Although mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis.

Several inhibitors of BCL2 proteins have been identified that induce apoptosis in a variety of tumor cells, indicating their potential in cancer therapy. We investigated the specificity of six putative BCL2 inhibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine and ABT-737). Using cells deficient either for Bax/Bak or caspase-9, we found that only ABT-737 specifically targeted BCL2 proteins and induced apoptosis by activation of caspase-9, as only ABT-737 induced apoptosis was completely inhibited in cells deficient for Bax/Bak or caspase-9. Our data show that only ABT-737 is a specific BCL2 inhibitor and all other compounds investigated were not specific for BCL2 proteins. Furthermore, investigations of the effects of these compounds in primary chronic lymphocytic leukemic cells showed that all compounds induced certain biochemical hallmarks of apoptosis, such as release of cytochrome c and caspase cleavage. However, they all caused strikingly different ultrastructural changes. ABT-737 induced all the characteristic ultrastructural changes of apoptosis together with early rupture of the outer mitochondrial membrane, whereas obatoclax, chlelerythrine and gossypol induced pronounced mitochondrial swelling with formation of phospholipid inclusions. Therefore, we conclude that biochemical measurements used earlier to define apoptosis like mitochondrial release of cytochrome c and caspase cleavage, are insufficient to distinguish between classic apoptosis and other forms of cell death.

Necroptotic signaling converges in the assembly of a cytosolic signaling platform, the necrosome, with the activation of its downstream effector, MLKL. RIPK1 and RIPK3, key components of the necrosome, act as signaling intermediates for the activation of MLKL. We report that RIPK3 and MLKL continuously shuttle between the nucleus and the cytoplasm, whereas RIPK1 is constitutively present in both compartments. During TNF-induced necroptosis, nuclear RIPK1 becomes ubiquitinated, after which nuclear MLKL becomes phosphorylated and oligomerized. Pharmacological inhibition of the nuclear export machinery leads to retention of RIPK3 and MLKL in the nucleus, prevents the nucleation of cytosolic RIPK3/MLKL oligomerization, and reduces cell death. Our results suggest that passage of necroptotic signaling components through the nucleus is a mechanism for regulating cytosolic necrosome formation and consequently necroptotic cell death.

BackgroundRecent studies have demonstrated that a molecular subtype of glioblastoma is characterized by overexpression of extracellular matrix (ECM)/mesenchymal components and shorter survival. Specifically, gene expression profiling studies revealed that matrix gla protein (MGP), whose function has traditionally been linked to inhibition of calcification of arteries and cartilage, is overexpressed in glioblastomas and associated with worse outcome.MethodsIn order to analyze the role of MGP in glioblastomas, we performed expression, migration and proliferation studies.ResultsReal-time PCR and ELISA assays confirmed overexpression of MGP in glioblastoma biopsy specimens and cell lines at mRNA and protein levels as compared to normal brain tissue. Immunohistochemistry verified positivity of glial tumor cells for MGP. RNAi-mediated knockdown of MGP in three glioma cell lines (U343MG, U373MG, H4) led to marked reduction of migration, as demonstrated by wound healing and transwell assays, while no effect on proliferation was seen.ConclusionOur data suggest that upregulation of MGP (and possibly other ECM-related components as well) results in unfavorable prognosis via increased migration.

LNM occurs in one quarter of all patients with T1 PTC, and also in the subset with microcarcinoma. Performing (131)I SPECT/CT, either with therapeutic or diagnostic radioactivities, directly after thyroidectomy should provide more accurate staging of T1 PTC, thus facilitating optimal therapeutic management.

BackgroundAdipose tissue is a promising source of mesenchymal stromal cells (MSCs) for the treatment of tendon disease. The goal of this study was to assess the effect of a single intralesional implantation of adipose tissue-derived mesenchymal stromal cells (AT-MSCs) on artificial lesions in equine superficial digital flexor tendons (SDFTs).MethodsDuring this randomized, controlled, blinded experimental study, either autologous cultured AT-MSCs suspended in autologous inactivated serum (AT-MSC-serum) or autologous inactivated serum (serum) were injected intralesionally 2 weeks after surgical creation of centrally located SDFT lesions in both forelimbs of nine horses. Healing was assessed clinically and with ultrasound (standard B-mode and ultrasound tissue characterization) at regular intervals over 24 weeks. After euthanasia of the horses the SDFTs were examined histologically, biochemically and by means of biomechanical testing.ResultsAT-MSC implantation did not substantially influence clinical and ultrasonographic parameters. Histology, biochemical and biomechanical characteristics of the repair tissue did not differ significantly between treatment modalities after 24 weeks. Compared with macroscopically normal tendon tissue, the content of the mature collagen crosslink hydroxylysylpyridinoline did not differ after AT-MSC-serum treatment (p = 0.074) while it was significantly lower (p = 0.027) in lesions treated with serum alone. Stress at failure (p = 0.048) and the modulus of elasticity (p = 0.001) were significantly lower after AT-MSC-serum treatment than in normal tendon tissue.ConclusionsThe effect of a single intralesional injection of cultured AT-MSCs suspended in autologous inactivated serum was not superior to treatment of surgically created SDFT lesions with autologous inactivated serum alone in a surgical model of tendinopathy over an observation period of 22 weeks. AT-MSC treatment might have a positive influence on collagen crosslinking of remodelling scar tissue. Controlled long-term studies including naturally occurring tendinopathies are necessary to verify the effects of AT-MSCs on tendon disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0564-8) contains supplementary material, which is available to authorized users.

SummaryBAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models.

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