Differential Impact of the SKP2/P27 Pathway On HSC Pool Size and Myeloid Progenitor Differentiation.

Abstract 380

S-phase kinase-associated protein 2 (SKP2) is the F-box subunit of the ubiquitin-ligase complex SCF^SKP2 that targets the cyclin-dependent kinases (CDKs) p27^Kip1, p21^Cip1 and p130 for ubiquitination and degradation. SKP2 protein levels are modulated during cell cycle and in response to mitogenic stimuli: its expression is essential for cell cycle entry whereas its downregulation is critical for cell cycle arrest. The fact that SKP2 controls the destruction of three CKIs underscores its central role as a cell cycle regulator. We have previously shown that Notch1 activation induces the transcriptional activation of SKP2, promoting downregulation of the CKIs and inducing accelerated cell cycle progression in hematopoietic cells. Thus, the Notch/SKP2/CKIs pathway links directly environmental cues to the cell cycle machinery and may play an important role in controlling stem cell pool size and expansion of hematopoietic progenitor cells, in steady-state and during stress conditions. However, it is unclear how SKP2 regulates these CKIs in distinct hematopoietic subpopulations and its function in normal hematopoiesis has not been investigated. Analysis of SKP2 expression on murine bone marrow (BM) hematopoietic cells showed high expression levels in the myeloid progenitors, erythroid cells, B-and T-cells; lower levels were found in Lin- cells, whereas LSK cells showed a little expression of SKP2, in agreement with their more quiescent status. To better define the role of SKP2 during hematopoietic differentiation we analyzed the effects of SKP2 deficiency in the BM compartment of SKP2 null mice at steady-state conditions. As anticipated, in SKP2 knockout mice loss of SKP2 expression correlated with p27^Kip1 accumulation and overall reduced cell cycle, both in vivo and in vitro. Loss and decreased levels of SKP2 in SKP2 ^-/- and SKP2^+/- mice correlated with a significant increase in stem cells (LSK, averages: 3.2% in WT vs 6.2% KO; p=0.007), suggesting that low rates of proliferation favor quiescence and decrease the egress of cells from the HSC pool to the more mature compartments. Further immunophenotypical analysis of the BM subsets revealed that SKP2 loss resulted in the specific decrease of granulocyte-monocyte (GMP) and megacaryocytic-erythroid (MEP; averages 16.5% vs 7.6%; p=0.045) progenitor pools. Interestingly, the decrease in GMPs was associated to increased (rather than decreased) percentages of maturing myeloid progenitors Gr1⁺Mac1⁺ (averages: 26% vs 33%; p=0.049). An increase in rate of myeloid differentiation in the absence of SKP2 was further confirmed by in vitro differentiation of SKP2^-/- progenitors and by overexpression of a SKP2 dominant negative into the PLB myeloid cell line.

In conclusion, this data suggests that SKP2 levels may play a distinct role in regulating quiescence in the pool of HSC and rate of proliferation and differentiation in myeloid progenitors.