Diseases of ribosome dysfunction (ribosomopathies), such as Diamond-Blackfan anemia and 5q- syndrome, are caused by deficiencies in one or more ribosomal proteins (RPs) and are characterized by impaired erythropoiesis. The p53 pathway has been identified as a central player in the ribosomopathy disease phenotypes, but the precise mechanism by which p53 is aberrantly induced in the setting of RP deficiency has not been fully elucidated. The current hypothesis for this induction is that, in the context of RP deficiency, cellular levels of free (non-ribosome bound) RPs rise, and a subset of RPs with MDM2-binding ability interact with and inhibit the negative regulatory activity of MDM2 on p53, resulting in p53 stabilization. RPs that have been implicated in this capacity include rpL5, rpL11, rpL23a, and rpS7. This hypothesis also suggests that overabundance of these free ribosomal proteins might be sufficient to induce p53 activity, even in the absence of ribosome protein deficiency. To test this specific aspect of this hypothesis, we generated lentiviral expression vectors carrying the full-length cDNAs for rpL5, rpL11, rpL23a, and rpS7, as well as ARF (which served as a positive control, as ARF is known to induce p53 by binding and inhibiting MDM2), and we used each virus to individually infect A549 cells. Despite increased levels of transcript and protein observed for each of the cDNAs expressed, only ARF overexpression resulted in p53 stabilization and p21 induction. Similarly, when RPs were overexpressed in each pairwise combination, none of the RP pairs tested induced p53 above baseline levels. We also tested all of the overexpression constructs in primary human cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs), and p53 pathway induction was only observed with overexpression of ARF. These data suggest that overabundance of these specific ribosomal proteins alone is insufficient for p53 pathway induction via MDM2 inhibition.
Ebert:Celgene: Consultancy; Genoptix: Consultancy.
Asterisk with author names denotes non-ASH members.