Constitutive MAP kinase activation in hematopoietic stem cells induces a myeloproliferative disorder

Myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPNs) are a group of myeloid neoplasms in which abnormal activation of the Ras signaling pathway is commonly observed. The PI3K/Akt pathway is a known target of Ras; however, activation of the PI3K/Akt pathway has been shown to lead to neoplastic transformation of not only myeloid but also lymphoid cells, suggesting that pathways other than the PI3K/Akt pathway should play a central role in pathogenesis of Ras-mediated MDS/MPN. The MEK/ERK pathway is another downstream target of Ras, which is involved in regulation of cell survival and proliferation. However, the role of the MEK/ERK pathway in the pathogenesis of MDS/MPN remains unclear. Here, we show that introduction of a constitutively activated form of MEK into hematopoietic stem cells (HSCs) causes hematopoietic neoplasms that are limited to MDS/MPNs, despite the multipotent differentiation potential of HSCs. Active MEK-mediated MDS/MPNs are lethal, but are not considered a frank leukemia because it cannot be transplanted into naïve animals. However, transplantation of MDS/MPNs co-expressing active MEK and an anti-apoptotic molecule, Bcl-2, results in T-cell acute lymphocytic leukemia (T-ALL), suggesting that longevity of cells may impact transplantability and alter disease phenotype. Our results clearly demonstrate the proto-oncogenic property of the MEK/ERK pathway in hematopoietic cells, which manifest in MDS/MPN development.     

造血干细胞嵌合体诱导移植免疫耐受

造血干细胞混合嵌合体是指两个不同基因型个体的骨髓造血干细胞共存的一种状态。在同种异体或异种移植的动物模型中,造血干细胞混合嵌合体巳成功地诱导出针对供者特异性的免疫耐受。现已证实造血干细胞具有否决活性,来自造血干细胞的否决细胞在诱导移植特异性免疫耐受中可能起重要作用。     

Bacterial infection of human hematopoietic stem cells induces monocytic differentiation

Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34(+) hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response.     

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.     

Thymosin activation of T-killer precursors for hematopoietic stem cells]

Lymph node cells of normal CBA mice, syngeneic radiation chimerae CBA leads to CBA and B-mice after incubation with thymosin (fraction 5) were transplanted to sublethally irradiated recipients (CBA X C57BL) F1; the number of endogenic colonies in the recipient's spleen was then recorded. Thymosin was shown to increase the killer activity of the lymph node cells of normal mice CBA, syngeneic chimerae CBA leads to CBA, but not of B-mice. As suggested, TU-cells' subpopulation served as target cells for thymosin.     

Inflammation induces myeloid-derived suppressor cells that facilitate tumor progression

Epidemiological and experimental observations support the hypothesis that chronic inflammation contributes to cancer development and progression; however, the mechanisms underlying the relationship between inflammation and cancer are poorly understood. To study these mechanisms, we have transfected the mouse 4T1 mammary carcinoma with the proinflammatory cytokine IL-1beta to produce a chronic inflammatory microenvironment at the tumor site. Mice with 4T1/IL-1beta tumors have a decreased survival time and elevated levels of immature splenic Gr1+CD11b+ myeloid-derived cells. These myeloid suppressor cells (MSC) are present in many patients with cancer and inhibit the activation of CD4+ and CD8+ T lymphocytes. 4T1/IL-1beta-induced MSC do not express the IL-1R, suggesting that the cytokine does not directly activate MSC. Neither T or B cells nor NKT cells are involved in the IL-1beta-induced increase of MSC because RAG2-/- mice and nude mice with 4T1/IL-1beta tumors also have elevated MSC levels. MSC levels remain elevated in mice inoculated with 4T1/IL-1beta even after the primary tumor is surgically removed, indicating that the IL-1beta effect is long lived. Collectively, these findings suggest that inflammation promotes malignancy via proinflammatory cytokines, such as IL-1beta, which enhance immune suppression through the induction of MSC, thereby counteracting immune surveillance and allowing the outgrowth and proliferation of malignant cells.     

Lipocalin-2 induces mitochondrial dysfunction in renal tubular cells via mTOR pathway activation

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Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.Key words: thrombopoietin, TPO, Mpl, hematopoietic stem cell, hematopoiesis, Jak2, MPLW515K, MPLW515L     

Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.     

Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth

Objectives:  The aim of this study was to determine whether normal human embryonic stem cells (hESC) would secrete factors that arrest growth of human epithelial cancer cell lines.
Materials and methods:  Cell proliferation was examined using the MTT assay then haemocytometer cell counts. Staining with propidium iodide followed by flow cytometry was used to detect cell cycle stages. Heat denaturation and molecular fractionation experiments were also performed.
Results:  We found that hESC conditioned medium (hESC CM) inhibited SKOV-3 and HEY cell proliferation. Similar results were also obtained when we used breast and prostate cancer cell lines, whereas little or no inhibitory effect was observed when human fibroblasts were tested. Moreover, a co-culture model confirmed that inhibition of cancer cell proliferation is mediated by soluble factors produced by hESCs. We also determined that the proportion of cancer cells in G₁ phase was increased by hESC CM treatment, accompanied by decrease in cells in S and G₂/M phases, suggesting that the factors slow progression of cancer cells by cell cycle inhibition. Heat denaturation and molecular fractionation experiments indicated a low molecular weight thermostable factor was responsible for these properties.
Conclusions:  Our findings provide evidence that the human embryonic microenvironment contains soluble factor(s) that are capable of inhibiting growth of cancer cells, and that exposure to such factors may represent a new cancer treatment strategy.     

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G₂/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.     

16,16-Dimethyl prostaglandin E2 induces radioprotection in murine intestinal and hematopoietic stem cells

Exogenous prostaglandins (PGs) have been shown to protect gastrointestinal mucosa, liver, and pancreas from several injurious agents, including the PG inhibitor, indomethacin. Previous studies from this laboratory showed exogenous administration of 16,16-dimethyl (dm) PGE2 also protected mouse intestinal stem cells from radiation injury. The present study extended that observation and demonstrated that PGs given to B6D2F1 mice 1 hr before irradiation increased the shoulder of the intestinal clonogenic cell survival curve. The D0 increased from 1.10 + 0.09 to 1.58 + 0.10 Gy. PGs increased the LD50/6 from 16.3 + 0.41 (95% confidence limits) in controls to 20.25 + 0.55 Gy. The 16,16-dm PGE2 increased the hematopoietic CFU-S survival in a qualitatively similar way; the extrapolation number (n) was increased from 1.03 (0.89-1.20) to 1.40 (1.27-1.54) and the D0 increased from 0.92 (0.87-0.98) to 1.14 (1.10-1.19) Gy. A large number of human tumors secrete a variety of PGs. Our results suggest that those tumors may be, in part, protected from radiation injury.     

Murine hematopoietic stem cells after acute and chronic administration of vincristine

The effects of vincristine on bone marrow hemopoietic stem cells were studied in mice. After the administration of a single dose of the drug there is a sharp decline in the content of CFUs. When the drug is given in small, weekly doses for 16 weeks, after an initial drop, the stem cell content rises at a normal level where it remains, although with wide oscillations. On the whole the effects of vincristine on b.m. CFUs are comparable with that observed after vinblastine administration.     

Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses

Harnessing the ability of cytotoxic T lymphocytes (CTLs) to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs) following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.     

Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells

Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, maintains bone mass in adulthood. We now report that IGF-1 released from the bone matrix during bone remodeling stimulates osteoblastic differentiation of recruited mesenchymal stem cells (MSCs) by activation of mammalian target of rapamycin (mTOR), thus maintaining proper bone microarchitecture and mass. Mice with knockout of the IGF-1 receptor (Igf1r) in their pre-osteoblastic cells showed lower bone mass and mineral deposition rates than wild-type mice. Further, MSCs from Igf1rflox/flox mice with Igf1r deleted by a Cre adenovirus in vitro, although recruited to the bone surface after implantation, were unable to differentiate into osteoblasts. We also found that the concentrations of IGF-1 in the bone matrix and marrow of aged rats were lower than in those of young rats and directly correlated with the age-related decrease in bone mass. Likewise, in age-related osteoporosis in humans, we found that bone marrow IGF-1 concentrations were 40% lower in individuals with osteoporosis than in individuals without osteoporosis. Notably, injection of IGF-1 plus IGF binding protein 3 (IGFBP3), but not injection of IGF-1 alone, increased the concentration of IGF-1 in the bone matrix and stimulated new bone formation in aged rats. Together, these results provide mechanistic insight into how IGF-1 maintains adult bone mass, while also providing a further rationale for its therapeutic targeting to treat age-related osteoporosis.     

Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy

Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin-treated mice, insulin- and TNF-α co-expressing bone marrow-derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high-fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co-expression of insulin and TNF-α in c-kit⁺Sca-1⁺lineage⁻ (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.     

mTOR and the differentiation of mesenchymal stem cells

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase, belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells. mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.     

Communications between bone cells and hematopoietic stem cells

The skeletal system, while characterized by a hard tissue component, is in fact an extraordinarily dynamic system, with disparate functions ranging from structural support, movement and locomotion and soft-organ protection, to the maintenance of calcium homeostasis. Amongst these functions, it has long been known that mammalian bones house definitive hematopoiesis. In fact, several data demonstrate that the bone microenvironment provides essential regulatory cues to the hematopoietic system. In particular, interactions between the bone-forming cells, or osteoblasts, and the most primitive Hematopoietic Stem Cells (HSC) have recently been defined. This review will focus mainly on the role of osteoblasts as HSC regulatory cells, discussing the signaling mechanisms and molecules currently thought to be involved in their modulation of HSC behavior. We will then review additional cellular components of the HSC niche, including endothelial cells and osteoclasts. Finally, we will discuss the potential clinical implications of our emerging understanding of the complex HSC microenvironment.     

Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation

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