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Rb-Mediated Stem Cell Control Delineated

Original article from STEM CELLS

"An Oct4-pRb Axis, Controlled by MiR-335, Integrates Stem Cell Self-Renewal and Cell Cycle Control"

The Retinoblastoma (pRb) protein has been linked to the regulation of the self-renewal capabilities of mouse embryonic stem cells (mESCs) (Dannenberg et al and Kim et al) and also their differentiation, through the modulation of the cell cycle program (Ballabeni et al, Savatier et al and White et al).   Overall these studies demonstrate that the inactivation of the pRb pathway inhibits mESC differentiation and consequently enhances the self-renewal potential of mESC.   Now further studies of pRb in mESCs by researchers from the group of Roberta Benetti at the Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Trieste, and Università degli Studi di Udine, Italy, have recently reported in Stem Cells the existence of a new regulatory circuit comprising miR-335, Oct4, and the pRb pathway which functions to control mESC self-renewal and differentiation (Schoeftner and Scarola et al).

Initial experiments using RNA interference (RNAi) in mESCs found that Oct4 knockdown (KD) led to a rapid reduction in the levels of phosphorylated pRb (p-pRb), but not total levels of pRb, while overexpression of Oct4 increased p-pRb and total protein level (due to a stabilising effect). pRb KD did not affect Oct4 mRNA or protein, but negatively affected mESC self-renewal as measured by Alkaline Phosphatase (AP) activity, the reduction of mRNA levels of other pluripotency-associated genes (e.g. Nanog, Sox2, and Gdf3), reduced mESC colony size, altered cell morphology, reduced cell proliferation rate and a reduction of cells in S phase and an increase of cells in G1 and G2/M phase of the cell cycle, a status associated with more differentiated cells.

Further analysis found that, upon differentiation, two genes known to affect p-pRb status (Nipp1 and Ccnf) through modulation of protein phosphatase 1 (PP1) were repressed, a finding also found upon Oct4 KD. Indeed, overexpression of Oct4 led to an increase in Ccnf and Nipp1 expression and chromatin immunoprecipitation experiments found Oct4 binding at Ccnf and Nipp1 promoter regions. Ccnf/Nipp1 KD led to a reduction in both p-pRb and total pRb levels alongside the appearance of characteristics indicative of mESC differentiation, while PP1β KD (one of three subunits of PP1) led to increased levels of p-pRb and total pRb. Furthermore, PP1β KD could rescue the reduction in p-pRb and total protein levels observed after Oct4 KD.

However, study of mRNA expression during normal differentiation demonstrated that Oct4 and pRb levels persist at detectable levels through to day 6 suggesting that post-transcriptional regulatory mechanisms could play a role in the state of the Oct4-pRb axis upon exit from the self-renewing state. Interestingly, both pRb and Oct4 were found to contain conserved target sites for miR-335 in their 3′ untranslated regions of mRNA, while miR-335 itself and its hosting gene (Mest) were shown to be greatly upregulated upon mESC differentiation. Transient transfection of mESCs with synthetic miR-335 reduced p-pRb, total pRb, Nipp1 and Oct4 protein levels in mESCs and ectopically elevated miR-335 levels reduced p-pRb phosphorylation and antagonized mESC self-renewal, which included alterations to the cell cycle. Antagonizing miR-335 under self-renewing conditions led to an increase in Oct4, Nipp1, and pRb expression and increased p-pRb, while antagonizing the upregulation of miR-335 prior to differentiation induction allowed mESCs to retain undifferentiated mESC characteristics. Additionally, miR-335 overexpression promoted the formation of contractile cardiomyocyte structures during embryoid body-mediated differentiation and teratomas with reduced size when subcutaneously injected into immune-compromised mice were also noted.

Together, the detailed experiments undertaken uncover a novel regulatory pathway; showing that Oct4 and miR-335 controls the activity of the pRb pathway in self-renewing mESCs; increased Oct4 leads to increased Nipp1 and Ccnf, which inhibits PP1 allowing an increase in p-pRb, establishing p-pRb as a characteristic marker of self-renewing mESCs. Under differentiation conditions however, this is reversed and the upregulation of miR-335 enhances differentiation due to the collapse of the Oct4-Nipp1/Ccnf-PP1-pRb axis. The authors also note that this research may also have implications for human cancers, where Rb status is modified, suggesting a possible important role for Oct4.

References

  • Ballabeni A et al. Cell cycle adaptations of embryonic stem cells. Proc Natl Acad Sci USA 2011; 108: 19252–19257.
  • Dannenberg JH et al. Ablation of the retinoblastoma gene family deregulates Gadb(1) control causing immortalization and increased cell turnover under growth-restricting conditions. Genes Dev 2000; 14: 3051–3064.
  • Kim Y et al. Cyclin-dependent kinase 2-associating protein 1 commits murine embryonic stem cell differentiation through retinoblastoma protein regulation. J Biol Chem 2009; 284: 23405–23414.
  • Savatier P et al. Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts. Oncogene 1994; 9: 809–818.
  • White J et al. Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation. Mol Biol Cell 2005; 16: 2018–2027.

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.