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.

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...

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.

Macrophages activated through Toll receptor triggering increase the expression of the A 2A and A 2B adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A 2A and A 2B receptors, we demonstrate that the A 2A receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A 2B receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1␣ is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A 2 receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.

Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.