Background Cancer patients have an approximately four-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF+) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF+ tumor-derived MVs (TMVs) activate platelets in vitro and in mice. Materials and Methods We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF+ TMVs on platelets and thrombosis. Results and Conclusions We found that both types of TF+ TMVs activated human platelets and induced aggregation in vitro in a TF- and thrombin-dependent manner. Further, injection of BxPc-3 TF+ TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF+ TMV-induced platelet activation contributes to thrombosis in cancer patients.

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BackgroundThe plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin–amphiphysin–Rvs (BAR)–pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair.MethodsWe used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice.ResultsHerein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin.ConclusionsOverall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0054-6) contains supplementary material, which is available to authorized users.

Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo. Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux.Macroautophagy is an evolutionarily conserved intracellular degradation/recycling process in which cytoplasmic cargo is non-selectively enveloped within double-membraned vesicles called autophagosomes that are transported to and fuse with lysosomes. Within these so-called autolysosomes, cytoplasmic cargo and the inner membrane are degraded so that the amino acids, fatty acids, and glucose released can be used to support cellular metabolism or to synthesize new proteins. Cardiomyocytes are both highly metabolically active cells and long-lived cells and as such are particularly dependent on macroautophagy (hereafter referred to as autophagy) for energy production and removal of damaged organelles or misfolded proteins. However, in some cases up-regulation of autophagy (or failure of autophagy suppression) can lead to detrimental cardiac remodeling.Autophagy is regulated in a stepwise fashion that involves the formation of distinct protein complexes composed of autophagy-related genes (Atg proteins) and their enzymatic binding partners. Nutrient deprivation-induced activation of the AMP kinase/Unc-51-like autophagy-activating kinase 1 (ULK) kinase cascade leads to the recruitment of core proteins VPS34, VPS15, and Beclin1 (complex I) to endoplasmic reticulum exit sites to form phosphatidylinositol 1,4,5-phosphate 3-kinase-dependent double membrane autophagosome precursors. Complex I then facilitates the recruitment of additional proteins, includi...

High constitutive expression of the c-myc oncogene in human melanoma leads to downregulation of expression of HLA Class I genes. The genes at the HLA-B locus are preferentially affected. To investigate the mechanism of downregulation, the activity of the main HLA Class I enhancer, enhancer A-region 1, was compared in a panel of c-myc transfectants with increasing myc expression. Gel retardation experiments demonstrated in all tested cell lines binding of the transcription factors KBF1 and NF-xB to the enhancer. However, no correlation between the levels of HLA Class I expression and binding to the enhancer could be established. Strikingly, the cell line with the highest c-myc expression showed more binding of KBF1 and NF-xB than the parental cell line. By using CAT reporter plasmids in transient transfection assays we investigated the in vivo function of enhancer A-region I in the c-myc transfectant panel. Again, c-myc expression had no effect at all on the activity of enhancer A. This study shows that HLA Class I expression is regulated by the c-myc oncogene at the level of transcription, but that the main HLA Class I enhancer is not involved in this process.