dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats

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.     

Jedi--a novel transmembrane protein expressed in early hematopoietic cells

Self-renewal and differentiation of hematopoietic stem and progenitor cells are defined by the ensembles of genes expressed by these cells. Here we report identification of a novel gene named Jedi, which is expressed predominantly in short- and long-term repopulating stem cells when compared to more mature bone marrow progenitors. Jedi mRNA encodes a transmembrane protein that contains multiple EGF-like repeats. Jedi and two earlier reported proteins, MEGF10 and MEGF11, share a substantial homology and are likely to represent a novel protein family. Studies of the potential role of Jedi in hematopoietic regulation demonstrated that the retrovirally mediated expression of Jedi in bone marrow cells decreased the number of myeloid progenitors in in vitro clonogenic assays. In addition, expression of Jedi in NIH 3T3 fibroblasts resulted in a decreased number of late and early myeloid progenitors in the non-adherent co-cultured bone marrow cells. Jedi shares a number of structural features with the Jagged/Serrate/Delta family of Notch ligands, and our experiments indicate that the extracellular domain of Jedi, similar to the corresponding domain of Jagged1, inhibits Notch signaling. On the basis of obtained results, we suggest that Jedi is involved in the fine regulation of the early stages of hematopoietic differentiation, presumably through the Notch signaling pathway.     

Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the Notch ligand Delta1

The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells. Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell. Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1). We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo. We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains. In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds. Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos.     

dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells

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.     

Molecular modeling and molecular dynamics simulation studies of Delta-Notch complex

Notch is a single-pass transmembrane receptor protein which is composed of a short extracellular region, a single-pass transmembrane domain and a small intracellular region. Notch ligand like Delta, member of the DSL protein family, is also single-pass transmembrane protein. It has been demonstrated that of the 36 EGF repeats of Notch, 11th and 12th are sufficient to mediate interactions with Delta. Crystal structure of mammalian Notch extracellular ligand binding domain contains 11 and 12 EGF-like repeats. Here a portion of the Delta protein of Drosophila, known to interact with Notch extracellular domain (ECD) has been modeled using homology modeling. The structure of the Delta-Notch complex was subsequently modeled by protein docking method using GRAMM. MD simulations of the modeled structures were performed. The structure for Delta-Notch complex has been proposed based on interaction energy parameter and planarity studies.     

The EGF-like homeotic protein dlk affects cell growth and interacts with growth-modulating molecules in the yeast two-hybrid system

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.     

Analysis of Delta-Notch interaction by molecular modeling and molecular dynamic simulation studies

Notch is a single-pass transmembrane receptor protein. Delta (member of the DSL protein family), a Notch ligand, is also single-pass transmembrane protein that can interact with Notch to form the Delta-Notch complex. It has been demonstrated that of the 36 Epidermal Growth Factor (EGF) repeats of Notch, 11th and 12th are sufficient to mediate interactions with Delta. Crystal structure of mammalian Notch1 extracellular ligand binding domain shows the presence of 11th and 12th EGF-like repeats. Here a portion of the Drosophila Delta protein, known to interact with Notch extracellular domain, has been modeled using homology modeling. The structure of the Delta-Notch complex was subsequently modeled by protein-docking method using GRAMM. Molecular dynamic simulations of the modeled structures were performed. The probable structures for Delta-Notch complex have been proposed based on interaction energy parameter and planarity studies.     

The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells

The EGF-like homeotic gene Dlk1 appears to function as an inhibitor of adipogenesis. Overexpression of Dlk1 prevents adipogenesis of 3T3-L1 cells. Dlk1-deficient mice are obese; however, adipose tissue still develops in Fc-dlk1 transgenic mice, suggesting that Dlk1 is not a strict inhibitor of adipogenesis. To clarify the role of Dlk1 in adipogenesis, we studied whether Dlk1 could act differently on this process depending upon the differentiation state of the precursor cells. We found that Dlk1 is a potentiator of adipogenesis for mesenchymal C3H10T1/2 cells. This potentiating effect can be triggered by overexpressing the entire protein or the extracellular EGF-like-containing region, but not by overexpressing the intracellular dlk1 sequence. In addition, coculture of C3H10T1/2 cells with other cells expressing Dlk1, but not with cells lacking Dlk1 expression, enhances their adipogenic response. Potentiation of adipogenesis by Dlk1 was associated with changes in the activation of ERK1/2 after IGFI/insulin induction. Finally, as reported with other cells, dlk1 functioned as a Notch signaling inhibitor in C3H10T1/2 cells, but inhibition of Notch1 expression prevented the potentiating effects of Dlk1 in adipogenesis. These data suggest that Dlk1 may potentiate or inhibit adipogenesis depending upon the cellular context, and that Notch1 expression and activation are important factors in this context.     

Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway

The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked bygamma-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jkappa-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jkappa, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jkappa-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype.     

Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.     

Expression of Notch receptors and ligands on immature and mature T cells

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.     

Microfibrillar proteins MAGP-1 and MAGP-2 induce Notch1 extracellular domain dissociation and receptor activation

Unlike most receptors, Notch serves as both the receiver and direct transducer of signaling events. Activation can be mediated by one of five membrane-bound ligands of either the Delta-like (-1, -2, -4) or Jagged/Serrate (-1, -2) families. Alternatively, dissociation of the Notch heterodimer with consequent activation can also be mediated experimentally by calcium chelators or by mutations that destabilize the Notch1 heterodimer, such as in the human disease T cell acute lymphoblastic leukemia. Here we show that MAGP-2, a protein present on microfibrils, can also interact with the EGF-like repeats of Notch1. Co-expression of MAGP-2 with Notch1 leads to both cell surface release of the Notch1 extracellular domain and subsequent activation of Notch signaling. Moreover, we demonstrate that the C-terminal domain of MAGP-2 is required for binding and activation of Notch1. Based on the high level of homology, we predicted and further showed that MAGP-1 can also bind to Notch1, cause the release of the extracellular domain, and activate signaling. Notch1 extracellular domain release induced by MAGP-2 is dependent on formation of the Notch1 heterodimer by a furin-like cleavage, but does not require the subsequent ADAM metalloprotease cleavage necessary for production of the Notch signaling fragment. Together these results demonstrate for the first time that the microfibrillar proteins MAGP-1 and MAGP-2 can function outside of their role in elastic fibers to activate a cellular signaling pathway.     

Dll4-selective Notch signaling induces ephrinB2 gene expression in endothelial cells

The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation. Here, we show that Notch stimulation instructively induces arterial characteristics in endothelial cells (EC). Forced expression of Notch intracellular domain (NICD, activated form of Notch) induced mRNA expression for a subset of arterial-specific markers such as ephrinB2, connexin40, and HERP1 only in EC but not other cell lines. In co-culture experiments using EC and either Dll4- or Jagged1-expressing cells, we found that Dll4 stimulation but not Jagged1 markedly induced ephrinB2 expression. An inducible expression of HERP1 and HERP2 by NICD has no measurable effects on expression of ephrinB2 and venous marker EphB4 although either HERP1 or HERP2 overexpression exerts potent inhibitory effects on EphB4 expression without ephrinB2 induction. We also found no functional interaction between Notch and TGF-beta-ALK1 signalings in an induction of ephrinB2 expression. These results suggest that Dll4-stimulated Notch signaling induces a part of arterial characteristics only in EC via HERP-independent mechanism. Our data provide new insight into the molecular mechanism of ligand-selective Notch activation during differentiation of arterial EC.     

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

Introduction

Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis.

Methods

Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb).

Results

Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss.

Conclusions

The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.     

Proteolytic processing of delta-like 1 by ADAM proteases

Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. Interactions between Dll1 and Notch in trans activate the Notch pathway, whereas Dll1 binding to Notch in cis inhibits Notch signaling. Dll1 undergoes proteolytic processing in its extracellular domain by ADAM10. In this work we demonstrate that Dll1 represents a substrate for several other members of the ADAM family. In co-transfected cells, Dll1 is constitutively cleaved by ADAM12, and the N-terminal fragment of Dll1 is released to medium. ADAM12-mediated cleavage of Dll1 is cell density-dependent, takes place in cis orientation, and does not require the presence of the cytoplasmic domain of ADAM12. Full-length Dll1, but not its N- or C-terminal proteolytic fragment, co-immunoprecipitates with ADAM12. By using a Notch reporter construct, we show that Dll1 processing by ADAM12 increases Notch signaling in a cell-autonomous manner. Furthermore, ADAM9 and ADAM17 have the ability to process Dll1. In contrast, ADAM15 does not cleave Dll1, although the two proteins still co-immunoprecipitate with each other. Asn-353 present in the catalytic motif of ADAM12 and other Dll1-processing ADAMs, but absent in ADAM15, is necessary for Dll1 cleavage. Dll1 cleavage is reduced in ADAM9/12/15(-/-) mouse embryonic fibroblasts (MEFs), suggesting that the endogenous ADAM9 and/or ADAM12 present in wild type MEFs contribute to Dll1 processing. Finally, the endogenous Dll1 present in primary mouse myoblasts undergoes cleavage in confluent, differentiating myoblast cultures, and this cleavage is decreased by ADAM12 small interfering RNAs. Our findings expand the role of ADAM proteins in the regulation of Notch signaling.     

The extracellular matrix protein MAGP-2 interacts with Jagged1 and induces its shedding from the cell surface

Elastic fibers are composed of the protein elastin and a network of 10-12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1. MAGP-2 was found complexed with the Jagged1 extracellular domain shed from 293T cells and COS-7 cells coexpressing full-length Jagged1 and MAGP-2. MAGP-2 shedding of the Jagged1 extracellular domain was decreased by the metalloproteinase hydroxamate inhibitor BB3103 implicating proteolysis in its release. Although MAGP-2 also interacted with the other DSL ligands, Jagged2 and Delta1, they were not found associated with MAGP-2 in the conditioned media, identifying differential effects of MAGP-2 on DSL ligand shedding. The related microfibrillar protein MAGP-1 was also found to interact with DSL ligands but, unlike MAGP-2, was unable to facilitate the shedding of Jagged1. Our findings suggest that in addition to its role in microfibrils, MAGP-2 may also affect cellular differentiation through modulating the Notch signaling pathway either by binding to cell surface DSL ligands or by facilitating release and/or stabilization of a soluble extracellular form of Jagged1.