Pericardial delta like non-canonical NOTCH ligand 1 (Dlk1) augments fibrosis in the heart through epithelial to mesenchymal transition.

BACKGROUND: Heart failure due to myocardial infarction (MI) involves fibrosis driven by epicardium-derived cells (EPDCs) and cardiac fibroblasts, but strategies to inhibit and provide cardio-protection remains poor. The imprinted gene, non-canonical NOTCH ligand 1 (Dlk1), has previously been shown to mediate fibrosis in the skin, lung and liver, but very little is known on its effect in the heart. METHODS: Herein, human pericardial fluid/plasma and tissue biopsies were assessed for DLK1, whereas the spatiotemporal expression of Dlk1 was determined in mouse hearts. The Dlk1 heart phenotype in normal and MI hearts was assessed in transgenic mice either lacking or overexpressing Dlk1. Finally, in/ex vivo cell studies provided knowledge on the molecular mechanism. RESULTS: Dlk1 was demonstrated in non-myocytes of the developing human myocardium but exhibited a restricted pericardial expression in adulthood. Soluble DLK1 was twofold higher in pericardial fluid (median 45.7 [34.7 (IQR)) µg/L] from cardiovascular patients (n = 127) than in plasma (median 26.1 µg/L [11.1 (IQR)]. The spatial and temporal expression pattern of Dlk1 was recapitulated in mouse and rat hearts. Similar to humans lacking Dlk1, adult Dlk1-/- mice exhibited a relatively mild developmental, although consistent cardiac phenotype with some abnormalities in heart size, shape, thorax orientation and non-myocyte number, but were functionally normal. However, after MI, scar size was substantially reduced in Dlk1-/- hearts as compared with Dlk1+/+ littermates. In line, high levels of Dlk1 in transgenic mice Dlk1fl/fl xWT1GFPCre and Dlk1fl/fl xαMHCCre/+Tam increased scar size following MI. Further mechanistic and cellular insight demonstrated that pericardial Dlk1 mediates cardiac fibrosis through epithelial to mesenchymal transition (EMT) of the EPDC lineage by maintaining Integrin ß8 (Itgb8), a major activator of transforming growth factor ß and EMT. CONCLUSIONS: Our results suggest that pericardial Dlk1 embraces a, so far, unnoticed role in the heart augmenting cardiac fibrosis through EMT. Monitoring DLK1 levels as well as targeting pericardial DLK1 may thus offer new venues for cardio-protection.

Different Expression Levels of DLK2 Inhibit NOTCH Signaling and Inversely Modulate MDA-MB-231 Breast Cancer Tumor Growth In Vivo.

NOTCH signaling is implicated in the development of breast cancer tumors. DLK2, a non-canonical inhibitor of NOTCH signaling, was previously shown to be involved in skin and breast cancer. In this work, we studied whether different levels of DLK2 expression influenced the breast cancer characteristics of MDA-MB-231 cells. We found that DLK2 overexpression inhibited NOTCH activation in a dose-dependent manner. Moreover, depending on the level of inhibition of NOTCH1 activation generated by different levels of DLK2 expression, cell proliferation, cell cycle dynamics, cell apoptosis, cell migration, and tumor growth in vivo were affected in opposite directions. Low levels of DLK2 expression produced a slight inhibition of NOTCH1 activation, and enhanced MDA-MB-231 cell invasion in vitro and cell proliferation both in vitro and in vivo. In contrast, MDA-MB-231 cells expressing elevated levels of DLK2 showed a strong inhibition of NOTCH1 activation, decreased cell proliferation, increased cell apoptosis, and were unable to generate tumors in vivo. In addition, DLK2 expression levels also affected some members of other cell signaling pathways implicated in cancer, such as ERK1/2 MAPK, AKT, and rpS6 kinases. Our data support an important role of DLK2 as a protein that can finely regulate NOTCH signaling and affect the tumor properties and growth dynamics of MDA-MB-231 breast cancer cells.

NOTCH4 Exhibits Anti-Inflammatory Activity in Activated Macrophages by Interfering With Interferon-γ and TLR4 Signaling.

NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Deletion of delta-like 1 homologue accelerates renal inflammation by modulating the Th17 immune response.

Preclinical studies have demonstrated that activation of the NOTCH pathway plays a key role in the pathogenesis of kidney damage. There is currently no information on the role of the Delta-like homologue 1 (DLK1), a NOTCH inhibitor, in the regulation of renal damage. Here, we investigated the contribution of DLK1 to experimental renal damage and the underlying molecular mechanisms. Using a Dlk1-null mouse model in the experimental renal damage of unilateral ureteral obstruction, we found activation of NOTCH, as shown by increased nuclear translocation of the NOTCH1 intracellular domain, and upregulation of Dlk2/hey-1 expression compared to wild-type (WT) littermates. NOTCH1 over-activation in Dlk1-null injured kidneys was associated with a higher inflammatory response, characterized by infiltration of inflammatory cells, mainly CD4/IL17A + lymphocytes, and activation of the Th17 immune response. Furthermore, pharmacological NOTCH blockade inhibited the transcription factors controlling Th17 differentiation and gene expression of the Th17 effector cytokine IL-17A and other related-inflammatory factors, linked to a diminution of inflammation in the injured kidneys. We propose that the non-canonical NOTCH ligand DLK1 acts as a NOTCH antagonist in renal injury regulating the Th17-mediated inflammatory response.

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.

The imprinted gene Delta like non-canonical notch ligand 1 (Dlk1) associates with obesity and triggers insulin resistance through inhibition of skeletal muscle glucose uptake.

BACKGROUND: The imprinted gene Delta like non-canonical Notch ligand 1 (Dlk1) is considered an inhibitor of adipogenesis, but its in vivo impact on fat mass indeed remains elusive and controversial. METHODS: Fat deposits were assessed by MRI and DXA scanning in two cohorts of non-diabetic men, whereas glucose disposal rate (GDR) was determined during euglycemic hyperinsulinemic clamp. Blood analyte measurements were used for correlation and mediation analysis to investigate how age, BMI, and fat percentage affect the relation between DLK1 and GDR. Confirmatory animal studies performed in normal (NC) and high fat diet (HFD) fed Dlk1+/+ and Dlk1-/- mice included DXA scanning, glucose tolerance tests (GTTs), blood measurements, and skeletal muscle glucose uptake studies by positron emission tomography (PET), histology, qRT-PCR, and in vitro cell studies. FINDINGS: Overall, DLK1 is positively correlated with fat amounts, which is consistent with a negative linear relationship between DLK1 and GDR. This relationship is not mediated by age, BMI, or fat percentage. In support, DLK1 also correlates positively with HOMA-IR and ADIPO-IR in these humans, but has no linear relationship with the early diabetic inflammation marker MCP-1. In Dlk1-/- mice, the increase in fat percentage and adipocyte size induced by HFD is attenuated, and these animals are protected against insulin resistance. These Dlk1 effects seem independent of gluconeogenesis, but at least partly relies on increased in vivo glucose uptake in skeletal muscles by Dlk1 regulating the major glucose transporter Glut4 in vivo as well as in two independent cell lines. INTERPRETATION: Thus, instead of an adipogenic inhibitor, Dlk1 should be regarded as a factor causally linked to obesity and insulin resistance, and may be used to predict development of type 2 diabetes. FUND: The Danish Diabetes Academy supported by the Novo Nordisk Foundation, The Danish National Research Council (#09-073648), The Lundbeck Foundation, University of Southern Denmark, and Dep. Of Clinical Biochemistry and Pharmacology/Odense University Hospital, the Swedish Research Council, the Swedish Diabetes Foundation, the Strategic Research Program in Diabetes at Karolinska Institute and an EFSD/Lilly grant.

Deletion of delta-like 1 homologue accelerates fibroblast-myofibroblast differentiation and induces myocardial fibrosis.

AIMS: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-ß1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-ß1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-ß1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFß-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFß/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFß signalling leads to chronic fibrosis.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

Deletion of Dlk1 increases the vulnerability to developing anxiety-like behaviors and ethanol consumption in mice.

Anxiety and alcohol use disorders (AUD) often present together, constituting a significant public health problem worldwide. In this study, we investigated the role of DLK1, a ligand of the Delta/NOTCH epidermal growth factor (EGF)-like protein family, reported to play a role in DA neurons differentiation in the striatum, as a neurobiological factor involved in the mechanisms regulating this psychiatric comorbidity. We exposed Dlk1 knockout mice (Dlk1-/- mice) to the open-field (OF), the light-dark box (LBD) and the elevated plus maze (EPM) tests, evaluating motivation to drink and ethanol consumption using the oral ethanol self-administration (OEA) paradigm. Quantitative real time polymerase chain reaction (qPCR) studies were carried out to evaluate alterations in targets closely related to DA neurotransmission in the reward system, tyrosine hydroxylase (Th) in the ventral tegmental area (VTA), and µ-opioid receptor (Oprm1) in the nucleus accumbens (NAc). No differences were observed in the total or peripheral distances travelled by Dlk1-/- compared to wild-type (WT) mice in OF. However, central distance travelled significantly decreased in Dlk1-/- mice. Deletion of Dlk1 increased anxiety-like behaviors in the LDB and EPM, and, Dlk1-/- mice also presented higher ethanol intake and motivation to drink (number of effective responses) in the OEA. In addition, Th and Oprm1 gene expression was reduced in the VTA and NAc of Dlk1-/- mice. We conclude that deletion of Dlk1 increases anxiety-related behaviors and vulnerability to ethanol consumption and modifies the gene expression of key targets closely related with DA neurotransmission involved in the reinforcing actions of ethanol.

Deletion of Dlk2 increases the vulnerability to anxiety-like behaviors and impairs the anxiolytic action of alprazolam.

The purpose of this study was to evaluate the role of the non-canonical DLK2 NOTCH ligand in the regulation of emotional behavior. To this aim, anxiety and depressive-like behaviors were examined in Dlk2 knock-out (Dlk2-/-) and its corresponding wild-type (WT) mice. Furthermore, relative gene expression analyses of corticotropin releasing hormone (Crh) in the paraventricular nucleus (PVN), glucocorticoid receptor (NR3C1) and FK506 binding protein 5 (FKBP5) in the hippocampus (HIPP), and the transcription factors Hes1, Hes5 and Hey1 in the PVN, HIPP and amygdala (AMY) were carried out in Dlk2-/- and WT mice under basal conditions and after exposure to restraint stress. The anxiolytic action of alprazolam and the relative gene expression levels of the GABA-A alpha 2 and gamma 2 receptor subunits (Gabra2 and Gabrg2) were also evaluated in the HIPP and AMY of WT and Dlk2-/- mice. The results reveal that deletion of Dlk2 increased anxiety and depressive-like behaviors and altered the vulnerability to restraint stress on Crh gene expression in the PVN, Nr3c1 and Fkbp5 gene expression in the HIPP, and Hes1, Hes5 and Hey1 gene expression in the PVN, HIPP and AMY. Interestingly, the administration of alprazolam failed to produce an anxiolytic action in Dlk2-/- mice. Indeed, Gabra2 and Gabrg2 gene expression levels were significantly affected under basal conditions and after stress exposure in Dlk2-/- mice compared with WT mice. In conclusion, the results suggest that DLK2 plays an important role in the regulation of emotional behaviors and relevant targets of the stress axis, NOTCH pathway and GABAergic neurotransmission. In addition, the deletion of Dlk2 blocked the anxiolytic response to alprazolam. Future studies are needed to determine the relevance of DLK2 as a potential therapeutic target for the treatment of neuropsychiatric disorders with anxiety or depressive-like behaviors.

Absence of Notch1 in murine myeloid cells attenuates the development of experimental autoimmune encephalomyelitis by affecting Th1 and Th17 priming.

Inhibition of Notch signalling in T cells attenuates the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Growing evidence indicates that myeloid cells are also key players in autoimmune processes. Thus, the present study evaluates the role of the Notch1 receptor in myeloid cells on the progression of myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, using mice with a myeloid-specific deletion of the Notch1 gene (MyeNotch1KO). We found that EAE progression was less severe in the absence of Notch1 in myeloid cells. Thus, histopathological analysis revealed reduced pathology in the spinal cord of MyeNotch1KO mice, with decreased microglia/astrocyte activation, demyelination and infiltration of CD4+ T cells. Moreover, these mice showed lower Th1 and Th17 cell infiltration and expression of IFN-γ and IL-17 mRNA in the spinal cord. Accordingly, splenocytes from MyeNotch1KO mice reactivated in vitro presented reduced Th1 and Th17 activation, and lower expression of IL-12, IL-23, TNF-α, IL-6, and CD86. Moreover, reactivated wild-type splenocytes showed increased Notch1 expression, arguing for a specific involvement of this receptor in autoimmune T cell activation in secondary lymphoid tissues. In summary, our results reveal a key role of the Notch1 receptor in myeloid cells for the initiation and progression of EAE.

Different expression levels of DLK1 inversely modulate the oncogenic potential of human MDA-MB-231 breast cancer cells through inhibition of NOTCH1 signaling.

NOTCH receptors participate in cancer cell proliferation and survival. Accumulated evidence indicates that, depending on the cellular context, these receptors can function as oncogenes or as tumor-suppressor genes. The epidermal growth factor-like protein delta-like homolog (DLK)1 acts as a NOTCH inhibitor and is involved in the regulation of normal and tumoral growth. In this work, we focused on the role of DLK1 in the control of breast cancer cell growth, a tumor type in which NOTCH receptors have been shown to play both opposite roles. We found that human DLK1 inhibits NOTCH signaling in MDA-MB-231 breast cancer cells. The proliferation rate and invasion capabilities of these cells depended on the level of NOTCH activation and signaling, as regulated by DLK1. High levels of DLK1 expression led to a significant decrease in NOTCH signaling, which was associated with a decrease in breast cancer cell proliferation and invasion. On the contrary, lower levels of NOTCH inhibition, caused by lower levels of DLK1 overexpression, led to enhanced in vitro MDA-MB-231 cell invasion, and to both in vitro and in vivo increased cell proliferation. The data presented in this work suggest that a fine regulation of NOTCH signaling plays an important role in the control of breast cancer cell proliferation and invasion.-Nueda, M.-L., Naranjo, A.-I., Baladrón V., Laborda, J. Different expression levels of DLK1 inversely modulate the oncogenic potential of human MDA-MB-231 breast cancer cells through inhibition of NOTCH1 signaling.

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.

Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals.

Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1(+/+) and Dlk1(-/-) mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1(+/+) embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10-15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.

DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR-activated murine macrophages.

Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-ß and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-ß signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.

DLK1 Regulates Whole-Body Glucose Metabolism: A Negative Feedback Regulation of the Osteocalcin-Insulin Loop.

The endocrine role of the skeleton in regulating energy metabolism is supported by a feed-forward loop between circulating osteoblast (OB)-derived undercarboxylated osteocalcin (Glu-OCN) and pancreatic ß-cell insulin; in turn, insulin favors osteocalcin (OCN) bioactivity. These data suggest the existence of a negative regulation of this cross talk between OCN and insulin. Recently, we identified delta like-1 (DLK1) as an endocrine regulator of bone turnover. Because DLK1 is colocalized with insulin in pancreatic ß-cells, we examined the role of DLK1 in insulin signaling in OBs and energy metabolism. We show that Glu-OCN specifically stimulates Dlk1 expression by the pancreas. Conversely, Dlk1-deficient (Dlk1(-/-) ) mice exhibited increased circulating Glu-OCN levels and increased insulin sensitivity, whereas mice overexpressing Dlk1 in OB displayed reduced insulin secretion and sensitivity due to impaired insulin signaling in OB and lowered Glu-OCN serum levels. Furthermore, Dlk1(-/-) mice treated with Glu-OC experienced significantly lower blood glucose levels than Glu-OCN-treated wild-type mice. The data suggest that Glu-OCN-controlled production of DLK1 by pancreatic ß-cells acts as a negative feedback mechanism to counteract the stimulatory effects of insulin on OB production of Glu-OCN, a potential mechanism preventing OCN-induced hypoglycemia.

The proteins DLK1 and DLK2 modulate NOTCH1-dependent proliferation and oncogenic potential of human SK-MEL-2 melanoma cells.

NOTCH receptors regulate cell proliferation and survival in several types of cancer cells. Depending on the cellular context, NOTCH1 can function as an oncogene or as a tumor suppressor gene. DLK1 is also involved in the regulation of cell growth and cancer, but nothing is known about the role of DLK2 in these processes. Recently, the proteins DLK1 and DLK2 have been reported to interact with NOTCH1 and to inhibit NOTCH1 activation and signaling in different cell lines. In this work, we focused on the role of DLK proteins in the control of melanoma cell growth, where NOTCH1 is known to exert an oncogenic effect. We found that human DLK proteins inhibit NOTCH signaling in SK-MEL-2 metastatic melanoma cells. Moreover, the proliferation rate of these cells was dependent upon the level of NOTCH activation and signaling as regulated by DLK proteins. In particular, high levels of NOTCH inhibition resulted in a decrease, whereas lower levels of NOTCH inhibition led to an increase in melanoma cell proliferation rates, both in vitro and in vivo. Finally, our data revealed additive NOTCH-mediated effects of DLK proteins and the γ-secretase inhibitor DAPT on cell proliferation. The data presented in this work suggest that a fine regulation of NOTCH signaling plays an important role in the control of metastatic melanoma cell proliferation. Our results open the way to new research on the role of DLK proteins as potential therapeutic tools for the treatment of human melanoma.