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pH is an important biomarker for many human diseases and great efforts are being made to develop new pH probes for bioimaging and biomedical applications. Here, the use of three different CdSe/ZnS QDs, functionalized with d-penicillamine and small peptides, as pH probes for fluorescence lifetime imaging microscopy (FLIM) is investigated. The fluorescence pH sensitivity of the nanoparticles is analyzed in different experimental media: aqueous solution, synthetic intracellular medium, and mesenchymal C3H10T1/2 and tumoral SK-MEL-2 cell lines. Different experiments along with theoretical calculations are conducted to unravel the mechanisms causing pH sensitivity of the nanoparticles and the effect of the length and composition of the peripheral branches on their photophysical properties. Absolute intracellular pH values measured in live cells with FLIM using a fluorescent probe based on a QD are reported here for the first time (intracellular pH values of 7.0 and 7.1 for C3H10T1/2 and SK-MEL-2 cells, respectively). These fluorescent nanoprobes can also be used to distinguish between different types of cells in cocultures on the basis of their different fluorescence lifetimes in dissimilar intracellular environments.

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 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.

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., Consejería de Educación, Cultura y Deporte of the Junta de Comunidades de Castilla-La Mancha, Spain (Grant SBPLY/17/180501/000301), Instituto Carlos III, Ministerio de Economía y Competitividad, Spain (Grant PI15/00991), and Ministerio de Ciencia e Innovación, Spain (Grant PID2019-109421RB-100)

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., Baladron 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 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.

dlk1 is an epidermal growth factor (EGF)-like homeotic protein containing an intracellular region, a single transmembrane domain, and an extracellular region possessing six EGF-like repeats and a protease-target sequence. dlk1 functions as a modulator of adipogenesis, and other differentiation processes. The molecular mechanisms by which dlk1 regulates these processes are unclear. It has been reported that different Dlk1 mRNA spliced variants, encoding for isoforms possessing the protease-target sequence or not, determine the production of membrane-associated or soluble, secreted extracellular dlk1 proteins that appear to affect adipogenesis of 3T3-L1 cells differently. In particular, only soluble variants inhibit this process. Some recent evidence suggest that dlk1 may modulate extracellular stimuli inducing differentiation. Thus, an enforced decrease of Dlk1 expression in BALB/c 3T3 cells, which results in an increase of their adipogenic potential in response to insulin-like growth factor 1 (IGF-1), modifies the kinetics and levels of activation of ERK1/2 triggered by it. In this work, we identified a strong and specific interaction between the protease-target dlk1 region and the non-IGF binding region of IGF binding protein 1 (IGFBP1), a protein that binds to IGFs and modulates their action. We also observed that the increased adipogenic potential of 3T3-L1 cells caused by diminishing Dlk1 expression through transfection with an antisense Dlk1 expression construct was inhibited by the presence of IGFBP1 in the differentiation medium. On the other hand, the presence of IGFBP1 in the culture medium slightly increased the adipogenic potential of control 3T3-L1 cells, expressing regular levels of Dlk1. These data suggest that membrane dlk1 variants bind to extracellular IGFBP1/IGF-1 complexes, which may favor the release of IGF-1 and increase the local concentration of free IGF-1 that can enhance IGF receptor signaling, leading to adipogenesis.

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDF-binding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A(2) (PLA(2))zeta and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA(2)/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A(2) activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.