IκΒα inhibits apoptosis at the outer mitochondrial membrane independently of NF-κB retention.

IκBα resides in the cytosol where it retains the inducible transcription factor NF-κB. We show that IκBα also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF-κB that accumulate high amounts of mitochondrial IκBα as a NF-κB target gene. 3T3 IκBα(-/-) cells also become protected from apoptosis when IκBα is specifically reconstituted at the OMM. Using various IκBα mutants, we demonstrate that apoptosis inhibition and NF-κB inhibition can be functionally and structurally separated. At mitochondria, IκBα stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IκBα is reduced in tumour cells with constitutively active NF-κB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IκBα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF-κB.

CKMT1 regulates the mitochondrial permeability transition pore in a process that provides evidence for alternative forms of the complex.

The permeability transition pore (PT-pore) mediates cell death through the dissipation of the mitochondrial membrane potential (ΔΨm). Because the exact composition of the PT-pore is controversial, it is crucial to investigate the actual molecular constituents and regulators of this complex. We found that mitochondrial creatine kinase-1 (CKMT1) is a universal and functionally necessary gatekeeper of the PT-pore, as its depletion induces mitochondrial depolarization and apoptotic cell death. This can be inhibited efficiently by bongkrekic acid, a compound that is widely used to inhibit the PT-pore. However, when the 'classical' PT-pore subunits cyclophilin D and VDAC1 are pharmacologically inhibited or their expression levels reduced, mitochondrial depolarization by CKMT1 depletion remains unaffected. At later stages of drug-induced apoptosis, CKMT1 levels are reduced, suggesting that CKMT1 downregulation acts to reinforce the commitment of cells to apoptosis. A novel high-molecular-mass CKMT1 complex that is distinct from the known CKMT1 octamer disintegrates upon treatment with cytotoxic drugs, concomitant with mitochondrial depolarization. Our study provides evidence that CKMT1 is a key regulator of the PT-pore through a complex that is distinct from the classical PT-pore.

Species differences of endothelial extracellular nucleotide metabolism and its implications for xenotransplantation.

There is a severe shortage of human organs available for transplantation and xenotransplantation - use of animal organs has long been suggested to overcome this problem. Recent advances in understanding rejection in xenotranplantation and development of genetically engineered pigs that reduced hyperacute rejection were fundamental steps forward but other unresolved mechanisms remain an obstacle. Endothelium is a major target for all rejection mechanisms in xenotransplantation. This is caused not only by location of these cells at the first line of contact but also because endothelium is a very variable cell type across different species. This variability affects not only its immune characteristics but also physiology and metabolism. Nucleotide metabolism is particularly variable in endothelial cells of different species. We attributed particular importance to one such difference - much lower activity of ecto-5'-nucleotidase (E5'N) in pig endothelial cells as compared to human. To study its significance our group developed pig endothelial cell line stably expressing human E5'N. This allowed us to determine that E5'N controls the rate of adenosine formation from extracellular nucleotides even with ATP as the substrate. Expression of human E5'N in pig cells attenuated several mechanisms involved in xenotransplant rejection such as cytotoxicity induced by human NK cells, human platelet aggregation or human platelet adherence to endothelium. We conclude that species differences of endothelial nucleotide metabolism could contribute to rejection following xenotransplantation. These studies suggests that expression of human ecto-5'-nucleotidase in pigs genetically engineered for xenotransplantation could help to prolong graft survival.

Expression of human ecto-5'-nucleotidase in pig endothelium increases adenosine production and protects from NK cell-mediated lysis.

Ecto-5'-nucleotidase (E5'N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. We evaluated whether expression of human E5'N on pig endothelial cells (EC) attenuates human NK cell-mediated cytotoxicity. A pig EC line was stably transfected with human E5'N and human NK cell adhesion and cytotoxicity toward pig EC cultures was measured by flow cytometry and intracellular enzyme release. E5'N activity in pig EC lysates increased from 0.68 +/- 0.07 to 1013 +/- 293 nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37 +/- 0.05 to >300 nmol/min/mg protein in non-transfected and transfected cells, respectively. The rate of adenosine production in transfected cells increased also with ATP as the extracellular substrate. Cytotoxicity of human NK cells was reduced from 10.7 +/- 0.4% and 11.1 +/- 1.1% with non-transfected pig EC to 5.2 +/- 0.2% and 5.0 +/- 0.2% in transfected cells with 50 microM and 250 microM AMP, respectively. Reduction of cytotoxicity in E5'N-transfected EC was abolished by the E5'N inhibitor and was mimicked in non-transfected EC by the addition of adenosine, demonstrating the key role of adenosine produced by E5'N in inhibiting NK cell cytotoxicity. We suggest that overexpression of E5'N in EC of transgenic pigs is a possible strategy to ameliorate rejection after xenotransplantation.