institute of Environmental Health Sciences, National Institutes of Health, CRC 4-2352, MSC 1301, 10, Center Drive, Bethesda, MD 20892-1301. Fax: 301-451-5588; email: E-mail: riderl@mail.nih.gov. * These authors contributed equally to this work. † Contributors to this study are listed in the appendix. NIH Public AccessAuthor Manuscript Medicine (Baltimore) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript metabolic abnormalities, which are common in generalized and partial LD and result in much of the LD-associated morbidity. Further study is warranted to investigate the pathogenesis of acquired LD in patients with DM.