Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells

CD34 is a transmembrane protein that is strongly expressed on hematopoietic stem/progenitor cells (HSCs); despite its importance as a marker of HSCs, its function is still poorly understood, although a role in cell adhesion has been demonstrated. To characterize the function of CD34 antigen on human HSCs, we examined, by both inhibition and overexpression, the role of CD34 in the regulation of HSC lineage differentiation. Our results demonstrate that CD34 silencing enhances HSC granulocyte and megakaryocyte differentiation and reduces erythroid maturation. In agreement with these results, the gene expression profile of these cells reveals the upregulation of genes involved in granulocyte and megakaryocyte differentiation and the downregulation of erythroid genes. Consistently, retroviral-mediated CD34 overexpression leads to a remarkable increase in erythroid progenitors and a dramatic decrease in granulocyte progenitors, as evaluated by clonogenic assay. Together, these data indicate that the CD34 molecule promotes the differentiation of CD34+ hematopoietic progenitors toward the erythroid lineage, which is achieved, at least in part, at the expense of granulocyte and megakaryocyte lineages.     

UHRF1 regulation of the Keap1–Nrf2 pathway in pancreatic cancer contributes to oncogenesis

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ～70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1‐mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2‐regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K‐Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2‐mediated cellular protection. Since UHRF1 over‐expression occurs in other cancers, its ability to regulate the Keap1–Nrf2 pathway may be critically important to the malignant behaviour of these cancers. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of αβ and γδ T lymphocytes. Surprisingly, using a new model of LAT‐deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat^Inv/Inv) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1⁺ cells. Thy1^dull cells expressed markers of NK lymphocytes and contained low frequency of TCR‐γ gene rearrangements without detectable TCR‐δ rearrangements. Thy1^high cells resembled immature CD44⁺CD25⁺ thymocytes and contained high frequency of non‐productive TCR‐γ and TCR‐δ rearrangements, indicating that cells displaying molecular signatures of commitment toward γδ T‐cell lineage can develop and populate lymphoid tissues of LAT‐deficient mice. Phenotypically similar Thy1^high cells were also found in lymph nodes of lymphocyte‐deficient (Rag2^?/?) mice but not in T lymphocyte proficient, heterozygous Lat^+/Inv mice suggesting that Thy1^high cells of LAT‐deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.     

Estrogen‐inducible sFRP5 inhibits early B‐lymphopoiesis in vivo,but not during pregnancy

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen‐induced soluble Frizzled‐related proteins (sFRPs), and particularly sFRP5, suppress B‐lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B‐lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B‐cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B‐lineage‐committed progenitors was observed in the BM of sfrp5‐null pregnant females. We concluded that, although high sFRP5 expression inhibits B‐lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho‐hematopoiesis in the maternal BM, but the maternal–fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.     

Notch1 expression in early lymphopoiesis influences B versus T lineage determination.

Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+ CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell.     

Nitro-fatty acids and cyclopentenone prostaglandins share strategies to activate the Keap1-Nrf2 system: a study using green fluorescent protein transgenic zebrafish

Nitro‐fatty acids are electrophilic fatty acids produced in vivo from nitrogen peroxide that have many physiological activities. We recently demonstrated that nitro‐fatty acids activate the Keap1‐Nrf2 system, which protects cells from damage owing to electrophilic or oxidative stresses via transactivating an array of cytoprotective genes, although the molecular mechanism how they activate Nrf2 is unclear. A number of chemical compounds with different structures have been reported to activate the Keap1‐Nrf2 system, which can be categorized into at least six classes based on their sensing pathways. In this study, we showed that nitro‐oleic acid (OA‐NO₂), one of major nitro‐fatty acids, activates Nrf2 in the same manner that of a cyclopentenone prostaglandin 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15d‐PGJ₂) using transgenic zebrafish that expresses green fluorescent protein (GFP) in response to Nrf2 activators. In transgenic embryos, GFP was induced in the whole body by treatment with OA‐NO₂, 15d‐PGJ₂ or diethylmaleate (DEM), but not with hydrogen peroxide (H₂O₂), when exogenous Nrf2 and Keap1 were co‐overexpressed. Induction by OA‐NO₂ or 15d‐PGJ₂ but not DEM was observed, even when a C151S mutation was introduced in Keap1. Our results support the contention that OA‐NO₂ and 15d‐PGJ₂ share an analogous cysteine code as electrophiles and also have similar anti‐inflammatory roles.     

A map for lineage restriction of progenitors during hematopoiesis: the essence of the myeloid-based model

Most hematology and immunology textbooks describe that the first branch point from the hematopoietic stem cells (HSCs) produces two progenitors, one for myelo-erythroid cells and the other for lymphoid cells including T and B cells. This model is based on the concept that the blood cell family can be subdivided into two major lineages, a myelo-erythroid lineage and a lymphoid lineage. Several alternative models have been proposed during the last three decades. We proposed the myeloid-based model in 2001, in which myeloid potential is retained in an early stage of branches toward erythroid, T-, and B-cell lineages. In this review, we focus on the point that cell differentiation models have two different facets: as a map of developmental potential and a cell fate map. These two are expressed in other words as a map for lineage restriction and a map for physiological production routes. We argue that a map of potential is first and foremost essential for the study of molecular mechanisms of lineage commitment, which is the least clarified aspect of cell differentiation. The validity of the myeloid-based model of hematopoiesis will be discussed in reference to these two issues, developmental potential and cell fate.     

The emerging role of the Nrf2–Keap1 signaling pathway in cancer

The Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2])–Keap1 (Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1) signaling pathway is one of the most important cell defense and survival pathways. Nrf2 can protect cells and tissues from a variety of toxicants and carcinogens by increasing the expression of a number of cytoprotective genes. As a result, several Nrf2 activators are currently being tested as chemopreventive compounds in clinical trials. Just as Nrf2 protects normal cells, studies have shown that Nrf2 may also protect cancer cells from chemotherapeutic agents and facilitate cancer progression. Nrf2 is aberrantly accumulated in many types of cancer, and its expression is associated with a poor prognosis in patients. In addition, Nrf2 expression is induced during the course of drug resistance. Collectively, these studies suggest that Nrf2 contributes to both intrinsic and acquired chemoresistance. This discovery has opened up a broad spectrum of research geared toward a better understanding of the role of Nrf2 in cancer. This review provides an overview of (1) the Nrf2–Keap1 signaling pathway, (2) the dual role of Nrf2 in cancer, (3) the molecular basis of Nrf2 activation in cancer cells, and (4) the challenges in the development of Nrf2-based drugs for chemoprevention and chemotherapy.     

Activated Notch Supports Development of Cytokine Producing NK Cells Which Are Hyporesponsive and Fail to Acquire NK Cell Effector Functions

Natural Killer (NK) cells are powerful effectors of cytotoxicity against “stressed” cells. They also produce cytokines and chemokines to activate the adaptive immune response. Understanding NK cell development and maturation may have implications for cancer therapy and for immunity against infections. We hypothesized that Notch signaling, critical for hematopoesis, would be involved in NK cell development. The role of constitutively activated Notch1 (ICN) on NK cell maturation was studied using human umbilical cord blood (UCB) progenitors cultured on a murine embryonic liver stroma cell line (EL08-1D2) and human cytokines. UCB CD34⁺/ICN⁺ sorted cells resulted in a population of CD7⁺ early lymphoid precursors and subsequent NK lineage commitment independent of stroma or IL-15. Early expression of L-selectin on ICN⁺ precursors suggested their homing competence. These precursors further committed to the NK lineage, and were capable of producing cytokines and chemokines such as interkeukin (IL)-13, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), yet poorly acquired NK inhibitory receptors and cytotoxic effector function. In the presence of stroma, ICN⁺ precursors also gave rise to a population of early T lineage committed cells characterized by expression of cytoplasmic CD3 γ, ε, and δ chains, RAG1/2, and production of IL-2, suggesting bona fide Th1 commitment. Importantly, signals from EL08-1D2 stroma were required for this development process. In conclusion, sustained Notch signaling can replace stroma in differentiation of a common CD7⁺ lymphoid precursor from UCB CD34⁺ progenitors and induce NK cell commitment. However, these NK cells are immature in their cytokine production profile, are hyporesponsive, and poorly acquire NK cell receptors involved in self-tolerance and effector function.     

Monoclonal antibodies identifying feline haemopoietic cell lineages

Monoclonal antibodies (MAbs) were prepared against feline bone marrow mononuclear cells. Immunogold immunofluorescence (IGIF), flow cytometry and fluorescence activated cell sorting (FACS) were used to determine the selective reactivity of four MAbs, designated FeMy, FeLy and FeEr1/Er2 with feline myeloid (granulocyte/macrophage), lymphoid, and erythroid lineage cells, respectively. Reactivity was also assessed to four feline lymphoma cell lines (3201, 3191, 3281, FL74). FeMy reacted with 74% of all myeloid lineage cells (88% of mature and 30% of early myeloid progenitors), 98% of blood neutrophils, 97% of eosinophils and 90% of monocytes. FACS of bone marrow using feMy yielded 89% myeloid lineage cells. FeLy reacted with 67–75% of lymphoid lineage marrow cells IGIF and flow cytometry. However, FeLy also recognised a surface molecule present on 30% of erythroid precursors, 86% of eosinophils, and three of four feline lymphoma cell lines. FACS of marrow cells using FeLy yielded 77% lymphoid cells (and 19% myeloid cells). FeErl and FeEr2 (which identified either the same or closely associated molecules) reacted with 55–66% of early erythroid and 90–95% of late erythroid lineage marrow cells but not with mature erythrocytes by immunogold immunofluorescence. Marrow FACS using FeErl and FeEr2 yielded 76–80% erythroid cells (and 18–21% myeloid progenitors). Wheres FeLy immunoprecipitated a 120 kDa molecule, neither FeMY nor FeErl and FeEr2 precipitated an identifiable molecule. The panel of MAbs described may be useful in immunophenotyping of feline haemopoietic neoplasia.