Adropin preserves the blood‐brain barrier through a Notch1/Hes1 pathway after intracerebral hemorrhage in mice

Adropin is expressed in the CNS and plays a crucial role in the development of stroke. However, little is currently known about the effects of adropin on the blood‐brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of adropin in collagenase‐induced ICH was investigated in mice. At 1‐h post‐ICH, mice were administered with recombinant human adropin by intranasal. Brain water +content, BBB permeability, and neurological function were measured at different time intervals. Proteins were quantified using western blot analysis, and the localizations of adropin and Notch1 were visualized via immunofluorescence staining. It is shown that adropin reduced brain water content and improved neurological functions. Adropin preserved the functionality of BBB by increasing N‐cadherin expression and reducing extravasation of albumin. Moreover, in vivo knockdown of Notch1 and Hes1 both abolished the protective effects of adropin. Taken together, our data demonstrate that adropin constitutes a potential treatment value for ICH by preserving BBB and improving functional outcomes through the Notch1 signaling pathway.

     

Adropin deficiency is associated with increased adiposity and insulin resistance

Adropin is a secreted peptide that improves hepatic steatosis and glucose homeostasis when administered to diet-induced obese mice. It is not clear if adropin is a peptide hormone regulated by signals of metabolic state. Moreover, the significance of a decline in adropin expression with obesity with respect to metabolic disease is also not clear. We investigated the regulation of serum adropin by metabolic status and diet. Serum adropin levels were high in chow-fed conditions and were suppressed by fasting and diet-induced obesity (DIO). High adropin levels were observed in mice fed a high-fat low carbohydrate diet, whereas lower levels were observed in mice fed a low-fat high carbohydrate diet. To investigate the role of adropin deficiency in metabolic homeostasis, we generated adropin knockout mice (AdrKO) on the C57BL/6J background. AdrKO displayed a 50%-increase in increase in adiposity, although food intake and energy expenditure were normal. AdrKO also exhibited dyslipidemia and impaired suppression of endogenous glucose production (EndoR(a)) in hyperinsulinemic-euglycemic clamp conditions, suggesting insulin resistance. While homo- and heterozygous carriers of the null adropin allele exhibited normal DIO relative to controls, impaired glucose tolerance associated with weight gain was more severe in both groups. In summary, adropin is a peptide hormone regulated by fasting and feeding. In fed conditions, adropin levels are regulated dietary macronutrients, and increase with dietary fat content. Adropin is not required for regulating food intake, however, its functions impact on adiposity and are involved in preventing insulin resistance, dyslipidemia, and impaired glucose tolerance.     

Adropin reduces paracellular permeability of rat brain endothelial cells exposed to ischemia-like conditions

Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16 h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40 kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100 ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions.     

Fbxw7-dependent degradation of Notch is required for control of "stemness" and neuronal-glial differentiation in neural stem cells

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.     

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.     

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.     

Receptor-like protein-tyrosine phosphatase α enhances cell surface expression of neural adhesion molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.     

Gegen Qinlian decoction maintains colonic mucosal homeostasis in acute/chronic ulcerative colitis via bidirectionally modulating dysregulated Notch signaling

BackgroundGegen Qinlian decoction (GQ) is a well-known traditional Chinese medicine that has been clinically proven to be effective in treating ulcerative colitis (UC). However, its therapeutic mechanism has not been fully elucidated. Notch signaling plays an essential role in the regeneration of the intestinal epithelium.PurposeThis study was designed to ascertain the mechanism by which GQ participates in the recovery of the colonic mucosa by regulating Notch signaling in acute and chronic UC models.MethodsAcute and chronic UC mice (C57BL/6) were established with 3 and 2% dextran sulfate sodium (DSS), respectively, and treated with oral administration of GQ. The expression of the Notch target gene Hes1 and the Notch-related proteins RBP-J, MAML and Math1 was analyzed by western blotting. PTEN mRNA levels were detected by qRT-PCR. Mucin production that is characteristic of goblet cells was determined by Alcian blue/periodic acid-Schiff staining and verified by examining MUC2 mRNA levels by qRT-PCR. Cell proliferation was assayed by immunohistochemistry analysis of Ki67. HT-29 and FHC cells and Toll-like receptor 4 knockout (TLR4^−/−) acute UC mice were also used in this study.ResultsGQ restored the injured colonic mucosa in both acute and chronic UC models. We found that Notch signaling was hyperactive in acute UC mice and hypoactive in chronic UC mice. GQ downregulated Hes1, RBP-J and MAML proteins and augmented goblet cells in the acute UC models, whereas GQ upregulated Hes1, RBP-J and MAML proteins in chronic UC mice, reducing goblet cell differentiation and promoting crypt base columnar (CBC) stem cell proliferation. Hes1 mRNA was suppressed in TLR4^−/− UC mice, and GQ treatment reversed this effect. In vitro, GQ reduced Hes1 protein in Notch-activated HT29 and FHC cells but increased Hes1 protein in Notch-inhibited cells.ConclusionsGQ restored the colonic epithelium by maintaining mucosal homeostasis via bidirectional regulation of Notch signaling in acute/chronic UC models.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

Pofut1 is required for the proper localization of the Notch receptor during mouse development

Protein O-fucosyltransferase 1 (Pofut1), which catalyzes the addition of O-linked fucose to the EGF domains of the Notch receptor, is indispensable for Notch signaling activation. However, the mechanism of action of Pofut1 in mice is still unclear. Mouse embryos lacking Pofut1 shows defects in valve formation and trabeculation in the cardiovascular system, which are almost identical abnormalities to those of the RBP-Jk mutants. In our current study, we have examined the epistatic relationship between the functions of Pofut1 and activated-Notch1 (NICD1) by taking advantage of the fact that forced expression of NICD1 results in myocardial defects. These defects were still evident in NICD1-expressing embryos irrespective of the presence or absence of Pofut1, which indicates that Pofut1 is required for Notch signaling upstream of NICD1. We further found that Pofut1-null cells do not possess normally localized Notch1 receptors, which may results in their lack of interaction with the Dll1 ligand in the presomitic mesoderm where Notch signaling plays a pivotal role. We propose that altered trafficking pathways may account for the abnormal accumulation of the Notch1 receptor in the endoplasmic reticulum in Pofut1-null mouse embryos.     

Alteration of serum and cardiac tissue adropin,copeptin, irisin and TRPM2 expressions in DOX treated male rats

Abstract

Doxorubicin (DOX) cardiotoxicity is a significant side effect in cancer survivors. DOX and its metabolites alter cardiac gene expression and affect metabolic energy-related peptides. Adropin, copeptin, irisin and TRPM2 are produced locally in the heart and play a role in energy homeostasis. We investigated the fates of adropin, copeptin, irisin and TRPM2 in serum and cardiac tissues of DOX treated rats. Animals were divided into three groups of six: 1) untreated controls, 2) DOX treated and 3) saline treated. The rats were fed a standard diet ad libitum for 14 days then were sacrificed and heart and serum samples were taken. Adropin, copeptin, irisin levels in tissue homogenates and serum were measured using ELISA. Immunoreactivity of heart tissue adropin, copeptin, irisin and TRPM2 also were investigated. The peptides increased in both serum and cardiac tissue homogenates in animals treated with DOX compared to the other groups. DOX increased adropin in endocardial and myocardial cells, but it decreased expression of copeptin. DOX did not affect endocardial irisin and TRPM2 expressions, but myocardial irisin and TRPM2 expressions were increased. Serum adropin, irisin and copeptin were increased in DOX treated rats. Cardiac adropin, copeptin, irisin and TRPM2 are affected by DOX and may play a role in DOX cardiotoxicity.     

TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. We identified, TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. Our genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz–Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, our results suggest that TRAF6 may function as a negative regulator of Notch signaling.     

Contribution of the neural cell recognition molecule NB‐3 to synapse formation between parallel fibers and Purkinje cells in mouse

The neural cell recognition molecule NB‐3, also referred to as contactin‐6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB‐3 to cerebellar development, focusing on lobule 3 where NB‐3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG‐1, L1, and NB‐3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB‐3‐immunoreactive zone did not overlap with TAG‐1‐immunoreactive zone. By contrast, the L1‐immunoreactive zone overlapped with both the TAG‐1‐ and NB‐3‐immunoreactive zones. NB‐3‐positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB‐3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB‐3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB‐3 knockout mice relative to wild‐type mice at P5 to P10. In parallel with these findings, caspase‐dependent cell death was significantly increased in the NB‐ 3‐deficient cerebellum at P15. Collectively, our results indicate that NB‐3 deficiency affects synapse formation during postnatal cerebellar development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion.     

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.     

Involvement of notch signaling in wound healing

The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with gamma-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.