Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

Tumour necrosis factor (TNF)-induced cell death involves the generation of ceramide by endosomal acid sphingomyelinase (A-SMase). This study reveals that in TNF-receptosomes, A-SMase is activated by caspase-7-mediated proteolytic cleavage downstream of caspase-8 activation

Role of Acid Sphingomyelinase-Induced Signaling in Melanoma Cells for Hematogenous Tumor Metastasis

Background: Hematogenous metastasis of malignant tumor cells is a multistep process that requires release of tumor cells from the local tumor mass, interaction of the tumor cells with platelets in the blood, and adhesion of either the activated tumor cells or the complexes of platelets and tumor cells to the endothelial cells of the target organ. We have previously shown that the interaction of melanoma cells with platelets results in the release of acid sphingomyelinase (Asm) from activated platelets. Secreted platelet-derived Asm acts on malignant tumor cells to cluster and activate integrins; such clustering and activation are necessary for tumor cell adhesion to endothelial cells and for metastasis. Methods: We examined the response of tumor cells to treatment with extracellular sphingomyelinase or co-incubation with wild-type and Asm-deficient platelets. We determined the phosphorylation and activation of several intracellular signaling molecules, in particular p38 kinase (p38K), phospholipase Cγ (PLCγ), ezrin, and extracellular signal-regulated kinases. Results: Incubation of B16F10 melanoma cells with Asm activates p38 MAP kinase (p38K), phospholipase Cγ (PLCγ), ezrin, and extracellular signal-regulated kinases. Co-incubation of B16F10 melanoma cells with wild-type or Asm-deficient platelets showed that the phosphorylation/activation of p38K is dependent on Asm. Pharmacological blockade of p38K prevents activation of β1 integrin and adhesion in vitro. Most importantly, inhibition of p38K activity in B16F10 melanoma cells prevents tumor cell adhesion and metastasis to the lung in vivo, a finding indicating the importance of p38K for metastasis. Conclusions: Asm, secreted from activated platelets after tumor cell-platelet contact, induces p38K phosphorylation in tumor cells. This in turn stimulates β1 integrin activation that is necessary for adhesion and subsequent metastasis of tumor cells. Thus, inhibition of p38K might be a novel target to prevent tumor metastasis

Comparative assessment of female mouse model of Graves' orbitopathy under different environments, accompanied by pro-inflammatory cytokine and T cell responses to thyrotropin hormone receptor antigen

We recently described a preclinical model of Graves' orbitopathy (GO), induced by genetic immunization of eukaryotic expression plasmid encoding human TSH-receptor (hTSHR) A-subunit by muscle electroporation in female BALB/c mice. The onset of orbital pathology is characterized by muscle inflammation, adipogenesis and fibrosis. Animal models of autoimmunity are influenced by their environmental exposures. This follow-up study was undertaken to investigate the development of experimental GO in two different locations, run in parallel under comparable housing conditions. Functional antibodies to TSHR were induced in TSHR A-subunit plasmid immunized animals, and antibodies to IGF-1 receptor α subunit were also present, while control animals were negative in both locations. Splenic T cells from TSHR A-subunit primed animals undergoing GO in both locations showed proliferative responses to purified TSHR antigen and secreted IFN-γ, IL-10, IL-6 and TNF-α cytokines. Histopathological evaluation showed orbital tissue damage in mice undergoing GO, manifest by adipogenesis, fibrosis and muscle damage with classic signs of myopathy. Although no inflammatory infiltrate was observed in orbital tissue in either location, the appearances were consistent with a 'hit and run' immune-mediated inflammatory event. A statistically significant increase of cumulative incidence of orbital pathology when compared to control animals was shown for both locations, confirming onset of orbital dysimmune myopathy. Our findings confirm expansion of the model in different environments, accompanied with increased prevalence of T cell derived pro-inflammatory cytokines, with relevance for pathogenesis. Wider availability of the model makes it suitable for mechanistic studies into pathogenesis and undertaking of novel therapeutic approaches

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation.

JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, β1 and β2, may contribute to CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1- (VCAM1-) and intercellular adhesion molecule 1-coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti-VLA-4 and anti-β2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-β2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen