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TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.

Diabetes is one of the world’s most important diseases and has taken on epidemic proportions worldwide. This multi-factorial disease is mainly characterised by hyperglycaemia and a low grade inflammation and increases the risk for developing several complications like nerve damage, high blood pressure, blindness and heart failure. Endothelial cells, which delineate the blood vessel, are the primary target for the development of complications. These specialised cells not only have a barrier function, preventing leakage from the blood vessel, but also function as an organ regulating blood flow, inflammation, blood clotting… Disturbance of any of these functions may lead to endothelial dysfunction and the development of vascular complications. Researchers all over the world are now trying to understand the development of these complications in an attempt to prevent them. It is now well established that hyperglycaemia itself induces endothelial dysfunction, for instance by modifying proteins. These modified proteins are called ‘advanced glycation end products’ or AGEs. Also, the mechanisms by which inflammation contributes to endothelial dysfunction are starting to become elucidated. For instance, the pro-inflammatory cytokine TNF is known to disturb the blood flow regulating function of endothelial cells. Although glucose was thought to be the main precursor of those AGEs, later studies have proven that it is in fact methylglyoxal, which is the main precursor. Methylglyoxal is a cytotoxic side product of the glycolysis and is normally detoxified by the glyoxalase system, which comprises glyoxalase1 and glyoxalase2. A previous study in our lab indicated that in a fibrosarcoma cell-line, TNF is able to induce the formation of specific methylglyoxal derived AGEs (MG-AGEs) by phosphorylating glyoxalase1. Given the importance of both TNF and AGEs in endothelial dysfunction, we wanted to investigate whether TNF could also phosphorylate glyoxalase1 in endothelial cells, and whether this phosphorylation was accompanied by the formation of specific MG-AGEs. Here we provide evidence that TNF is also capable of inducing AGE formation in endothelial cells, just like hyperglycaemia does, by upregulating the glycolytic flux which produces the AGE precursor methylglyoxal. In addition we provide evidence that TNF modifies specific proteins. Although we also observed phosphorylation of glyoxalase1 upon TNF treatment, phosphorylation of this enzyme seems not necessary to induce the formation of MG-AGEs in these cells. Additionally we identified the transcription factor NF-κB as a possible new AGE modified protein. Our data indicate that methylglyoxal modification of this protein abolishes its DNA binding capacity, resulting in decreased transcription. Since NF-κB is known to induce the transcription of several genes, protecting cells from TNF induced cell death, our data indicate that methylglyoxal modification of NF-κB could sensitise endothelial cells to TNF induced apoptosis, resulting in vascular leakage and the development of vascular complications.