The 26 S proteasome is a high molecular mass proteinase complex that is built by at least 32 different protein subunits. Such protease complexes in bacteria and yeast are systems that undergo a highly sophisticated network of gene expression regulation. However, regulation of mammalian proteasome gene expression has been neglected so far as a possible control mechanism for the amount of proteasomes in the cell. Here, we show that treatment of cells with proteasome inhibitors and the concomitant impairment of proteasomal enzyme activity induce a transient and concerted up-regulation of all mammalian 26 S proteasome subunit mRNAs. Proteasome inhibition in combination with inhibition of transcription revealed that the observed up-regulation is mediated by coordinated transcriptional activation of the proteasome genes and not by post-transcriptional events. Our experiments also demonstrate that inhibitor-induced proteasome gene activation results in enhanced de novo protein synthesis of all subunits and in increased de novo formation of proteasomes. This phenomenon is accompanied by enhanced expression of the proteasome maturation factor POMP. Thus, our experiments present the first evidence that the amount of proteasomes in mammalia is regulated at the transcriptional level and that there exists an autoregulatory feedback mechanism that allows the compensation of reduced proteasome activity.

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The proteasome inhibitor bortezomib has been approved as a cytostatic drug for the therapy of multiple myeloma, and is currently being tested in clinical trials for a variety of other malignancies. At the same time, a growing number of animal studies suggest that proteasome inhibitors may also prove to be valuable remedies for the treatment of non-tumorous diseases. In this review, we will revisit the current applications of proteasome inhibitors in clinical research according to the cellular effects of proteasome inhibitors as poisons, which induce apoptosis, or as remedies, which modulate cellular function and protect from cell death. We postulate that the correct distinction of a poison from a remedy depends on cell type and on the degree of proteasome inhibition. Dose-dependent and differential inhibition of the proteasome may affect specific sets of substrates, thereby conferring substrate specificity. According to this idea, we suggest that inhibition of the proteasome to a defined degree may offer a promising tool in achieving desired therapeutic effects in various diseases.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Endothelial nitric oxide synthase (eNOS) is the key enzyme of vascular homeostasis involved in the pathophysiology of several cardiovascular diseases. The aim of our study was to investigate whether eNOS expression and activity are regulated by the proteasome. Bovine pulmonary artery endothelial cells (CPAE cells) were treated with the proteasome inhibitor MG132. MG132 (50-250 nmol/L) dose-dependently increased mRNA and protein levels of eNOS. Comparable results were obtained with other specific proteasome inhibitors, whereas the nonproteasomal calpain and cathepsin inhibitor ALLM had no effect. Efficacy of proteasome inhibition was evidenced by accumulation of poly-ubiquitinylated proteins and by measuring proteasomal activity in cell extracts. Cycloheximide prevented up-regulation of eNOS protein, indicating that post-translational stabilization of eNOS is not involved. eNOS activity was increased up to 2.8-fold (MG132 100 nmol/L, 48 h). Incubation of rat aortic rings with MG132 significantly enhanced endothelial-dependent vasorelaxation. Single MG132 treatment (100 nmol/L) induced long-term effects in CPAE cells, with increases of eNOS protein and activity for up to 10 days. Our results indicate that low-dose proteasome inhibition enhances eNOS expression and activity, and improves endothelial function.

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Atherosclerosis is a chronic inflammatory disease accompanied by the expression of endothelial adhesion molecules. Phloretin is a plant-derived phytochemical that is mainly present in apples. Because phloretin is reported to promote antioxidative activities, we investigated the effects of phloretin on cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) in human umbilical vein endothelial cells (HUVECs). Phloretin prevented TNF-alpha-stimulated upregulation of VCAM-1, ICAM-1, and E-selectin expression in a concentration-dependent manner. To the same extent as for TNF-alpha, phloretin also inhibited IL-1beta-induced upregulation in expression of all 3 adhesion molecules. Inhibition of cytokine-induced adhesion molecule expression for VCAM-1, ICAM-1, and E-selectin was detected already at the level of mRNA. Preincubation with phloretin dose-dependently attenuated TNF-alpha-stimulated adhesion of monocytic THP-1 cells to HUVECs and human aortic endothelial cells. Phloretin did not affect TNF-alpha-stimulated activation of nuclear factor kappaB (NF-kappaB) but inhibited activation of interferon regulatory factor 1, a transcription factor involved in the regulation of endothelial cell adhesion molecule expression. In human platelets, phloretin diminished adenosine diphosphate (ADP) and thrombin receptor-activating peptide-stimulated expression of the activated form of the GPIIb/IIIa complex and reduced platelet aggregation stimulated by ADP. Thus phloretin may have beneficial effects in the onset and progression of cardiovascular diseases.

Telomerase is a promising target for human cancer gene therapy. Its inhibition allows telomere shortening to occur in cancer cells, which in turn is thought to trigger delayed senescence and / or apoptosis. We tested whether telomerase inhibition might have additional, immediate effects on tumor cell growth. Ovarian cancer cell lines with widely differing telomere lengths were efficiently transduced with an adenovirus expressing a ribozyme directed against the T motif of the catalytic subunit of human telomerase, hTERT. Three days after transduction, telomerase activity was significantly reduced and massive cell loss was induced in mass cultures from all four ovarian cancer cell lines tested, whereas transduction of telomerase -negative human fibroblasts did not attenuate their growth. The kinetics of induction of cell death in cancer cells was not significantly dependent on telomere length, and telomeres did not shorten measurably before the onset of apoptosis. The data suggest the existence of a ''fast -track'' mechanism by which diminution of telomerase can interfere with cancer cell growth and induce cell death, presumably by apoptosis. This phenomenon might be a consequence of the telomere capping function provided by telomerase in tumor cells. Uncapping of telomeres by ribozyme -mediated inhibition of telomerase bears therapeutic potential for ovarian cancer.