Involvement of the ubiquitin-mediated proteolytic system in the signaling pathway induced by ceramide in primary astrocyte cultures.
The selective degradation of abnormal or short half-life proteins in eukaryotic cells proceeds through the ubiquitin-mediated proteolytic system (UbPS). The signals that tag the proteins for their ubiquitination are well known. In the present study, our aim was to investigate the relationship between the action of ceramide and the changes in the expression of certain mRNAs of the Ub pathway and in the activation of the UbPS in cultured astrocytes (ASTs). Changes in the expression of components that are known to be substrates of the UbPS and that participate in the regulation of the cell death process were also studied. Addition of different concentrations of C2 ceramide to cultured ASTs produced an increase in the expression of the Ub gene and in the gene that encodes E1, one of the enzymes involved in the ubiquitination process, without any changes on cell viability. Immunocytochemical studies showed an increase in the expression of Bcl-2 with no changes in cytochrome c. Also, there was an increase in the nuclear reactivity of NFkappaB, suggesting a translocation of this factor towards the nucleus. Western blots showed a decrease in IkappaB and its phosphorylated form as well as an increase in Bcl-2 with no changes in cytochrome c. All of these compounds appear to be acting as possible modulators of AST responses to C2 ceramide. Our results suggest that in AST primary cultures, C2 ceramide, at the concentrations used in this study, does not produce apoptosis. However, it induces an activation of the UbPS, probably as a consequence of an activation of phosphatases and kinases, or through the generation of reactive oxygen species, which act as triggering signals of the UbPS. The fundamental role of NFkappaB and Bcl-2 as antiapoptotic factors is discussed.
Calatayud
C A
CA
Departamento de Química Biológica and Instituto de Química y Fisicoquímica Biológica (IQUIFIB), UBA-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Pasquini
L A
LA
Pasquini
J M
JM
Soto
E F
EF
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D020672:I-kappa B Proteins; C064769:N-acetylsphingosine; D016328:NF-kappa B; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D013110:Sphingosine