Rho GTPase Activating Protein p190-B Regulates Hematopoietic Stem Cell Fate Decision

Abstract 575

Hematopoietic stem cells (HSC) are undifferentiated cells that are capable of self-renewal and production of all mature blood and immune cells. The molecular mechanisms that determine HSC self renewal and cell fate decision still remain poorly understood. We have previously shown that p190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, is a critical regulator of HSC self renewal. In these studies, while p190-B-deficiency did not alter HSC cell cycle kinetics or survival either in vivo and in vitro, the loss of p190-B conferred enhanced serial transplantation capacity and also preserved the repopulating capacity of HSC/Ps during ex vivo cytokine-induced culture compared to WT HSC/Ps. Together, these data suggest that p190-B modulates HSC self renewal decisions during cell division (Xu et al, Blood 2009).

In the current study, we tested the hypothesis that p190-B modulates the balance of asymmetric/symmetric self renewal divisions. To do so, single LSK cells isolated from fetal livers were cultured in the presence of cytokines known to promote self renewal. After 1–2 divisions, the media was replaced with cytokines that promote both proliferation and differentiation. The kinetics of the first 1–2 divisions of WT and p190-B^–/– LSK was similar (n=450). However, after 10 days, the size of clones derived from p190-B^−/− LSK cells were larger. Importantly, the frequency of clones exhibiting multipotent lineage differentiation was also higher from p190-B^−/− LSK compared with WT (18 multipotent clones out of 43 (42%) vs 10 out of 39 (26%)). Therefore, p190-B may modulate the balance of asymmetric/symmetric HSC self renewal divisions. One fundamental mechanism of asymmetric cell division is the asymmetric inheritance of cell fate determinants, which is controlled by the asymmetric distribution of the cytoskeleton and polarity cues. Because Rho GTPases are key regulators of cytoskeleton polarity, we next examined cell shape and polarity (of WT and p190-B^–/– LSK cells). WT cells exhibited an asymmetric shape with two distinct cell poles, including one pole enriched with microtubules. In contrast, p190-B^–/– LSK appeared round with a homogenous distribution of microtubules. Using image flow spectral analysis (Imagestream), we analyzed the distribution of numb, a conserved cell fate determinant, which is unequally distributed during stem cell division in order to generate distinct daughter cells. Significantly more p190-B^–/– LSK showed a symmetric numb distribution than WT LSK (41±5.8% vs 29±4.6%, n>1000 cells from 3 independent experiments, p<0.02). To determine the importance of this cell shape change, we examined the p38^MAPK signaling pathway, since p190-B is known to regulate p38^MAPK activity (Sordella et al, Dev Cell 2002) and p38^MAPK has been implicated HSC self renewal decision (Ito et al, Nat Med 2006). Flow cytometry analysis revealed that p38^MAPK activity increased in WT LSK-CD150+ cells during serial transplantation. p190-B-deficiency prevents the elevation of p38^MAPK activity in HSC in secondary transplant recipients (mean fluorescence intensity in HSC isolated from non transplanted animals: 1182±439; from secondary recipients of WT HSC: 2218±680=; from secondary recipients of p190-B^−/− HSC: 1540±711**, n=7, *p<0.01 compared to non transplanted mice; ** p<0.01 compared to WT and ns compared to non transplanted mice). Remarkably, inhibition of p38^MAPK activity in WT LSK led to alterations in shape and polarity that was similar to p190-B^–/– LSK. Furthermore, p38^MAPK inhibition significantly increased LSK competitive repopulation activity after 7 days in culture, compared to vehicle treated LSK cells (chimera 53±1.7 vs 23±18%, n=4, p<0.02).

Together, these data suggest that p190-B regulates HSC/P self renewal by modulating cell fate decision as the cell divide and that HSC/P shape integrity is critical for this process. This study implies that modulating cell polarity via Rho GTPase may provide rational to devise new therapeutic approaches to clinical HSC transplantation protocols.