br Results br Discussion We have conducted experiments to

Results
Discussion We have conducted experiments to clarify whether Pten regulates HSCs cell autonomously or non-cell autonomously. Our data confirm that Pten regulates HSCs via both cell-autonomous and non-cell-autonomous mechanisms (Figure 1). The data also show that the cell-autonomous effects of Pten microcystin lr are significantly greater than the non-cell-autonomous effects (Figure 1). While our analyses of Pten;Csf3;Mx1-Cre mice do not support an obligate role for G-CSF in HSC mobilization (Figure 2C), we have found that Pten-deficient HSCs are hyper-sensitive to the mobilizing and depleting effects of G-CSF (Figures 2D and 2E). This hyper-sensitivity does not appear to reflect enhancement of normal physiologic mobilization mechanisms. Rather, G-CSF enhances PI3K/mTOR pathway activation in HSCs that lack Pten (Figure 2H). This could occur through direct engagement of the G-CSF receptor or through indirect activation of other cytokines in the microenvironment. Pten-deficient HSCs also hyper-activate mTORC1/S6K and mobilize in response to IFNα (Figure 3). Together, these data suggest that Pten modulates the HSC response to inflammatory cytokines, and pIpC contributes to the HSC proliferation, mobilization, and self-renewal phenotypes that have been widely described using Pten;Mx1-Cre mice (Kalaitzidis et al., 2012; Lee et al., 2010; Magee et al., 2012; Signer et al., 2014; Yilmaz et al., 2006; Zhang et al., 2006). Our data potentially link two major modes of HSC regulation—the PI3K pathway and pro-inflammatory cytokines—as common causes of HSC depletion during illness or aging. The consequences of PI3K pathway activation in HSCs have been extensively studied (Gan et al., 2008; Kharas et al., 2010; Lee et al., 2010; Magee et al., 2012; Siegemund et al., 2015; Signer et al., 2014), but the signals that activate the pathway—either in native or stressed conditions—have not been well characterized. Likewise, inflammatory signals have drawn scrutiny for their putative role in bone marrow failure, HSC aging, and pre-leukemic clonal evolution (Baldridge et al., 2010, 2011; Essers et al., 2009; Walter et al., 2015), but the downstream mechanisms by which these signals deplete HSCs have not been fully resolved. Our data suggest a model in which inflammatory cytokines hyper-activate the PI3K pathway in HSCs leading to increased protein synthesis and tumor-suppressor expression, which ultimately depletes the HSC pool. Ongoing experiments will test whether mTORC1 or mTORC2 inactivation can preserve the function of cytokine-stimulated HSCs. If so, mTOR inhibitors may have a role in sustaining the HSC pool in patients with inflammation and otherwise tenuous HSC function (e.g., bone marrow transplant patients with complicating graft-versus-host disease or infections).
Experimental Procedures
Author Contributions S.N.P., A.S.C., J.V., and J.A.M. performed all experiments except the analysis of serum G-CSF levels and cell-cycle assays (performed by D.A.M. and L.G.S.) and analyses of Pten/Csf3 compound mutant mice (performed by R.A.J.S.). S.N.P., A.S.C., R.A.J.S., L.G.S., and J.A.M. designed experiments and interpreted the data. J.A.M. directed the study and wrote the manuscript.
Acknowledgments This work was supported by grants from the Children\'s Discovery Institute of Washington University and St. Louis Children\'s Hospital (L.G.S. and J.A.M.), the St. Baldrick\'s Foundation (J.A.M.), and the Department of Defense (CA130124, J.A.M.). A.S.C. is supported by a training grant to the Department of Pediatrics (5T32HD043010-12). J.A.M. and L.G.S. are scholars of the Child Health Research Center for Excellence in Developmental Biology at Washington University (K12-HD076224). Analyses of Pten/Csf3 compound mutant mice were conducted in Sean Morrison\'s laboratory. We thank John Dipersio and Julie Ritchey for providing AMD3100 and for performing the splenectomies.
Introduction