Diamond Blackfan anemia (DBA) is one of a class of disorders linked to defects in ribosome biogenesis. While classified as a bone marrow failure syndrome, some DBA patients exhibit craniofacial anomalies. To date, all cases of DBA have been linked to mutations in genes encoding ribosomal proteins, however, orofacial anomalies (cleft palate) were found only in patients with RPL5 mutations. Rpl5 and Rpl11 bind cooperatively, possibly with 5S rRNA, to the murine double minute (Mdm2) protein, a critical negative regulator of the p53 tumor suppressor. This interaction inhibits Mdm2 function, leading to p53 activation and enhanced apoptosis. Rpl11 has also been shown to act as a feedback inhibitor of the c-myc oncoprotein. Our goal is to use mouse embryonic stem (ES) cells heterozygous for Rpl5 or Rpl11 to create neural crest cell models of DBA. To account for the finding that mutations in RPL5 but not RPL11 are associated with cleft palates in DBA patients, we hypothesize that the mutations differ in their effects on p53 and c-myc regulation. Both of these mutations are expected to disrupt ribosome assembly, and haploinsufficiency for Rpl5 and Rpl11 should increase the levels of free Rpl11 or Rpl5, respectively. When liberated in this fashion, Rpl5 may interact with Mdm2 to regulate p53 activation, whereas Rpl11 may regulate both p53 and c-myc. We hypothesize that inhibition of c-myc function through its interaction with Rpl11 is the proximate trigger for loss of neural crest cells, thus accounting for the selective effect of RPL5 mutations on orofacial development. Further, we propose that RNA interference (RNAi) could be used to reduce the amount of free Rpl11 in cells haploinsufficient for RPL5 with consequent rescue of neural crest cell development. To test this hypothesis, our specific aims include: 1. Compare neural crest-like cell development in two murine ES cell lines with mutations in either Rpl5 or Rpl11. Assess whether haploinsufficiency for Rpl5 and Rpl11 leads to increased free levels of Rpl11 or Rpl5, respectively, and whether the cells haploinsufficient for Rpl5 selectively inhibit c-myc activity. 2. Use RNA interference methods to reduce levels of free Rpl11 or Rpl5 in these haploinsufficient neural crest-like cells to determine the relative importance of the p53 and c-myc pathways in modulating neural crest development. Assess whether modulation of nucleolar stress pathways can lead to rescue of neural crest development. We believe that these experiments will lead to a better understanding of the role of ribosome biogenesis in craniofacial development and provide the basis for therapeutic interventions to prevent or treat craniofacial disorders, pursuant to the mission of the NIDCR and the """"""""Translational Application of Gene Silencing Strategies to Oral and Craniofacial Disorders"""""""" Request for Applications.
The neural crest is a specialized population of stem cells that generates most of the bone, cartilage, connective and peripheral nerve tissue in the head in the developing embryo, and abnormalities in the neural crest can lead to craniofacial defects like cleft palate. Our proposal explores the role of defective ribosome production in neural crest development and provides the basis for therapeutic interventions to prevent or treat craniofacial disorders that are caused by defective ribosome assembly. We propose using genetic engineering to modulate the levels of ribosomal proteins in mouse embryonic stem cells, which has clear relevance to the application of human embryonic stem cells to treat neural crest-related disorders.
| Burwick, Nicholas; Shimamura, Akiko; Liu, Johnson M (2011) Non-Diamond Blackfan anemia disorders of ribosome function: Shwachman Diamond syndrome and 5q- syndrome. Semin Hematol 48:136-43 |
