| The nucleolus has long been recognized as the site of ribosomal biogenesis and plays a major role in coordinating cellular growth with proliferation. As a resident nucleolar protein, the tumor suppressor p19ARF is thus ideally situated to play a dual role in surveilling against inappropriate growth signals and initiating cell cycle arrest. While ARF expression is nearly undetectable in normal tissues, it is potently induced by oncogenic stress. As a result of enforced expression of ARF, the tumor suppressor protein p53 is stabilized and promotes cell cycle arrest. Mutations or epigenetic alterations of the Ink4a/Arf locus are second only to p53 mutations in cancer cells, and in some cancers, alterations in both are observed.;To determine the roles of ARF outside of its context as a tumor suppressor, I first examined the nucleolar morphology of cells lacking ARF. I noticed that Arf-/- cells displayed increased nucleolar area and ultrastructual abnormalities, suggesting that basal ARF regulates key nucleolar functions. Concordantly, ribosomal biosynthesis and protein synthesis rates were dramatically elevated in the absence of Arf. Bone marrow-derived osteoclasts from Arf-null mice, which are post-mitotic cells whose activities depend on their protein synthesis rates, exhibited an enhancement of cell number and of TRAP protein production, demonstrating a physiological function for basal ARF. Moreover, knockdown of NPM, a nucleolar protein important for ribosomal biogenesis and export that is antagonized by ARF, blocked the increases in Arf -/- osteoclast activity, demonstrating that these gains require NPM.;In parallel with the above studies I have demonstrated nucleolar phenotypes in Arf-/- mice. Histological examination of tissues demonstrates morphological aberrations in the nucleolus similar to those seen in vitro. Furthermore, these mice also exhibit increases in both osteoclast and osteoblast activity arguing for a central role for basal ARF in maintaining bone homeostasis.;I have further identified a novel target for ARF's function in the nucleolus, the p68 RNA helicase. Loss of Arf results in enrichment of nucleolar p68. Wild type osteoclasts transduced with excess p68 also partially mimic the ARF-null phenotype. Collectively, these data point to a vital role for ARF in maintaining proper nucleolar function. |
