Dongsu Park

An essential role for Akt1 in dendritic cell function and tumor immunotherapy

Current dendritic cell (DC) vaccine preparations involving ex vivo differentiation and maturation produce short-lived, transiently active DCs that may curtail T-cell responses in vivo. We demonstrate that Akt1, downregulation of which decreases DC lifespan, is critical for proinflammatory signal–mediated DC survival and maturation. Lipopolysaccharide or CD40 signaling stabilizes Akt1, promoting both activation and Bcl-2–dependent survival of DCs. Expression of a potent allele encoding a lipid raft–targeted Akt1, MF-ΔAkt, is sufficient for maturation and survival of murine bone marrow–derived DCs in vivo. MF-ΔAkt–transduced DCs enhanced T-cell proliferation, activation and long-term memory responses, enabling eradication of large pre-established lymphomas and aggressive B16 melanomas. Human myeloid DCs expressing constitutively active MF-ΔhAkt also survived significantly longer and promoted antigen-specific T-cell responses. Thus, Akt1 is a critical regulator of DC lifespan, and its manipulation in DCs can improve the clinical efficacy of DC-based tumor vaccines.

The hematopoietic stem cell niche

Hematopoietic stem cells (HSCs) possess the ability to self-renew and to differentiate to mature progeny along multiple different hematopoietic lineages. The function of HSCs depends upon the signals from surrounding cells found within the highly specialized microenvironment termed the hematopoietic stem cell niche. Understanding and exploiting the HSC niche is a goal of basic scientists and clinicians alike. Recent studies have focused on defining the cellular components and molecular factors critical to this microenvironment. Here we review recent findings, discuss unresolved questions, and examine the clinical implications of our current knowledge of the HSC niche.

Lipid raft-targeted Akt promotes axonal branching and growth cone expansion via mTOR and Rac1, respectively

The molecular mechanisms by which extracellular guidance cues regulate axonal morphology are not fully understood. Recent findings suggest that increased activity of the protein kinase Akt promotes dendritic branching and elongation in hippocampal neurons. We tested whether expression of constitutively active Akt (CA‐Akt) in primary sensory neurons would promote axonal branching and whether targeting CA‐Akt to lipid rafts, common sites of Akt function, would differentially regulate axonal morphology. Biolistic transduction of sensory neurons induced a rapid expression of CA‐Akt, resulting in increased axonal branching, cell hypertrophy, and growth cone expansion. Additionally, we found that targeting of CA‐Akt to lipid rafts significantly potentiated growth cone expansion compared with expression of CA‐Akt throughout the neuron. Because lipid rafts are concentrated within the growth cone, this finding suggests that signaling of expansion is likely regulated locally. We found that CA‐Akt‐mediated growth cone expansion, but not axonal branching, was attenuated by coexpression of dominant‐negative Rac1. In contrast, blockade of mammalian target of rapamycin (mTOR) prevented axonal branching and hypertrophy in response to CA‐Akt, but not growth cone expansion. These data indicate that Akt activity can regulate growth cone expansion via localized Rac1 signaling and regulate axonal branching and soma size via activation of mTOR.

Activation of Wnt Signaling by Chemically Induced Dimerization of LRP5 Disrupts Cellular Homeostasis

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2Kb promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63+ cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.

Stress Hematopoiesis Is Regulated by the Krüppel‐Like Transcription Factor ZBP‐89

Previous studies have shown that ZBP‐89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP‐89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89−/− HSPC also resulted in a myeloid‐to‐B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89−/− HSPC are reproduced in vitro in the ZBP‐89‐silenced multipotent hematopoietic cell line FDCP‐Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA‐1 in ZBP89‐deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP‐89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA‐1. These data identify an important role for ZBP‐89 in regulating stress hematopoiesis in adult mouse bone marrow. Stem Cells 2014;32:791–801

Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells

Periosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different locations are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible Mx1 and NestinGFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of Mx1 and NestinGFP expression, while endogenous P-SSCs can be isolated by positive selection of Mx1, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and osteocalcinGFP (a mature osteoblast marker). Comparative gene expression analysis with FACS-sorted BM-SSCs, P-SSCs, Osterix+ preosteoblasts, CD51+ stroma cells and CD45+ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51+ cells are highly heterogeneous and have little overlap with SSCs. This was further supported by the microarray cluster analysis, where the two SSC populations clustered together but are separate from the CD51+ cells. However, when comparing SSC population to controls, we found several genes that are uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset in vivo.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

The Skeletal Stem Cell

Abstract Tissues in mammals are generally maintained by one of two schemas, mature cell replacement by mature cell division or mature cell replenishment from a more immature stem or progenitor population. It is the latter model that applies to bone homeostasis and we will review here the definition of the skeletal stem cell, its relationship to descendent semi-committed osteoprogenitors, and the terminally differentiated mesenchymal cells (MC) that make up the mature bone. In addition, we will discuss the migration of mesenchymal osteoprogenitors, their kinetics in regulation of life-long bone production, and the interaction of bone cells with hematopoietic cells in the bone marrow.

1132. Role of Akt in Dendritic Cell (DC) Survival and Application of an Akt |[ldquo]|LIFE SWITCH|[rdquo]| in DC-Based Tumor Vaccines

Top of pageAbstract The lifespan of activated dendritic cells (DCs) in lymphoid organs is critical to the regulation of immunity. Although pro-inflammatory cytokines and pathogens are important in maintaining the survival of activated DCs through the regulation of Bcl-2 family members, the molecular mechanisms from pro-survival signals to downstream anti-apoptotic molecules are not completely defined. Here we show that DC death from GM-CSF deprivation induces the rapid down-regulation of Akt, but that treatment of DCs with LPS or anti-CD40 antibody reverses this down-regulation, thereby promoting DC survival. In addition, we found that the down-regulation of Akt correlates with Bcl-2 expression levels but not with Bcl-xL. DCs treated with Akt-1 siRNA showed greatly reduced Akt expression level, resulting in rapid DC death. Furthermore, a functionally optimized Akt (MF-DAkt) protected DCs against cell death signals from PI3-K inhibition and GM-CSF deprivation. Finally, MF-|[Delta]|Akt-transduced DCs displayed significantly increased lifespan in vitro and in vivo, induced maturation, allogeneic and antigen-specific T cell proliferative responses, and enhanced IFN-|[gamma]| production, resulting in the rapid eradication of pre-established tumors in mice. Thus, these data suggest that Akt is a critical regulator of DC lifespan and functions as a |[ldquo]|life switch|[rdquo]| that may contribute to the development of a DC-based tumor vaccine. (See Figure)