Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.

Neurons require trophic support during neural circuit formation; however, how the cellular milieu contributes to neuronal survival remains unclear. We found that layer V cortical neurons require support from microglia for survival during postnatal development. Specifically, we found that microglia accumulated close to the subcerebral and callosal projection axons in the postnatal brain. Inactivation of microglia by minocycline treatment or transient ablation of microglia in CD11b-DTR transgenic mice led to increased apoptosis, specifically in layer V subcerebral and callosal projection neurons. CX3CR1 in microglia was required for the survival of layer V neurons. Microglia consistently promoted the survival of cortical neurons in vitro. In addition, we identified microglia-derived IGF1 as a trophic factor that maintained neuronal survival. Our results highlight a neuron-glia interaction that is indispensable for network formation during a specific period in the developing brain.

Kidney fibrosis is the final common pathway of all progressive chronic kidney diseases, of which diabetic nephropathy is the leading cause. Endothelial-to-mesenchymal transition (EndMT) has emerged as one of the most important origins of matrix-producing fibroblasts. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been introduced into the market as antidiabetes drugs. Here, we found that the DPP-4 inhibitor linagliptin ameliorated kidney fibrosis in diabetic mice without altering the blood glucose levels associated with the inhibition of EndMT and the restoration of microRNA 29s. Streptozotocin-induced diabetic CD-1 mice exhibited kidney fibrosis and strong immunoreactivity for DPP-4 by 24 weeks after the onset of diabetes. At 20 weeks after the onset of diabetes, mice were treated with linagliptin for 4 weeks. Linagliptin-treated diabetic mice exhibited a suppression of DPP-4 activity/protein expression and an amelioration of kidney fibrosis associated with the inhibition of EndMT. The therapeutic effects of linagliptin on diabetic kidneys were associated with the suppression of profibrotic programs, as assessed by mRNA microarray analysis. We found that the induction of DPP-4 observed in diabetic kidneys may be associated with suppressed levels of microRNA 29s in diabetic mice; linagliptin restored microRNA 29s and suppressed DPP-4 protein levels. Using cultured endothelial cells, we found that linagliptin inhibited TGF-β2–induced EndMT, and such anti-EndMT effects of linagliptin were mediated through microRNA 29 induction. These results indicate the possible novel pleiotropic action of linagliptin to restore normal kidney function in diabetic patients with renal impairment.

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18. 3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B. halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 sigma factors which belong to the extracytoplasmic function family, 10 are unique to B. halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.

Little is known about the organizational and functional connectivity of the corticospinal (CS) circuits that are essential for voluntary movement. Here, we map the connectivity between CS neurons in the forelimb motor and sensory cortices and various spinal interneurons, demonstrating that distinct CS-interneuron circuits control specific aspects of skilled movements. CS fibers originating in the mouse motor cortex directly synapse onto premotor interneurons, including those expressing Chx10. Lesions of the motor cortex or silencing of spinal Chx10 interneurons produces deficits in skilled reaching. In contrast, CS neurons in the sensory cortex do not synapse directly onto premotor interneurons, and they preferentially connect to Vglut3 spinal interneurons. Lesions to the sensory cortex or inhibition of Vglut3 interneurons cause deficits in food pellet release movements in goal-oriented tasks. These findings reveal that CS neurons in the motor and sensory cortices differentially control skilled movements through distinct CS-spinal interneuron circuits.

Common molecular machineries between hematopoietic stem cell (HSC) maintenance and leukemia prevention have been highlighted. The tumor suppressor Fbxw7 (F-box and WD-40 domain protein 7), a subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle. We demonstrate that inactivation of Fbxw7 in hematopoietic cells causes premature depletion of HSCs due to active cell cycling and p53-dependent apoptosis. Interestingly, Fbxw7 deletion also confers a selective advantage to cells with suppressed p53 function, eventually leading to development of T-cell acute lymphoblastic leukemia (T-ALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis.Supplemental material is available at http://www.genesdev.org.Received October 1, 2007; revised version accepted February 22, 2008. Stem cells in various organ tissues are governed by general genetic programs that maintain their common features, including self-renewal and multipotency. Recent studies have demonstrated that it is crucial for hematopoietic stem cells (HSCs) to be quiescent for protection against oxidative stress and to sustain self-renewal capacity Tothova et al. 2007). Most HSCs remain quiescent when located in the stem cell niche (Calvi et al. 2003;Zhang et al. 2003;Arai et al. 2004). Once HSCs are released from the niche, they enter the cell cycle and start to proliferate. Cell cycle kinetics of HSCs are strictly controlled by various systems to sustain blood cell production throughout life (Cheng et al. 2000). These findings indicate that precise regulation of the cell cycle in stem cells is essential to maintain stem cell phenotype. The ubiquitin-proteasome system plays a critical role in controlling physiologic events-such as cell cycle progression, apoptosis, signal transmission, and repair of DNA damage-through protein degradation ) independently reported that Fbxw7-deficient mice die at embryonic day 10.5 and exhibit deficiencies in hematopoietic and vascular development, suggesting that Fbxw7 functions in hematopoiesis. Mutations in FBXW7 have been detected in certain human malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) (Spruck et al. 2002; EkholmReed et al. 2004;Mao et al. 2004;Maser et al. 2007). Recent reports have highlighted the existence of molecules operating differentially in the self-renewal of both normal tissue stem cells and cancer stem cells (Yilmaz et al. 2006;Zhang et al. 2006). In this study, we addressed this issue by examining the effect of Fbxw7 deletion on the maintenance of HSCs and leukemogenesis. Here, we demonstrate that inactivation of Fbxw7 in bone marrow (BM) HSCs leads to premature depletion of normal HSCs due to active cell cycling and promotes T-ALL due to a compromised p53 response. Thus, Fbxw7 acts as a critical fail-safe against premature loss of HSCs and development of T-ALL (Supplemental Fig. S1). Results and DiscussionWe examined Fbxw7 expression by quantitative RT-PCR in various hematopoietic l...

The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM ؊ Sca-1 ؉ cells, osteoblast-enriched ALCAM ؉ Sca-1 ؊ , and ALCAM -Sca-1 ؊ cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM ؉ Sca-1 ؊ cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing-and cell adhesion-related genes in HSCs. Microarray analysis showed that ALCAM ؊ Sca-1 ؉ fraction highly expressed cytokine-related genes, whereas the ALCAM ؉ Sca-1 ؊ fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic marker low/؊ subpopulation in ALCAM ؉ Sca-1 ؊ fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow. (Blood. 2010;116(9):1422-1432) IntroductionDuring postnatal life, bone marrow (BM) supports both selfrenewal and differentiation of hematopoietic stem cells (HSCs) in specialized niches. These niches, which are composed of cellular components located around stem cells, facilitate the signaling networks that control the balance between self-renewal and differentiation. [1][2][3][4][5] Long-term HSCs (LT-HSCs) are retained in a quiescent state in the BM, where they are anchored to specialized niches along the endosteum (the border between the bone and the BM) and in perivascular sites adjacent to the endothelium. Cytokines, chemokines, adhesion molecules, proteolytic enzymes, neurotransmitters, and transcription factors regulate the balance between quiescence and activation (proliferation and migration) of HSCs. 4,6 In particular, Angiopoietin-1 (Angpt1), 5 Kit-ligand (Kitl), 7,8 chemokine (C-X-C motif) ligand 12 (Cxcl12), 9-12 thrombopoietin (Thpo), 13,14 Wnt, 15 Jagged1 (Jag1), 16 osteopontin (OPN), 17, [19][20][21] are known to be involved in the niche regulation of HSCs in BM, and cooperative regulation among cytokine signals and cell adhesion molecules is required for the maintenance of HSCs. We previously reported that Tie2/Angpt1 and Mpl/Thpo signaling between HSCs and the endosteal (osteoblastic) niche cells plays a critical role in the enhancement of cell-to-cell and cell-to-extracellular matrix interactions of HSCs with niche cells and in the maintenance of cell-cycle quiescence of HSCs. Therefore, the functionality of the endosteal niche cells is critical for maintaining a sufficient rese...

Introduction:The information regarding therapeutically relevant genomic alterations in small cell lung cancer (SCLC) is not well developed. We analyzed the SCLC genome using an integrative approach to stratify the targetable alterations.Methods:We performed whole exon sequencing (n = 51) and copy number analysis (n =47) on surgically resected tumors and matched normal tissue samples from treatment-naive Japanese SCLC patients.Results:The demographics of the 51 patients included in this study were as follows: median age, 67 years (range, 42–86 years); female, 9 (18%); history of smoking, 50 (98%); and pathological stage I/II/III/IV, 28/13/9/1, respectively. The average number of nonsynonymous mutations was 209 (range, 41–639; standard deviation, 130). We repeatedly confirmed the high prevalence of inactivating mutations in TP53 and RB1, and the amplification of MYC family members. In addition, genetic alterations in the PI3K/AKT/mTOR pathway were detected in 36% of the tumors: PIK3CA, 6%; PTEN, 4%; AKT2, 9%; AKT3, 4%; RICTOR, 9%; and mTOR, 4%. Furthermore, the individual changes in this pathway were mutually exclusive. Importantly, the SCLC cells harboring active PIK3CA mutations were potentially targetable with currently available PI3K inhibitors.Conclusions:The PI3K/AKT/mTOR pathway is distinguishable in SCLC genomic alterations. Therefore, a sequencing-based comprehensive analysis could stratify SCLC patients by potential therapeutic targets.