Ephrin-B2 reverse signaling is required for axon pathfinding and cardiac valve formation but not early vascular development

AbstractVascular development begins with the formation of a primary vascular plexus that is rapidly remodeled by angiogenesis into the interconnected branched patterns characteristic of mature vasculature. Several receptor tyrosine kinases and their ligands have been implicated to control early development of the vascular system. These include the vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) that bind VEGF, the Tie-1 and Tie-2 receptors that bind the angiopoietins, and the EphB4 receptor that binds the membrane-anchored ligand ephrin-B2. Targeted mutations in the mouse germline have revealed essential functions for these molecules in vascular development. In particular, protein-null mutations that delete either EphB4 or ephrin-B2 from the mouse have been shown to result in early embryonic lethality due to failed angiogenic remodeling. The venous expression of EphB4 and arterial expression of ephrin-B2 has lead to the speculation that the interaction of these two molecules leads to bidirectional signaling into both the receptor-expressing cell and the ligand-expressing cell, and that both forward and reverse signals are required for proper development of blood vessels in the embryo. Indeed, targeted removal of the ephrin-B2 carboxy-terminal cytoplasmic tail by another group was shown to perturb vascular development and result in the same early embryonic lethality as the null mutation, leading the authors to propose that ephrin-B2 reverse signaling directs early angiogenic remodeling of the primary vascular plexus [Cell 104 (2001) 57]. However, we show here that the carboxy-terminal cytoplasmic domain of ephrin-B2, and hence reverse signaling, is not required during early vascular development, but it is necessary for neonatal survival and functions later in cardiovascular development in the maturation of cardiac valve leaflets. We further show that ephrin-B2 reverse signaling is required for the pathfinding of axons that form the posterior tract of the anterior commissure. Our results thus indicate that ephrin-B2 functions in the early embryo as a typical instructive ligand to stimulate EphB4 receptor forward signaling during angiogenic remodeling and that later in embryonic development ephrin-B2 functions as a receptor to transduce reverse signals involved in cardiac valve maturation and axon pathfinding

Voluntary Exercise Reduces Alzheimer’s-like Pathology After Inflammation in Mice

Current global statistics estimate that 44.4 million people are afflicted with dementia, and that 50%-75% of these patients suffer from Alzheimer’s disease (AD; Prince et al. 2013). AD, a progressive disorder categorized by neuronal and behavioral deterioration, is the 6th leading cause of death in America (Alz facts and figure 2012). One hallmark pathology of AD is the presence of amyloid-beta (Aβ) in the brain, which can limit cell-to-cell communication, leading to cognitive deficits, and neuronal cell death. Although the exact origins of this disease still remain unknown, one possible catalyst of AD pathology is inflammation. Our lab has previously shown that 7 consecutive peripheral injections of a bacterial mimetic led to systemic inflammation, increased levels of Ab in the brain, and cognitive dysfunction (Kahn et al., 2012; Weintraub et al., 2013). Currently there are very few effective treatments that diminish AD symptomology. One documented way to decrease inflammation without the use of pharmaceuticals is through regular physical exercise (Cho et al., 2003; Cotman & Berchtold, 2002; Cotman et al., 2007). The present study tested the hypothesis that voluntary exercise would decrease the level of brain Ab following inflammation. Interestingly, we found that two weeks of voluntary wheel running after inflammation led to a reduction of Ab when compared to sedentary recovery. These results indicate that exercise may be an effective modality to reduce AD-like pathology, and that these effects appear to be facilitated by higher versus lower levels of exercise, as measured by total distance run

EphB Receptors Coordinate Migration and Proliferation in the Intestinal Stem Cell Niche

SummaryMore than 1010 cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche

Dissociation of EphB2 Signaling Pathways Mediating Progenitor Cell Proliferation and Tumor Suppression

SummarySignaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth

Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein beta during inhibition of adipogenesis by ceramide.

To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, suggesting that it was not affecting nuclear import. CRM1 (more recently named 'exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPbeta at 24 h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis

A Synthetic Formula Amino Acid Diet Leads to Microbiome Dysbiosis, Reduced Colon Length, Inflammation, and Altered Locomotor Activity in C57BL/6J Mice

The effects of synthetic, free-amino acid diets, similar to those prescribed as supplements for (phenylketonuria) PKU patients, on gut microbiota and overall health are not well understood. In the current, multidisciplinary study, we examined the effects of a synthetically-derived, low-fiber, amino acid diet on behavior, cognition, gut microbiome composition, and inflammatory markers. A cohort of 20 male C57BL/6J mice were randomly assigned to either a standard or synthetic diet (n = 10) at post-natal day 21 and maintained for 13 weeks. Sequencing of the 16S rRNA gene from fecal samples revealed decreased bacterial diversity, increased abundance of bacteria associated with disease, such as Prevotella, and a downward shift in gut microbiota associated with fermentation pathways in the synthetic diet group. Furthermore, there were decreased levels of short chain fatty acids and shortening of the colon in mice consuming the synthetic diet. Finally, we measured TNF-α, IL-6, and IL-10 in serum, the hippocampus, and colon, and found that the synthetic diet significantly increased IL-6 production in the hippocampus. These results demonstrate the importance of a multidisciplinary approach to future diet and microbiome studies, as diet not only impacts the gut microbiome composition but potentially systemic health as well