Modulation of interleukin signalling and gene expression in cardiac myocytes by endothelin-1

The related inflammatory cytokines, interleukin- (IL-) 1β and IL-33, are both implicated in the response of the heart to injury. They also activate mitogen-activated protein kinases (MAPKs) in cardiac myocytes. The hypertrophic Gq protein-coupled receptor agonist endothelin-1 is a potentially cardioprotective peptide and may modulate the inflammatory response. Endothelin-1 also stimulates (MAPKs) in cardiac myocytes and promotes rapid changes in expression of mRNAs encoding intercellular and intracellular signalling components including receptors for IL-33 (ST2) and phosphoprotein phosphatases. Prior exposure to endothelin-1 may specifically modulate the response to IL-33 and, more globally, influence MAPK activation by different stimuli. Neonatal rat ventricular myocytes were exposed to IL-1β or IL-33 with or without pre-exposure to endothelin-1 (5 h) and MAPK activation assessed. IL-33 activated ERK1/2, JNKs and p38-MAPK, but to a lesser degree than IL-1β. Endothelin-1 increased expression of soluble IL-33 receptors (sST2 receptors) which may prevent binding of IL-33 to the cell-surface receptors. However, pretreatment with endothelin-1 only inhibited activation of p38-MAPK by IL-33 with no significant influence on ERK1/2 and a small increase in activation of JNKs. Inhibition of p38-MAPK signalling following pretreatment with endothelin-1 was also detected with IL-1β, H2O2 or tumour necrosis factor α (TNFα) indicating an effect intrinsic to the signalling pathway. Endothelin-1 pretreatment suppressed the increase in expression of IL-6 mRNA induced by IL-1β and decreased the duration of expression of TNFα mRNA. Coupled with the general decrease in p38-MAPK signalling, we conclude that endothelin-1 attenuates the cardiac myocyte inflammatory response, potentially to confer cardioprotection.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)(2)] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38(MAPK)/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N`]copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage

Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development.

Spezifische Inhibition des Wnt-Signalwegs und die Analyse der Konsequenzen auf zellbiologischer Ebene

Der kanonische Wnt Signalweg ist durch Regulation einer Vielzahl von Zielgenen in unterschiedliche Prozesse wie Entwicklung, Wachstum und Differenzierung involviert. Fehlregulation des Signalwegs kann zur Tumorentstehung führen. Die exakte Rolle des Wnt Signalwegs und seiner Zielgene in der karzinogenen Kaskade ist noch nicht genau bekannt. In dieser Arbeit sollte die Beteiligung der Wnt Zielgene c-MYC, CCND1 (kodiert Cyclin D1) und VEGF an der Karzinogenese untersucht werden. Um die Funktionen der Wnt Zielgene und ihre zellulären Effekte unabhängig voneinander untersuchen zu können, wurden die Mengen der entsprechenden Transkriptionsprodukte durch siRNA (short interfering RNA) gezielt verringert. Die Konsequenzen der Inaktivierung wurden in Kolon- und Zervixkarzinomzelllinien untersucht, wobei die zellulären Parameter Proliferation, Apoptose, Metabolismus sowie Migration und Adhäsion untersucht wurden. Dabei konnte beobachtet werden, dass der Wnt Signalweg mit seinen Zielgenen Cyclin D1 und c-MYC die Proliferation mit dem Energiemetabolismus von Tumorzellen verknüpft. Darüber hinaus konnte gezeigt werden, dass Cyclin D1 an der Regulation der zelluläre Migration und Adhäsion beteiligt ist, während VEGF die Apoptose abhängig vom zellulären Kontext inhibiert. Diese Ergebnisse liefern erste Hinweise auf die funktionelle Rolle der verschiedenen Zielgene im Prozess der Karzinogenese in Tumoren mit aktiviertem Wnt Signalweg. Damit ist diese Arbeit ein möglicher Ausgangspunkt für Studien mit dem Ziel der gezielten therapeutischen Beeinflussung des Wnt Signalwegs auf Ebene der Zielgene.

Effetti dell'inibizione di Tanchirasi in linee cellulari di Medulloblastoma umano

Tanchirasi (TNKS) è un membro della superfamiglia delle PARP (Poli ADP-Ribosio Polimerasi). TNKS è coinvolta nella stabilizzazione della subunità catalitca del complesso proteico DNA-PK (protein chinasi DNA-dipendente), la DNA-PKcs. Questa proteina è fondamentale per il corretto funzionamento del meccanismo di riparo del DNA chiamato "Saldatura Non Omologa delle Estremità" (NHEJ). La deplezione di TNKS induce una degradazione della DNA-PKcs e una maggiore sensibilità alle radiazioni ionizzanti (RI). TNKS è inoltre un regolatore negativo di axina e di conseguenza un attivatore del pathway di WNT; l'inibizione quindi di TNKS induce anche una inibizione del pathway di WNT. Alterazioni in questo signalling si riscontrano frequentemente nel Medulloblastoma (MB), il tumore cerebrale embrionale più comune dell'infanzia. La radioterapia post-operatoria risulta essere molto efficacia in questa neoplasia, ma causa gravi effetti collaterali e un terzo dei pazienti presenta radioresistenza intrinseca. Un'importante sfida per la ricerca è quindi l'aumento della radiosensibilità tumorale. In questo lavoro, abbiamo studiato gli effetti dell'inibizione farmacologica di TNKS in linee cellulari di MB umano, mediante la small molecule XAV939, potente e specifico inibitore di TNKS. Il trattamento con XAV939 induce una consistente inibizione della capacità proliferativa e clonogenica, non correlata ad un aumento della mortalità cellulare, indicando una bassa tossicità legata alla molecola. Il co-trattamento di XAV939 e RI (γ-ray, dose 2 Gy) causa una ulteriore inibizione della proliferazione cellulare e della capacità di formare colonie. Abbiamo inoltre constatato, mediante Neutral Comet Assay, una minore efficacia nel riparo del DNA in cellule irradiate trattate con XAV939, indicando un effettivo aumento della radiosensibilità in cellule di MB trattate con l'inibitore di TNKS. L'aumentata mortalità cellulare in cellule tumorali trattate con XAV939 e RI ha confermato la nostra ipotesi. Il nostro studio in vitro indica come TNKS possa essere un utile target terapeutico per rendere più efficace l'attuale terapia contro il MB.

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.

Targeting phosphoinositide 3-kinase signalling in lung cancer

Lung cancer is the leading cause of cancer-related mortality worldwide and more than 1 million people annually die in consequence of lung cancer. Although an improvement in lung cancer treatment could be achieved, especially in the last decade, the development of additional therapeutic strategies is urgently required in order to provide improved survival benefit for patients. Lung cancer formation is caused by genetic modifications commonly caused by tobacco smoking. Numerous studies have demonstrated the role of extracellular growth factors in lung cancer cell proliferation, metastasis, and chemoresistance. Mutations and amplifications in molecules related to receptor tyrosine signalling, such as EGFR, ErbB2, c-Met, c-Kit, VEGFR, PI3K, and PTEN are only some of the alterations known to contribute to the development of lung cancer. The phosphoinositide 3-kinase (PI3K) pathway, fundamental for cell development, growth, and survival, is known to be frequently altered in neoplasia, including carcinomas of the lung. Based on the high frequency of alterations, which include mutations and amplifications, leading to over-activation of certain upstream/downstream mediators, targeting components of the PI3K signalling pathway is considered to be a promising therapeutic approach in cancer treatment. In this article we will summarize the current knowledge about the involvement of PI3K signalling in lung cancer and discuss the development of targeted therapies involving PI3K pathway inhibitors.

Fritz: a secreted frizzled-related protein that inhibits Wnt activity

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations

BACKGROUND: Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor. METHODS: After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed. RESULTS: The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased. CONCLUSION: Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

Nutrient-responsive mTOR signalling grows on Sterile ground

The control of cell growth, that is cell size, is largely controlled by mTOR (the mammalian target of rapamycin), a large serine/threonine protein kinase that regulates ribosome biogenesis and protein translation. mTOR activity is regulated both by the availability of growth factors, such as insulin/IGF-1 (insulin-like growth factor 1), and by nutrients, notably the supply of certain key amino acids. The last few years have seen a remarkable increase in our understanding of the canonical, growth factor-regulated pathway for mTOR activation, which is mediated by the class I PI3Ks (phosphoinositide 3-kinases), PKB (protein kinase B), TSC1/2 (the tuberous sclerosis complex) and the small GTPase, Rheb. However, the nutrient-responsive input into mTOR is important in its own right and is also required for maximal activation of mTOR signalling by growth factors. Despite this, the details of the nutrient-responsive signalling pathway(s) controlling mTOR have remained elusive, although recent studies have suggested a role for the class III PI3K hVps34. In this issue of the Biochemical Journal, Findlay et al. demonstrate that the protein kinase MAP4K3 [mitogen-activated protein kinase kinase kinase kinase-3, a Ste20 family protein kinase also known as GLK (germinal centre-like kinase)] is a new component of the nutrient-responsive pathway. MAP4K3 activity is stimulated by administration of amino acids, but not growth factors, and this is insensitive to rapamycin, most likely placing MAP4K3 upstream of mTOR. Indeed, MAP4K3 is required for phosphorylation of known mTOR targets such as S6K1 (S6 kinase 1), and overexpression of MAP4K3 promotes the rapamycin-sensitive phosphorylation of these same targets. Finally, knockdown of MAP4K3 levels causes a decrease in cell size. The results suggest that MAP4K3 is a new component in the nutrient-responsive pathway for mTOR activation and reveal a completely new function for MAP4K3 in promoting cell growth. Given that mTOR activity is frequently deregulated in cancer, there is much interest in new strategies for inhibition of this pathway. In this context, MAP4K3 looks like an attractive drug target since inhibitors of this enzyme should switch off mTOR, thereby inhibiting cell growth and proliferation, and promoting apoptosis.

Mechanisms regulating the p120-catenin/Kaiso pathway

The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.