A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation

The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.

EphrinB-EphB Signaling Induces Hyperalgesia through ERK5/CREB Pathway in Rats

Background: There are numerous studies implicating that EphB receptors and ephrinB ligands play important roles in modulating the transduction of spinal nociceptive information. EphrinB-EphB signaling may contribute to hyperalgesia via various kinds of downstream molecules, the mechanisms of which have not been completely understood. Objective: The aim of the present study was to identify whether ephrinB-EphB signaling could contribute to hyperalgesia through ERK5/CREB pathway. Study Design: Controlled animal study. Setting: University laboratory. Methods: This study attempted to detect the changes of pain behaviors and the protein level of p-ERK5 and p-CREB by activating EphB receptors in the spinal cord of rats. To further confirm our hypothesis, we designed LV-siRNA for knockdown of spinal ERK5. When ERK5 was inhibited, we recorded the changes of spinal p-CREB expression and the pain behaviors of rats after activating EphB receptors. We also confirmed this conclusion in rat CCI model. Statistical analyses were performed using GraphPad Prism 5. Results: Intrathecal injection of ephrinB2-Fc in rats evoked thermal hyperalgesia and mechanical allodynia, along with activation of ERK5 and CREB in the spinal cord. Knockdown of ERK5 inhibited ephrinB2-Fc-induced CREB activation and hyperalgesia. Blocking EphB receptors prevented CCI-induced neuropathic pain and spinal ERK5/CREB activation. Limitations: More underlying mechanisms that underlie the relationship between ephrinBEphB signaling and ERK5/CREB pathway will need to be explored in future studies. Conclusions: Our study suggests that ERK5/CREB pathway plays important roles in the transduction of nociceptive information associated with ephrinB-EphB signaling. This study provides further understanding of the downstream mechanisms of ephrinB-EphB signaling and helps to explore new targets for treating pathological pain. Key words: EphrinB-EphB signaling, MAPK, ERK5, CREB, hyperalgesia, pain, CCI, NMDA

Tiam–Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion

Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB+ cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB+ cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion.

EphrinB1 Activation As a Potential New Treatment Option in AML

Abstract 5235 Aberrant Ephrin signaling has been shown to be an important pathway that contributes to the pathogenesis of many solid tumors (Surawska et al. Cytokine & Growth factor reviews 2004). Deregulated ephrin receptor (Eph) and ligand (Efn) expression is often associated with poor prognosis in solid tumors. Ephrin receptor and ligand overexpression can result in tumorigenesis through induced tumor growth, tumor cell survival, angiogenesis and metastasis (Surawska et al. Cytokine & Growth Factor Reviews 2004; Campbell et al. Curr. Isues Mol. Biol. 2008; Chen et al. Cancer Research 2008). In normal cells Eph receptors and ligands play key roles in vascular patterning, where they function in endothelial cell migration, and proliferation (Adams et al, Genes Dev. 1999; Zhang et al., Blood 2001). Thus far particularly EphB4 receptor and ephrin-B2 ligand have been implicated in the process of normal angiogenesis. In acute myeloid leukemia (AML) patients it was found that bone marrow biopsies at diagnosis exhibited enhanced microvessel density (MVD) (de Bont ES et al., BJH 2001; Byrd JC et al., Blood 2002; Padro et al., Blood 2000). Normal hematopoietic stem cells (HSCs) express the following mRNA transcripts ephrin receptors EphA1, EphA2, EphB2, and EphB4 and ephrin ligands EfnA3, EfnA4, and EfnB2. Moreover, overexpression of EphB4 receptor in HSCs (from cord blood) resulted in enhanced differentiation towards megakaryocytes (Wang et al. Blood 2002). In AML cell lines there is a common co-expression on protein level observed between EphB4 receptor and ephrin-B2 ligand. Recently, an aberrant DNA methylation of ephrin receptors and ligands was described in acute lymphocytic and myelocytic leukemia cell lines (Kuang et al. Blood 2010). In addition, restoration of EphB4 expression in an acute lymphoid leukemia cell line resulted in reduced proliferation and apoptotic cell death. These data suggests that the ephrin signaling pathway might play an important role in leukemia. In a previous study we have found high kinase activity of EphB receptors and high phosphorylation levels of EphB receptors in AML samples, as measured using kinase arrays and proteome profiler arrays. In this study, we have found extensive membrane expression of EphB1 on AML cell lines and primary AML blasts. To identify the role of Ephrin signaling in AML, two AML cell lines THP-1 and HL60 with an EphB1 membrane expressing cell percentage of 70% and 20% respectively were chosen for stimulation with Ephrin-B1 ligand. Treatment of these cell lines with Ephrin-B1 ligand resulted in a decreased proliferation 30% in THP-1 cells versus 22% in HL60 cells and increased apoptosis 23% in THP-1 cells and 4% in HL60 cells. Of note, the most prominent effect of Ephrin-B1 stimulation was found in THP-1 cells, this cell line contained a higher percentage of EphB1 membrane expressing cells. We further investigated the mechanism through which EphB1 reduces leukemic cell growth and induces leukemic cell death in THP-1 cells. Westernblot analysis of cell cycle regulators showed that expression of the anti-apoptotic protein BCL2 is reduced upon Ephrin-B1 ligand stimulation and the expression of the pro-apoptotic protein BAX is induced. In addition, mRNA expression of the cell cycle inhibitor of cell cycle progression p21 was found to be 2,5 fold upregulated in ephrin-B1 ligand treated cells compared to untreated control cells. MGG stainings of Ephrin-B1 treated cells revealed multiple cells with two nuclei in both THP-1 and HL60 cells. These results indicate that a high percentage of AML cells express EphB1 receptor on the membrane and that stimulation of these cells with Ephrin-B1 ligand results in reduced leukemic growth and increased cell death. EphrinB1 activation in AML deserves further investigation considering EphB1 as a putative new treatment option for AML patients. Disclosures: No relevant conflicts of interest to declare.