NOTCH3 signaling is essential for NF-κB activation in TLR-activated 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.

NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells.

The NOTCH family of receptors and ligands is involved in numerous cell differentiation processes, including adipogenesis. We recently showed that overexpression of each of the four NOTCH receptors in 3T3-L1 preadipocytes enhances adipogenesis and modulates the acquisition of the mature adipocyte phenotype. We also revealed that DLK proteins modulate the adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite way, despite their function as non-canonical inhibitory ligands of NOTCH receptors. In this work, we used multipotent C3H10T1/2 cells as an adipogenic model. We used standard adipogenic procedures and analyzed different parameters by using quantitative-polymerase chain reaction (qPCR), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), luciferase, Western blot, and metabolic assays. We revealed that C3H10T1/2 multipotent cells show higher levels of NOTCH receptors expression and activity and lower Dlk gene expression levels than 3T3-L1 preadipocytes. We found that the overexpression of NOTCH receptors enhanced C3H10T1/2 adipogenesis levels, and the overexpression of NOTCH receptors and DLK (DELTA-like homolog) proteins modulated the conversion of cells towards a brown-like adipocyte phenotype. These and our prior results with 3T3-L1 preadipocytes strengthen the idea that, depending on the cellular context, a precise and highly regulated level of global NOTCH signaling is necessary to allow adipogenesis and determine the mature adipocyte phenotype.

Author Correction: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

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DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

The role of NOTCH signaling in adipogenesis is highly controversial, with data indicating null, positive or negative effects on this differentiation process. We hypothesize that these contradictory results could be due to the different global NOTCH signaling levels obtained in different experimental settings, because of a specific modulation of NOTCH receptors' activity by their ligands. We have previously demonstrated that DLK1 and DLK2, two non-canonical NOTCH1 ligands that inhibit NOTCH1 signaling in a dose-dependent manner, modulate the adipogenesis process of 3T3-L1 preadipocytes. In this work, we show that over-expression of any of the four NOTCH receptors enhanced adipogenesis of 3T3-L1 preadipocytes. We also determine that DLK proteins inhibit not only the activity of NOTCH1, but also the activity of NOTCH2, 3 and 4 receptors to different degrees. Interestingly, we have observed, by different approaches, that NOTCH1 over-expression seems to stimulate the differentiation of 3T3-L1 cells towards a brown-like adipocyte phenotype, whereas cells over-expressing NOTCH2, 3 or 4 receptors or DLK proteins would rather differentiate towards a white-like adipocyte phenotype. Finally, our data also demonstrate a complex feed-back mechanism involving Notch and Dlk genes in the regulation of their expression, which suggest that a precise level of global NOTCH expression and NOTCH-dependent transcriptional activity of specific targets could be necessary to determine the final phenotype of 3T3-L1 adipocytes.

The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling.

The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB-dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target.

The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities.

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.

The novel gene EGFL9/Dlk2, highly homologous to Dlk1, functions as a modulator of adipogenesis.

The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.