Abstract
Mutations in gene encoding Ribosomal protein S19 (rps19) have been identified in 25% of patients affected by Diamond-Blackfan anemia, the rare congenital erythroblastopenia. Recently, mutations in yet another gene encoding for a ribosomal protein, the rps24 gene, have been found in approximately 3% of DBA cases. These findings imply a role for impaired ribosomal assembly due to mutant ribosomal proteins in the pathophysiology of DBA. We have previously implicated the ubiquitin-proteasome pathway (UPP) in degrading unstable RPS19 mutant proteins. Three different proteasome inhibitors, lactacystin, MG132 and bortezomib were shown to be able to restore both RPS19 protein expression level and nucleolar localization of several mutants (V15F, G127E, L131P, and truncated RPS19 containing more than 80 aminoacids). Bortezomib, the only proteasome inhibitor currently approved for use in humans, is a Nuclear factor-κB (NF-κB) pathway inhibitor. Indeed, UPP inhibition by proteasome inhibitors is the result of either a direct inhibition of the 20S core of the proteasome or an indirect inhibition of NF-κB pathway through the IκB UPP degradation pathway. At steady state, NF-κB binds to IκB, the natural NF-κB inhibitor in the cytoplasm. Under stress or stimulation, IκB is phosphorylated and degraded by the UPP. NF-κB, free of its inhibitor is translocated to the nucleus and acts as an efficient transcription factor regulating expression of a number of genes. In the present study, we analyzed the effects of direct inhibition of NF-κκB pathway on expression level and subcellular localization of several RPS19 mutants using a Migr-NF-κB super-repressor retroviral vector, corresponding to IκB, in which two amino acids have been mutated in order to prevent phosphorylation of IκB. Following retroviral infection, IκB can not be degraded and hence NF-κB can not translocate to the nucleus. Following transient transfection of Cos-7 with mutant GFP- RPS19, which are degraded by UPP and retrovirus infection, we found that RPS19 decreased expression level was restored to normal. Specific inhibitors of p50 and p65 (NF-κB subunits) failed to restore RPS19 expression levels suggesting that the degradation of unstable RPS19 is dependent solely on IκB. By gel shift assay, we were able to show that p50-p65 heterodimers, p50 homodimers and p65 NF-κB subunits bound to rps19 gene promoter upstream of the ATG start site. These NF-κB subunits have been found to repress rps19 gene promoter activity (Da Costa and al., Blood 2003). In conclusion, NF-κB pathway regulates RPS19 expression level by two different mechanisms:
IκB regulated proteasomal degradation of the unstable mutant RPS19 proteins; and transcriptional repression of rps19 gene promoter by p50 homodimers, p50-p65 heterodimers and p65, which act as transcription factors.
These findings suggest potential therapeutic strategies for DBA involving modulation of NF-κB pathway.
Targeted Disruption of the Ribosomal Protein S19 Gene Is Lethal Prior to Implantation