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Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue cancer in children. The prognosis of patients with relapsed or metastatic disease remains poor. ERMS genomes show few recurrent mutations, suggesting that other molecular mechanisms such as epigenetic regulation might play a major role in driving ERMS tumor biology. In this study, we have demonstrated the diverse roles of histone deacetylases (HDACs) in the pathogenesis of ERMS by characterizing effects of HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) in vitro and in vivo. TSA and SAHA suppress ERMS tumor growth and progression by inducing myogenic differentiation as well as reducing the self-renewal and migratory capacity of ERMS cells. Differential expression profiling and pathway analysis revealed downregulation of key oncogenic pathways upon HDAC inhibitor treatment. By gain-of-function, loss-of-function, and chromatin immunoprecipitation (ChIP) studies, we show that Notch1- and EphrinB1-mediated pathways are regulated by HDACs to inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Our study demonstrates that aberrant HDAC activity plays a major role in ERMS pathogenesis. Druggable targets in the molecular pathways affected by HDAC inhibitors represent novel therapeutic options for ERMS patients.

Mutations in X-linked ephrin-B1 in humans cause craniofrontonasal syndrome (CFNS), a disease that affects female patients more severely than males. Sorting of ephrin-B1-positive and -negative cells following X-inactivation has been observed in ephrin-B1(+/-) mice; however, the mechanisms by which mosaic ephrin-B1 expression leads to cell sorting and phenotypic defects remain unknown. Here we show that ephrin-B1(+/-) mice exhibit calvarial defects, a phenotype autonomous to neural crest cells that correlates with cell sorting. We have traced the causes of calvarial defects to impaired differentiation of osteogenic precursors. We show that gap junction communication (GJC) is inhibited at ectopic ephrin boundaries and that ephrin-B1 interacts with connexin43 and regulates its distribution. Moreover, we provide genetic evidence that GJC is implicated in the calvarial defects observed in ephrin-B1(+/-) embryos. Our results uncover a novel role for Eph/ephrins in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues.

Craniofrontonasal syndrome (CFNS) is an X-linked malformation syndrome with variable phenotype that is caused by mutations in the ephrin-B1 gene ( EFNB1 ). Over 50% of EFNB1 mutations result in premature termination codons that may elicit mRNA degradation by the nonsense-mediated decay pathway. To assess the effects of various mutations at the transcript level, expression of EFNB1 mRNA was studied by RT-PCR in fibroblast cultures established from CFNS female patients. Compared to the wild-type and two missense mutation alleles, severe depletion of transcripts was observed for mutant alleles harbouring either splice site mutation c.407-2A>T at the exon 2/3 boundary or frameshift mutation c.377_384delTCAAGAAG in exon 2. In contrast, escape from mRNA decay was observed for mutation c.614_615delCT, which generates a premature termination codon close to the 3′-end of the penultimate exon 4 disobeying the ‘50–55 bp’ rule. These results suggest differential degradation of mutant EFNB1 transcripts by the nonsense-mediated mRNA decay pathway. Although the clinical phenotypes of the patients were not highly suggestive of a phenotype–genotype correlation, the two female patients were diagnosed with diaphragmatic hernia harbouring putative ephrin-B1 truncating mutations. Previously, disease manifestation in heterozygous females had been attributed mainly to cellular interference of divergent cell populations expressing wild-type or mutant EFNB1 , depending on the pattern of X-inactivation. Upon clonal expansion of patient cells with either the wild-type or mutant EFNB1 on the active X-chromosome, we were able to separate mutant and wild-type EFNB1 -expressing cells in vitro , further supporting the concept of cellular interference in CFNS.

Background Mutations of EFNB1 cause the X-linked malformation syndrome craniofrontonasal syndrome (CFNS). CFNS is characterized by an unusual phenotypic pattern of inheritance, because it affects heterozygous females more severely than hemizygous males. This sex-dependent inheritance has been explained by random X-inactivation in heterozygous females and the consequences of cellular interference of wild type and mutant EFNB1 -expressing cell populations. EFNB1 encodes the transmembrane protein ephrin-B1, that forms bi-directional signalling complexes with Eph receptor tyrosine kinases expressed on complementary cells. Here, we studied the effects of patient-derived EFNB1 mutations predicted to give rise to truncated ephrin-B1 protein or to disturb Eph/ephrin-B1 reverse ephrin-B1 signalling. Five mutations are investigated in this work: nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT, splice-site mutation c.406 + 2T > C and two missense mutations p.P54L and p.T111I. Both missense mutations are located in the extracellular ephrin domain involved in Eph-ephrin-B1 recognition and higher order complex formation. Methods Nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT and splice-site mutation c.406+2T > C were detected in the primary patient fibroblasts by direct sequencing of the DNA and were further analysed by RT-PCR and Western blot analyses. The impact of missense mutations p.P54L and p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was analysed in a cell culture model using NIH 3T3 fibroblasts. These cells were transfected with the constructs generated by in vitro site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy. Results and Discussion Nonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT escape nonsense-mediated RNA decay (NMD), splice-site mutation c.406+2T > C results in either retention of intron 2 or activation of a cryptic splice site in exon 2. However, c.614_615delCT and c.406+2T > C mutations were found to be not compatible with production of a soluble ephrin-B1 protein. Protein expression of the p.R66X mutation was predicted unlikely but has not been investigated. Ectopic expression of p.P54L ephrin-B1 resists Eph-receptor mediated cell cluster formation in tissue culture and intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. Cells expressing p.T111I protein show similar responses as wild type expressing cells, however, phosphorylation of Tyr324 and Tyr329 is reduced. Conclusions Pathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference.

gamma-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid beta-peptide (Abeta) and the APP intracellular domain. Abeta is widely believed to have a causative role in Alzheimer's disease pathogenesis, and therefore modulation of gamma-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of gamma-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to gamma-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs). Here, we investigated gamma-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Abeta40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Abeta were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Abeta40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased. In summary our data suggests that gamma-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of gamma-secretase inhibitors.

Germinal centers (GCs) are the site of antibody affinity maturation. The GC response fundamentally depends on contact-dependent signal exchange between antigen-specific T and B lymphocytes. Lu et al. uncovered a repulsive guidance system that inhibits GC recruitment and retention of T follicular helper (T FH ) cells while simultaneously promoting their helper activities locally (see the Perspective by Moschovakis and Forster). This system comprises the GC-specific transmembrane ephrin B1 (EFNB1) molecule and two EFNB1 receptors—EPHB4 and EPHB6—expressed by activated T cells, including T FH cells. In the absence of EFNB1 on GC B cells or when EPHB6 was suppressed on T cells, inappropriately large numbers of T cells were recruited to and retained in the GC as a result of relaxed repulsion of T FH cells. Science , this issue p. eaai9264 ; see also p. 703 A repulsive guidance system is involved in immunological T cell recruitment to the sites of antibody affinity maturation. Follicular T helper (T FH ) cells orchestrate the germinal center (GC) reaction locally. Local mechanisms regulating their dynamics and helper functions are not well defined. Here we found that GC-expressed ephrin B1 (EFNB1) repulsively inhibited T cell to B cell adhesion and GC T FH retention by signaling through T FH -expressed EPHB6 receptor. At the same time, EFNB1 promoted interleukin-21 production from GC T FH cells by signaling predominantly through EPHB4. Consequently, EFNB1-null GCs were associated with defective production of plasma cells despite harboring excessive T FH cells. In a competitive GC reaction, EFNB1-deficient B cells more efficiently interacted with T FH cells and produced more bone-marrow plasma cells, likely as a result of gaining more contact-dependent help. Our results reveal a contact-dependent repulsive guidance system that controls GC T FH dynamics and effector functions locally.

Background: Z-360 is an orally active cholecystokinin-2 (CCK2)/gastrin receptor antagonist currently under development as a therapeutic drug for pancreatic cancer. It was previously reported that Z-360 treatment in combination with gemcitabine prolonged the survival period in a lethal pancreatic cancer xenograft model in mice. In a phase Ib/IIa clinical study, Z-360 treatment displayed a trend of reduced pain in patients with advanced pancreatic cancer in combination with gemcitabine including analgesics such as opioids. Here, we investigated the mechanism of analgesic action of Z-360 in a severe cancer-induced pain model in mice, which is considered to be opioid-resistant, by examining ephrin B1 gene expression, N-methyl-D-aspartate receptor NR2B subunit phosphorylation, and interleukin-1β (IL-1β) production. Results: In a mouse model of cancer-induced pain, ephrin B1 gene expression in dorsal root ganglia (DRGs) and the phosphorylation of NR2B in the spinal cord were induced. Z-360 treatment inhibited both ephrin B1 gene expression and the phosphorylation of NR2B. In addition, IL-1β production increased in the cancer-inoculated hind paw of mice, but could be suppressed by treatment with Z-360. Moreover, we observed that the CCK1 receptor antagonist devazepide similarly suppressed up-regulation of ephrin B1 gene expression and IL-1β production, and that the intraperitoneal injection of sulfated CCK-8 induced the production of IL-1β in the cancer-inoculated region. Conclusions: We have identified a novel pain cascade, in which IL-1β production in cancer-inoculated regions induces ephrin B1 gene expression in DRGs and then ephrin B1 enhances the tyrosine phosphorylation of NR2B via Eph B receptor in the spinal cord. Notably, Z-360 relieves cancer-induced pain by preventing this pain cascade through the suppression of IL-1β production, likely via the blockade of CCK1 receptor. The pre-clinical results presented here support the analgesic action of Z-360 in pancreatic cancer patients with severe, opioid-resistant pain. Pre-clinical and clinical results have demonstrated that Z-360 combined with gemcitabine represents a promising pancreatic cancer therapy approach with characteristic analgesic effects in addition to the prolongation of survival.

Prolactin (PRL) is known to participate in the lactation-induced maternal bone loss, presumably by inducing the release of receptor activator of nuclear factor-κB ligand (RANKL), a potent osteoclastogenic factor from osteoblasts. Since maternal bone resorption was too massive to be solely explained by RANKL and osteoclasts did not express PRL receptors (PRLR), the involvement of some other osteoblast-derived osteoclastogenic modulators was anticipated. Herein, the authors used quantitative real-time PCR to investigate the mRNA expressions of various osteoclastogenic factors in osteoblast-like UMR106 cells directly exposed to PRL for 48 h. These cells were found to express PRLR and respond to 300 ng/ml PRL by increasing RANKL mRNA expression. This PRL concentration (comparable to plasma PRL levels in lactation) also induced the upregulation of monocyte chemoattractant protein (MCP)-1, cyclooxygenase (Cox)-2, and ephrin-B1, whereas a higher concentration (500 ng/ml) was required to upregulate tumor necrosis factor (TNF)-α and interleukin (IL)-1. However, 100-500 ng/ml PRL affected neither the cell proliferation, the cell viability nor the mRNA expressions of macrophage colony-stimulating factor, IL-6, ephrin type-B receptor 4 and ephrin-B2. In conclusion, besides RANKL overexpression, PRL upregulated the expressions of other osteoclastogenic modulators, i.e., MCP-1, Cox-2, TNF-α, IL-1, and ephrin-B1, thus, further explaining how PRL induced bone loss in lactating mothers.