Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE141401 | GEO | 2019/12/04
REPOSITORIES: GEO
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Project description:Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.
| S-EPMC7305688 | biostudies-literature
Project description:Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture
| PRJNA593274 | ENA
Project description:Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in C. elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.
2024-01-29 | PXD041786 | Pride
Project description:Objectives: Eph/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). Methods: We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from SSc patients and healthy controls, which was followed by in vivo analysis of fibroblast-specific EphB2 deficient mice. Results: Expression of EphB2 is upregulated in SSc skin tissue and explanted SSc dermal fibroblasts compared to healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-β (TGFβ) cytokines upregulate EphB2 in dermal fibroblasts via non-canonical TGFβ/SMAD signaling and silencing EphB2 in dermal fibroblasts is sufficient to dampen TGFβ-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. Conclusion: Our data implicate EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.
2024-04-10 | GSE253089 | GEO
Project description:Human epidermal keratinocytes were treated with 25 ng.ml EphB2 or EFNA4, both as-Fc conjugates (Sigma). Human epidermal keratinocytes are treated with 25 ng/ml EphB2 or EFNA4 Fc conjugates in a 48hr time course.
2011-03-01 | E-GEOD-26523 | biostudies-arrayexpress
Project description:The role of Eph/ephrin signaling in numerous biological processes has been established. However, Eph/ephrin signaling has been shown to have complex role in tumor progression. The role of EphB2 receptor in the progression of cutaneous squamous cell carcinoma (cSCC) has not been studied before. EphB2 is significantly overexpressed in cSCC cells compared with normal human epidermal keratinocytes (NHEKs). We used microarray based global gene expression profiling to study the effect of EphB2 knockdown on the expression of different genes in cSCC cells. cSCC cell lines (n=3) were transfected with either control or EphB2 siRNA (75 nM) and incubated for 72 hours. Total RNA was extracted and processed expression profiling with GeneChip 3’ IVT Express Kit according to manufacturer’s protocol.
2015-08-27 | E-GEOD-66361 | biostudies-arrayexpress
Project description:Local cerebral hypoperfusion causes ischemic stroke while driving multiple cell-specific responses including inflammation, glutamate-NMDAR-mediated neurotoxicity, edema formation and angiogenesis. Despite the relevance of these pathophysiological mechanisms for disease progression and outcome, molecular determinants controlling the onset of these processes are only partially understood. In this context, our study intended to investigate the functional role of EphB2 – a receptor tyrosine kinase that is crucial for synapse function and binds to membrane-associated ephrin-B ligands. Cerebral ischemia was induced in EphB2-deficient mice by transient middle cerebral artery occlusion followed by different times (6, 12, 24 and 48 h) of reperfusion. Histological, neurofunctional and transcriptome analyses indicated an increase in EphB2 phosphorylation under these conditions and attenuated progression of stroke in EphB2-deficient mice. Moreover, while infiltration of microglia/macrophages and astrocytes into the peri-infarct region was not altered, expression of the pro-inflammatory mediators MCP-1 or IL-6 was decreased in these mice. In fact, in vitro analyses indicated that binding of EphB2 to astrocytic ephrin-B ligands stimulates NF-κB-mediated cytokine expression via the MAPK pathway. Further magnetic resonance imaging of the EphB2-deficient ischemic brain revealed a lower level of neuronal cytotoxic edema formation within 6 h upon onset of reperfusion. On the mechanistic level, absence of neuronal EphB2 decreased the mitochondrial Ca2+ load upon activation of NMDAR but not AMPAR. Moreover, activation of EphB2 by ephrin-B2 enhanced the NMDA-evoked excitotoxic neuronal cell death in vitro while neuron-specific loss of ephrin-B2 reduced the extent of cerebral tissue damage in the acute phase of ischemic stroke. Collectively, EphB2 may promote the immediate response to an ischemia-reperfusion event in the central nervous system by (i) pro-inflammatory activation of astrocytes via ephrin-B-dependent signalling and (ii) amplification of the NMDA-evoked neuronal excitotoxicity.
2019-02-13 | GSE120565 | GEO
Project description:Current therapeutic strategies for treating non-alcoholic steatohepatitis (NASH) have failed to alleviate liver fibrosis, which is a devastating feature leading to hepatic dysfunction. Here, we integrated single-nucleus transcriptomics and epigenomics to characterize all major liver cell types during NASH development in mice and humans. The bifurcation of hepatocyte trajectory with NASH progression was conserved between mouse and human. At the non-alcoholic fatty liver (NAFL) stage, hepatocytes exhibited metabolic adaptation, whereas at the NASH stage, a subset of hepatocytes was enriched for the signatures of cell adhesion and migration, which was mainly demarcated by receptor tyrosine kinase EphB2. EphB2, acting as a downstream effector of Notch signaling in hepatocytes, was sufficient to induce cell-autonomously inflammation. Knockdown of Ephb2 in hepatocytes ameliorated inflammation and fibrosis in NASH. Thus, EphB2 expressing hepatocytes contribute to NASH progression and may serve as a potential therapeutic target.
2023-02-08 | GSE189600 | GEO
Project description:Human epidermal keratinocytes were treated with 25 ng.ml EphB2 or EFNA4, both as-Fc conjugates (Sigma).
2011-03-01 | GSE26523 | GEO
Project description:UnlabelledBeyond the well-defined role of the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinases in developmental processes, cell motility, cell trafficking/adhesion, and cancer, nothing is known about their involvement in liver pathologies. During blood-stage rodent malaria infection we have found that EphB2 transcripts and proteins were up-regulated in the liver, a result likely driven by elevated surface expression on immune cells including macrophages. This was significant for malaria pathogenesis because EphB2(-/-) mice were protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate control mice. This protection was correlated with a defect in the inflammatory potential of hepatocytes from EphB2(-/-) mice resulting in a reduction in adhesion molecules, chemokine/chemokine receptor RNA levels, and infiltration of leukocytes including macrophages/Kupffer cells, which mediate liver fibrosis during rodent malaria infections. These observations are recapitulated in the well-established carbon tetrachloride model of liver fibrosis in which EphB2(-/-) carbon tetrachloride-treated mice showed a significant reduction of liver fibrosis compared to carbon tetrachloride-treated littermate mice. Depletion of macrophages by clodronate-liposomes abrogates liver EphB2 messenger RNA and protein up-regulation and fibrosis in malaria-infected mice.ConclusionDuring rodent malaria, EphB2 expression promotes malaria-associated liver fibrosis; to our knowledge, our data are the first to implicate the EphB family of receptor tyrosine kinases in liver fibrosis or in the pathogenesis of malaria infection.
| S-EPMC4549176 | biostudies-literature