ABSTRACT
The function of the proteasome has been linked to various pathologies, including cancer and neurodegeneration. Proteasomal inhibition can lead to death in a variety of cell types, however the manner in which this occurs is unclear, and may depend on the particular cell type. In this work we have extended previous findings pertaining to the effects of pharmacological proteasomal inhibitors on PC12 cells, by examining in more detail the induced death pathway. We find that cell death is apoptotic by ultrastructural criteria. Caspase 9 and 3 are processed, cytochrome c is released from the mitochondria and a dominant negative form of caspase 9 prevents death. Furthermore, Bax undergoes a conformational change and is translocated to the mitochondria in a caspase-independent fashion. Total cell levels of Bax however do not change, whereas levels of the BH3-only protein Bim increase with proteasomal inhibition. Transient overexpression of bcl-xL or, to a lesser extent, of bcl-2, significantly decreased apoptotic death and prevented Bax conformational change. We conclude that death elicited by proteasomal inhibition of PC12 cells follows a classical "intrinsic" pathway. Significantly, antiapoptotic bcl-2 family members prevent apoptosis by inhibiting Bax conformational change. Increased levels of Bim may contribute to cell death in this model.
Subject(s)
Apoptosis , Proteasome Inhibitors , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Caspase 9/metabolism , Cytochromes c/metabolism , Gene Expression/drug effects , Genes, Dominant , Membrane Proteins/metabolism , Mitochondria/drug effects , Mitochondria/enzymology , Mitochondria/metabolism , PC12 Cells , Protein Processing, Post-Translational/drug effects , Protein Structure, Quaternary/drug effects , Protein Transport/drug effects , Proto-Oncogene Proteins/metabolism , Rats , bcl-X Protein/geneticsABSTRACT
Ubiquitinated inclusions and selective neuronal cell death are considered the pathological hallmarks of Parkinson's disease and other neurodegenerative diseases. Recent genetic, pathological and biochemical evidence suggests that dysfunction of ubiquitin-dependent protein degradation by the proteasome might be a contributing, if not initiating factor in the pathogenesis of these diseases. In neuronal cell culture models inhibition of the proteasome leads to cell death and formation of fibrillar ubiquitin and alpha-synuclein-positive inclusions, thus modeling some aspects of Lewy body diseases. The processes of inclusion formation and neuronal cell death share some common mechanisms, but can also be dissociated at a certain level.
Subject(s)
Inclusion Bodies/physiology , Neurons/physiology , Ubiquinone/metabolism , Alzheimer Disease/pathology , Animals , Cell Death/genetics , Cell Death/physiology , Humans , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neurodegenerative Diseases/pathology , Neurons/pathology , Parkinson Disease/pathology , Synucleins , alpha-SynucleinABSTRACT
Adrenomedullin is a biologically active peptide released from the vascular wall, which increases blood flow through its vasorelaxant effects and prevents platelet activation by stimulation of nitric oxide synthesis. The present study demonstrates that activated platelets suppress adrenomedullin secretion from vascular endothelial cells by releasing a factor that was identified as transforming growth factor (TGF)-beta1. Adrenomedullin levels were reduced by up to 40% and this effect was completely abrogated by the addition of latency-associated protein (LAP) or TGF-beta1-neutralizing antibody. Inhibition of adrenomedullin secretion in response to platelet aggregation may be an important mechanism in the induction of hemostasis.
