Early postnatal decabromodiphenyl ether exposure reduces thyroid hormone and astrocyte density in the juvenile mouse dentate gyrus.

Decabromodiphenyl ether (decaBDE) is a flame retardant that was widely-applied to many consumer products for decades. Consequently, decaBDE and other members of its class have become globally-distributed environmental contaminants. Epidemiological and animal studies indicate that decaBDE exposure during critical periods of brain development produces long-term behavioral impairments. The current study was designed to identify potential neuroendocrine mechanisms for learning and response inhibition deficits observed by our lab in a previous study. C57BL6/J mouse pups were given a single daily oral dose of 0 or 20 mg/kg decaBDE from day 1 to 21. Serum thyroid hormone levels and astrocyte-specific staining in three regions of the hippocampus were measured on day 22. DecaBDE exposure significantly reduced serum triiodothyronine, thyroxine, and astrocyte density in the subgranular zone but not the hilus or granular layer in both male and female mice. The reduction of thyroid hormone and/or glia activity could impair hippocampal development, leading to behavior dysfunction.

Resistance to visceral obesity is associated with increased locomotion in mice expressing an endothelial cell-specific fibroblast growth factor 1 transgene.

Overdevelopment of visceral adipose is positively correlated with the etiology of obesity-associated pathologies including cardiovascular disease and insulin resistance. However, identification of genetic, molecular, and physiological factors regulating adipose development and function in response to nutritional stress is incomplete. Fibroblast Growth Factor 1 (FGF1) is a cytokine expressed and released by both adipocytes and endothelial cells under hypoxia, thermal, and oxidative stress. Expression of Fibroblast Growth Factor 1 (FGF1) in adipose is required for normal depot development and remodeling. Loss of FGF1 leads to deleterious changes in adipose morphology, metabolism, and insulin resistance. Conversely, diabetic and obese mice injected with recombinant FGF1 display improvements in insulin sensitivity and a reduction in adiposity. We report in this novel, in vivo study that transgenic mice expressing an endothelial-specific FGF1 transgene (FGF1-Tek) are resistant to high-fat diet-induced abdominal adipose accretion and are more glucose-tolerant than wild-type control animals. Metabolic chamber analyses indicate that suppression of the development of visceral adiposity and insulin resistance was not associated with alterations in appetite or resting metabolic rate in the FGF1-Tek strain. Instead, FGF1-Tek mice display increased locomotor activity that likely promotes the utilization of dietary fatty acids before they can accumulate in adipose and liver. This study provides insight into the impact that genetic differences dictating the production of FGF1 has on the risk for developing obesity-related metabolic disease in response to nutritional stress.

Motor deficits, impaired response inhibition, and blunted response to methylphenidate following neonatal exposure to decabromodiphenyl ether.

Decabromodiphenyl ether (decaBDE) is an applied brominated flame retardant that is widely-used in electronic equipment. After decades of use, decaBDE and other members of its polybrominated diphenyl ether class have become globally-distributed environmental contaminants that can be measured in the atmosphere, water bodies, wildlife, food staples and human breastmilk. Although it has been banned in Europe and voluntarily withdrawn from the U.S. market, it is still used in Asian countries. Evidence from epidemiological and animal studies indicate that decaBDE exposure targets brain development and produces behavioral impairments. The current study examined an array of motor and learning behaviors in a C57BL6/J mouse model to determine the breadth of the developmental neurotoxicity produced by decaBDE. Mouse pups were given a single daily oral dose of 0 or 20mg/kg decaBDE from postnatal day 1 to 21 and were tested in adulthood. Exposed male mice had impaired forelimb grip strength, altered motor output in a circadian wheel-running procedure, increased response errors during an operant differential reinforcement of low rates (DRL) procedure and a blunted response to an acute methylphenidate challenge administered before DRL testing. With the exception of altered wheel-running output, exposed females were not affected. Neither sex had altered somatic growth, motor coordination impairments on the Rotarod, gross learning deficits during operant lever-press acquisition, or impaired food motivation. The overall pattern of effects suggests that males are more sensitive to developmental decaBDE exposure, especially when performing behaviors that require effortful motor output or when learning tasks that require sufficient response inhibition for their successful completion.

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.

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.

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.

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 IL1alpha. 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 IL1alpha presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders.

Selective expression of an aP2/Fatty Acid Binding Protein 4-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, beta-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 beta-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.

The non-transmembrane form of Delta1, but not of Jagged1, induces normal migratory behavior accompanied by fibroblast growth factor receptor 1-dependent transformation.

The interactions between Notch (N) receptors and their transmembrane ligands, Jagged1 (JI) and Delta1 (Dl1), mediate signaling events between neighboring cells that are crucial during embryonal development and in adults. Since the non-transmembrane extracellular form of J1 acts as an antagonist of N activation in NIH 3T3 mouse fibroblast cells and induces fibroblast growth factor 1 (FGF1)-dependent transformation (Small, D., Kovalenko, D., Soldi, R., Mandinova, A., Kolev, V., Trifonova, R., Bagala, C., Kacer, D., Battelli, C., Liaw, L., Prudovsky, I., and Maciag, T. (2003) J. Biol. Chem. 278, 16405-16413), we examined the potential redundant functions of the two subfamilies of Notch ligands and report that while the soluble (s) forms of both Dl1 and J1 act as N signaling antagonists in NIH 3T3 cells, they do display disparate functions. While sJ1 induced an attenuation of cell motility which is accompanied by a decrease in actin stress fibers and an increase in adherence junctions, sDl1 does not. However, sJ1, like sDl1, induces a NIH 3T3 cell tranformed phenotype mediated by FGF signaling. Because the inhibition of N signaling by sJ1 and sDl1 is rescued by dominant-negative Src expression, we suggest that there may be cooperation between the Notch and Src signaling pathways.

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.

Notch activation suppresses fibroblast growth factor-dependent cellular transformation.

Aberrant activations of the Notch and fibroblast growth factor receptor (FGFR) signaling pathways have been correlated with neoplastic growth in humans and other mammals. Here we report that the suppression of Notch signaling in NIH 3T3 cells by the expression of either the extracellular domain of the Notch ligand Jagged1 or dominant-negative forms of Notch1 and Notch2 results in the appearance of an exaggerated fibroblast growth factor (FGF)-dependent transformed phenotype characterized by anchorage-independent growth in soft agar. Anchorage-independent growth exhibited by Notch-repressed NIH 3T3 cells may result from prolonged FGFR stimulation caused by both an increase in the expression of prototypic and oncogenic FGF gene family members and the nonclassical export of FGF1 into the extracellular compartment. Interestingly, FGF exerts a negative effect on Notch by suppressing CSL (CBF-1/RBP-Jk/KBF2 in mammals, Su(H) in Drosophila and Xenopus, and Lag-2 in Caenorhabditis elegans)-dependent transcription, and the ectopic expression of constitutively active forms of Notch1 or Notch2 abrogates FGF1 release and the phenotypic effects of FGFR stimulation. These data suggest that communication between the Notch and FGFR pathways may represent an important reciprocal autoregulatory mechanism for the regulation of normal cell growth.

The myoglobin gene of the Antarctic icefish, Chaenocephalus aceratus, contains a duplicated TATAAAA sequence that interferes with transcription.

Six of the 16 known species of Antarctic icefish (family Channichthyidae) have lost the ability to express cardiac myoglobin (Mb) via at least four independent events during radiation of these species. We report here that the lesion in Chaenocephalus aceratus Mb is a duplicated TATAAAA element that blocks transcription. This lesion is distinct from those of other icefish species that do not express cardiac Mb. The C. aceratus Mb gene is nearly identical to that of Chionodraco rastrospinosus, a closely related Mb-expressing icefish species, with one exception. A 15-bp segment is present in C. aceratus but absent from C. rastrospinosus; this insertion is located 648 bp upstream from the reference transcription start site of C. rastrospinosus and includes the sequence TATAAAA, which bound HeLa cell transcription factor IID (TFIID) and icefish nuclear proteins in gel-retardation assays. Reporter constructs containing the 'full-length' C. aceratus Mb promoter were not expressed in transient expression assays in oxidative skeletal muscle of live icefish. By contrast, constructs employing the nearly identical 'full-length' C. rastrospinosus Mb promoter were efficiently expressed in parallel assays in the same tissue. Truncated constructs of C. aceratus Mb that did not contain the 15-bp duplication were expressed at very low levels. These data confirm a third independent mechanism of Mb loss among channichthyid species, indicate that C. aceratus aerobic muscle is capable of expressing functional Mb genes and demonstrate that duplication of the muscle-specific TATAAAA sequence in an inappropriate context can result in loss of a gene's expression, resulting in significant physiological consequences.