Deena Small

F3/Contactin Acts as a Functional Ligand for Notch during Oligodendrocyte Maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.

Members of the Jagged/Notch Gene Families Are Expressed in Injured Arteries and Regulate Cell Phenotype via Alterations in Cell Matrix and Cell-Cell Interaction

The Jagged/Notch signaling pathways control cell fate determination and differentiation, and their dysfunction is associated with human pathologies involving cardiovascular abnormalities. To determine the presence of these genes during vascular response to injury, we analyzed expression of Jagged1, Jagged2, and Notch1 through 4 after balloon catheter denudation of the rat carotid artery. Although low levels of Jagged1, Jagged2, and constitutive expression of Notch1 were seen in uninjured endothelium, expression of all was significantly increased in injured vascular cells. High Jagged1 expression was restricted to the regenerating endothelial wound edge, whereas Notch transcripts were abundant in endothelial and smooth muscle cells. To understand the basis for Jagged/Notch control of cellular phenotype, we studied an in vitro model of NIH3T3 cells transfected with a secreted form of the extracellular domain of Jagged1. We report that the soluble Jagged1 protein caused decreased cell-matrix adhesion and cell migration defects. Cadherin-mediated intercellular junctions as well as focal adhesions were modified in soluble Jagged1 transfectants, demonstrating that cell-cell contacts and adhesion plaques may be targets of Jagged/Notch activity. We suggest that Jagged regulation of cell-cell and cell-matrix interactions may contribute to the control of cell migration in situations of tissue remodeling in vivo.

Secretion without Golgi

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non‐classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1α. Stress‐induced transmembrane translocation of these proteins requires the assembly of copper‐dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non‐classical release of FGF1 and IL1α presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders. J. Cell. Biochem. 103: 1327–1343, 2008.

Selective expression of an aP2/Fatty Acid Binding Protein4-Cre transgene in non-adipogenic tissues during embryonic development

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, β-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of β-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

The intracellular domain of Notch ligand Delta1 induces cell growth arrest

Notch signaling involves proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands, Delta or Jagged; and the resultant soluble intracellular domain of Notch stimulates a cascade of transcriptional events. The Delta1 ligand also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. We demonstrate that the expression of the intracellular domain of Delta1 results in a non‐proliferating senescent‐like cell phenotype which is dependent on the expression of the cell cycle inhibitor, p21, and is abolished by co‐expression of constitutively active Notch1. These data suggest a new intracellular role for Delta1.

Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes

We previously found that soluble forms of the Notch ligands Jagged1 and Delta1 induced fibroblast growth factor receptor-dependent cell transformation in NIH3T3 fibroblasts. However, the phenotypes of these lines differed, indicating distinct functional differences among these Notch ligands. In the present study, we used allografts to test the hypothesis that NIH3T3 fibroblasts that express soluble forms of Delta1 and Jagged1 accelerate tumorigenicity in vivo. With the exception of the full-length Jagged1 transfectant, all other cell lines, including the control, generated tumors when injected subcutaneously in athymic mice. Suppression of Notch signaling by the soluble ligands significantly increased tumor onset and growth, whereas full-length Jagged1 completely suppressed tumor development. In addition, there were striking differences in tumor pathology with respect to growth kinetics, vascularization, collagen content, size and number of necrotic foci, and invasiveness into the underlying tissue. Further, the production of angiogenic factors, including vascular endothelial growth factor, also differed among the tumor types. Lastly, both Jagged1- and Delta1-derived tumors contained phenotypically distinct populations of lipid-filled cells that corresponded with increased expression of adipocyte markers. The divergence of tumor phenotype may be attributed to ligand-specific alterations in Notch receptor responses in exogenous and endogenous cell populations within the allographs. Our findings demonstrate distinct functional properties for these Notch ligands in the promotion of tumorigenicity in vivo.

A Novel Vertebrate Myoglobin Gene Containing Three A+T-Rich Introns Is Conserved Among Antarctic Teleost Species Which Differ in Myoglobin Expression

Abstract. This report presents the first teleost myoglobin (Mb) genomic DNA sequence, several features of which are distinct from mammalian Mb genes. We have isolated and compared genomic clones of three closely related Antarctic icefish: Chionodraco rastrospinosus, which expresses Mb mRNA and functional polypeptide; Champsocephalus gunnari, which transcribes the Mb gene but does not produce polypeptide due to a 5-base pair (bp) frameshift insertion; and Chaenocephalus aceratus, which lacks both Mb protein and mRNA. The single-copy icefish Mb gene contains three introns: two at positions identical to those found in mammalian Mb and a novel intron located in the 5′ untranslated region three nucleotides upstream from the initiator codon. All three introns are shorter than those found in mammalian Mb genes and exhibit a considerably higher A+T content. The entire Mb transcriptional unit is intact in C. aceratus, indicating that the failure to express this gene is not due to aberrations in the coding region, splice junctions, polyadenylation signals, or core promoter elements. The three icefish Mb sequences display an extreme degree of identity in the transcriptional unit and putative promoter region. In contrast, sequences 65 bp downstream from the polyadenylation site bear no homology among the three species, demonstrating that rapid sequence change has occurred in the 1 million years since the divergence of these species.

Effect of soluble Jagged1-mediated inhibition of Notch signaling on proliferation and differentiation of an adipocyte progenitor cell model

Adipose tissue development is dependent on multiple signaling mechanisms and cell-cell interactions that regulate adipogenesis, angiogenesis and extracellular remodeling. The Notch signaling pathway is an important cell-fate determinant whose role in adipogenesis is not clearly defined. To address this issue, we examined the effect of inhibition of Notch signaling by soluble-Jagged1 in the 3T3-L1 preadipocyte line. In vitro, soluble-Jagged1 expression in 3T3-L1 cells altered cell morphology, increased the rate of cell proliferation and induced an early transcriptional response to differentiation stimuli. However, these cells did not form mature adipocytes due to their inability to exit the cell-cycle in response to serum-starvation and glucocorticoid-induced cell-cycle arrest. In contrast, subcutaneous allografts of soluble-Jagged1 cells formed larger fat pads containing lipid-filled adipocytes with improved neovascularization compared with controls. Since adipogenesis is tightly associated with angiogenesis, we evaluated the influence of soluble-Jagged1 on endothelial cells by culturing them in cell-free conditioned media from preadipocytes. Soluble Jagged1-mediated inhibition of Notch signaling increased levels of secreted cytokines, potentially contributing to the improved cell growth and proliferation observed in these cultures. Our findings demonstrate an initial requirement of Notch signaling inactivation for preadipocyte cell commitment and support the hypothesis that cell-to-cell crosstalk between the preadipocytes and endothelial cells is required for neovascularization and remodeling of the tissue to promote hyperplasia and hypertrophy of differentiating adipocytes.

Regulation of non-classical FGF1 release and FGF-dependent cell transformation by CBF1-mediated notch signaling†

FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non‐classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML. The expression of a dominant negative (dn) form of CBF1 in 3T3 cells induces transcription of FGF1 and sphingosine kinase 1 (SphK1), which is a component of FGF1 export pathway. dnCBF1 expression stimulates the stress‐independent release of transduced FGF1 from NIH 3T3 cells and endogenous FGF1 from A375 melanoma cells. NIH 3T3 cells transfected with dnCBF1 form colonies in soft agar and produce rapidly growing highly angiogenic tumors in nude mice. The transformed phenotype of dnCBF1 transfected cells is efficiently blocked by dn forms of FGF receptor 1 and S100A13, which is a component of FGF1 export pathway. FGF1 export and acceleration of cell growth induced by dnCBF1 depend on SphK1. Similar to dnCBF1, dnMAML transfection induces FGF1 expression and release, and accelerates cell proliferation. The latter effect is strongly decreased in FGF1 null cells. We suggest that the regulation of FGF1 expression and release by CBF1‐mediated Notch signaling can play an important role in tumor formation. J. Cell. Physiol. 226: 3064–3075, 2011.

Novel Cross-Talk between Three Cardiovascular Regulators: Thrombin Cleavage Fragment of Jagged1 Induces Fibroblast Growth Factor 1 Expression and Release

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular domain. The sJ1 form produced as a result of thrombin cleavage inhibits Notch-mediated CBF1/Suppressor of Hairless [(Su(H)]/Lag-1-dependent transcription and induces FGF1 expression and release. The overexpression of Jagged1 in PAR1 null cells results in a rapid thrombin-induced export of FGF1. These data demonstrate the existence of novel cross-talk between thrombin, FGF, and Notch signaling pathways, which play important roles in vascular formation and remodeling.