Summary Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53515C (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2−/− mice. Mdm2−/− p53515C/515C mice, however, die by postnatal day 13 due to hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2−/− p53515C/515C mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS, and cell cycle arrest, senescence and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized due to ROS, and its loss increased cell cycling, and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

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Microglial activation plays a central role in poststroke inflammation and causes secondary neuronal damage; however, it also contributes in debris clearance and chronic recovery. Microglial pro- and antiinflammatory responses (or so-called M1-M2 phenotypes) coexist and antagonize each other throughout the disease progress. As a result of this balance, poststroke immune responses alter stroke outcomes. Our previous study found microglial expression of interferon regulatory factor 5 (IRF5) and IRF4 was related to pro- and antiinflammatory responses, respectively. In the present study, we genetically modified the IRF5 and IRF4 signaling to explore their roles in stroke. Both in vitro and in vivo assays were utilized; IRF5 or IRF4 small interfering RNA (siRNA), lentivirus, and conditional knockout (CKO) techniques were employed to modulate IRF5 or IRF4 expression in microglia. We used a transient middle cerebral artery occlusion model to induce stroke and examined both acute and chronic stroke outcomes. Poststroke inflammation was evaluated with flow cytometry, RT-PCR, MultiPlex, and immunofluorescence staining. An oscillating pattern of the IRF5-IRF4 regulatory axis function was revealed. Down-regulation of IRF5 signaling by siRNA or CKO resulted in increased IRF4 expression, enhanced M2 activation, quenched proinflammatory responses, and improved stroke outcomes, whereas down-regulation of IRF4 led to increased IRF5 expression, enhanced M1 activation, exacerbated proinflammatory responses, and worse functional recovery. Up-regulation of IRF4 or IRF5 by lentivirus induced similar results. We conclude that the IRF5-IRF4 regulatory axis is a key determinant in microglial activation. The IRF5-IRF4 regulatory axis is a potential therapeutic target for neuroinflammation and ischemic stroke.

IntroductionThe survival of an organism is dependent upon the ability of hematopoietic stem cells (HSCs) to replenish the blood compartment on a daily basis. To accomplish this task, HSCs must maintain a fine balance between 3 possible fates: self-renewal, differentiation, or senescence. Although the decision process that determines the fate of an HSC clone remains to be completely defined, several molecules are already known to play a role in this process, including components of the HSC microenvironment or niche. This niche consists of both extracellular matrix molecules and cells, such as osteoblasts and stromal cells, which produce cytokines and chemokines important for the maintenance of the HSC pool. [1][2][3][4][5] These microenvironmental or external cues engage receptors on HSCs, leading to the activation of signaling pathways governing cell proliferation, self-renewal, differentiation, mobilization, and bone marrow (BM) retention. 5 Some of these pathways, such as those initiated by stem cell factor (SCF/c-kitL), 6 stromal cellderived factor 1 (SDF-1/CXCL12), 7 and thrombopoietin (TPO), 8 result in the activation of phosphatidylinositol 3Ј kinase (PI3K) and the formation of phosphatidyl inositol 3,4,5-trisphosphate (PIP3). Therefore, the SH2 domain-containing inositol 5Ј-phosphatase 1 (SHIP) may influence these pathways in HSCs. 9,10 SHIP is a 145-kDa protein primarily expressed by cells of the hematopoietic system, 11 including HSCs, 12 that can associate with various adapter proteins, scaffold proteins, or receptors following activation of hematopoietic cells. 9,13 Formation of these complexes enables SHIP to hydrolyze the 5Јphosphate on PIP3, 11,14 thus preventing membrane recruitment and activation of pleckstrin homology domain-containing kinases that serve as effectors of PI3K signaling. This activity permits SHIP to limit the survival, activation, differentiation, and/or proliferation of hematopoietic cells. 10 Thus, we hypothesized that SHIP might also influence these processes in the HSC compartment. Previous studies reported that SHIP Ϫ/Ϫ whole bone marrow (WBM) cells do not reconstitute lethally irradiated mice as well as wild-type (WT) WBM in a noncompetitive setting. 15 Furthermore it was reported that SHIP Ϫ/Ϫ WBM has comparable numbers of competitive repopulating units (CRUs) relative to WT littermates in a limiting-dilution assay, which uses compromised competitor cells. 15 However, because these analyses were performed with WBM rather than purified HSCs, they did not assess whether SHIP Ϫ/Ϫ HSCs are defective for repopulation in a WT environment. Thus, it is not possible from these previous studies to conclude that SHIP plays a direct role in signaling pathways active in the HSC compartment.To study the effects of SHIP on HSCs we used SHIP Ϫ/Ϫ mice generated by a Cre-lox mutation strategy. 16 We found that SHIP Ϫ/Ϫ mice contain significantly more HSCs in their bone marrow and spleen as measured by flow cytometry. We also observe that a greater proportion of SHIP Ϫ/Ϫ HSCs enter the cell ...