Dragan Marinkovic

Foxo3 is required for the regulation of oxidative stress in erythropoiesis

Erythroid cells accumulate hemoglobin as they mature and as a result are highly prone to oxidative damage. However, mechanisms of transcriptional control of antioxidant defense in erythroid cells have thus far been poorly characterized. We observed that animals deficient in the forkhead box O3 (Foxo3) transcription factor died rapidly when exposed to erythroid oxidative stress-induced conditions, while wild-type mice showed no decreased viability. In view of this striking finding, we investigated the potential role of Foxo3 in the regulation of ROS in erythropoiesis. Foxo3 expression, nuclear localization, and transcriptional activity were all enhanced during normal erythroid cell maturation. Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of p21(CIP1/WAF1/Sdi1) (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role.

Foxo3 Is Essential for the Regulation of Ataxia Telangiectasia Mutated and Oxidative Stress-mediated Homeostasis of Hematopoietic Stem Cells

Unchecked accumulation of reactive oxygen species (ROS) compromises maintenance of hematopoietic stem cells. Regulation of ROS by the tumor suppressor protein ataxia telangiectasia mutated (ATM) is critical for preserving the hematopoietic stem cell pool. In this study we demonstrate that the Foxo3 member of the Forkhead Box O (FoxO) family of transcription factors is essential for normal ATM expression. In addition, we show that loss of Foxo3 leads to defects in hematopoietic stem cells, and these defects result from an overaccumulation of ROS. Foxo3 suppression of ROS in hematopoietic stem cells is mediated partly by regulation of ATM expression. We identify ROS-independent modulations of ATM and p16INK4a and ROS-mediated activation of p53/p21CIP1/WAF1/Sdi1 tumor suppressor pathways as major contributors to Foxo3-null hematopoietic stem cells defects. Our studies demonstrate that Foxo3 represses ROS in part via regulation of ATM and that this repression is required for maintenance of the hematopoietic stem cell pool.

Reduction of marginal zone B cells in CD22-deficient mice.

CD22 is a B cell‐specific member of the immunoglobulin superfamily and binds to sialic acid. CD22 inhibits B cell receptor signaling. Mice deficient for CD22 show a largely normal B cell development. Here, we have performed a detailed analysis of the splenic B cell population and found that the subset of marginal zone (MZ) B cells was selectively reduced in CD22‐deficient mice. CD22‐deficient mice showed a lack of TNP‐ficoll capturing cells in the MZ and a reduced response to TNP‐ficoll, particularly when the antigen was applied intravenously. CD22‐deficient B cells showed both enhanced motility as well as enhanced chemotaxis to certain chemokines. The altered chemokine responsiveness or the higher signaling capacity of CD22‐deficient B cells may lead to the compromised MZ B cell compartment, as both processes have previously been shown to affect MZ composition.

The Yaa Mutation Promoting Murine Lupus Causes Defective Development of Marginal Zone B Cells

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.

c-MYC activation impairs the NF-κB and the interferon response: Implications for the pathogenesis of Burkitt's lymphoma

Deregulation of the proto‐oncogene c‐myc is a key event in the pathogenesis of many tumors. A paradigm is the activation of the c‐myc gene by chromosomal translocations in Burkitt lymphoma (BL). Despite expression of a restricted set of Epstein–Barr viral (EBV) antigens, BL cells are not recognized by antigen‐specific cytotoxic T cells (CTLs) because of their inability to process and present HLA class I‐restricted antigens. In contrast, cells of EBV‐driven posttransplant lymphoproliferative disease (PTLD) are recognized and rejected by EBV‐specific CTLs. It is not known whether the poor immunogenicity of BL cells is due to nonexpression of viral antigens, overexpression of c‐myc, or both. To understand the basis for immune recognition and escape, we have compared the mRNA expression profiles of BL and EBV‐immortalized cells (as PTLD model). Among the genes expressed at low level in BL cells, we have identified many genes involved in the NF‐κB and interferon response that play a pivotal role in antigen presentation and immune recognition. Using a cell line in which EBNA2 and c‐myc can be regulated at will, we show that c‐MYC negatively regulates STAT1, the central player linking the Type‐I and Type‐II interferon response. Switching off c‐myc expression leads to STAT1 induction through a direct and indirect mechanism involving induction of Type‐I interferons. c‐MYC thus masks an interferon‐inducing activity in these cells. Our findings imply that immune escape of tumor cells is not only a matter of in vivo selection but may be additionally promoted by activation of a cellular oncogene.

Reversible lymphomagenesis in conditionally c-MYC expressing mice

It is well documented that deregulation of MYC leads to tumor development, yet many aspects of this process are only partially understood. We have established a transgenic mouse model in which c‐MYC is conditionally expressed in lymphoid cells using the tetracycline‐regulated system of gene regulation. Mice with continuously expressed transgenic c‐MYC died of invasive T‐ or B‐cell lymphomas within 4 months. Lymphomas developing in transgenic mice were c‐MYC dependent since doxycycline treatment led to tumor regression. Using transplantation of established tumor cell lines labeled with GFP, we followed the fate of neoplastic cells in recipients upon MYC inactivation. This approach allowed us to elucidate both apoptosis and differentiation as mechanisms of tumor elimination. Comparative genomic hybridization (CGH) and FISH analyses were performed in order to analyze possible chromosomal aberrations induced by c‐MYC. We observed that overexpression of c‐MYC is sufficient to induce recurrent patterns of genomic instability. The main observation was a gain of genomic material that corresponded to chromosome 15 in several T‐cell tumors, which could be identified as trisomy.

B Cell–specific Transgenic Expression of Bcl2 Rescues Early B Lymphopoiesis but Not B Cell Responses in BOB.1/OBF.1-deficient Mice

Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in B cell differentiation. Numbers of immature transitional B cells in the bone marrow are reduced and fewer B cells reach the periphery. Furthermore, germinal center B cells are absent and marginal zone (MZ) B lymphocytes are markedly reduced. Increased levels of B cell apoptosis in these mice prompted us to analyze expression and function of antiapoptotic proteins. Bcl2 expression is strongly reduced in BOB.1/OBF.1-deficient pre–B cells. When BOB.1/OBF.1-deficient mice were crossed with Bcl2-transgenic mice, B cell development in the bone marrow and numbers of B cells in peripheral lymphoid organs were normalized. However, neither germinal center B cells nor MZ B cells were rescued. Additionally, Bcl2 did not rescue the defects in signaling and affinity maturation found in BOB.1/OBF.1-deficient mice. Interestingly, Bcl2-transgenic mice by themselves show an MZ B cell defect. Virtually no functional MZ B cells were detected in these mice. In contrast, mice deficient for Bcl2 show a relative increase in MZ B cell numbers, indicating a previously undetected function of Bcl2 for this B cell compartment.

BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.

ABSTRACT Marginal-zone (MZ) B cells represent a first line of defense against particulate blood-borne antigens. Together with the B1 cells, they are responsible for the early response against type II T-independent antigens. The molecular pathways controlling the development of MZ B cells are only poorly understood. We found that these cells are virtually absent in mice deficient in the BOB.1/OBF.1 coactivator. Loss of these B cells was demonstrated by the lack of cells showing the appropriate cell surface phenotype but also by histological analyses and tri-nitro-phenol-Ficoll capturing. The lack of these cells is a B-cell-intrinsic defect, as shown by bone marrow complementation experiments. We also show that the expression of BOB.1/OBF.1 in peripheral B cells is required for the development of MZ B lymphocytes. Our analysis of BOB.1/OBF.1-deficient splenic B cells reveals alterations in cell motility, tumor necrosis factor receptor expression, and B-cell receptor (BCR) signaling. These changes could contribute to the loss of MZ B lymphocytes by altering the maturation of the cells. Interestingly, development of and BCR signaling in B1 B cells are completely normal in BOB.1/OBF.1 mutant mice.

The IKK2/NF-κB pathway suppresses MYC-induced lymphomagenesis

Deregulated c-MYC is found in a variety of cancers where it promotes proliferation as well as apoptosis. In many hematologic malignancies, enhanced NF-kappaB exerts prosurvival functions. Here we investigated the role of NF-kappaB in mouse and human c-MYC-transformed lymphomas. The NF-kappaB pathway is extinguished in murine lymphoma cells, and extrinsic stimuli typically inducing NF-kappaB activity fail to activate this pathway. Genetic activation of the NF-kappaB pathway induces apoptosis in these cells, whereas inhibition of NF-kappaB by an IkappaBalpha superrepressor provides a selective advantage in vivo. Furthermore, in human Burkitt lymphoma cells we find that NF-kappaB activation induces apoptosis. NF-kappaB up-regulates Fas and predisposes to Fas-induced cell death, which is caspase-8 mediated and can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-kappaB-induced apoptosis, and persistent inactivity of NF-kappaB signaling is a prerequisite for MYC-mediated tumorigenesis. We could also show that low immunogenicity and Fas insensitivity of MYC-driven lymphoma cells are reversed by activation of NF-kappaB. Our observations provide a molecular explanation for the described absence of the NF-kappaB signaling in Burkitt lymphoma and question the applicability of NF-kappaB inhibitors as candidates for treatment of this cancer.

B Cell Hyperresponsiveness and Expansion of Mature Follicular B Cells but Not of Marginal Zone B Cells in NFATc2/c3 Double-Deficient Mice

Marginal zone (MZ) B cells and peritoneal B-1 cells provide a first defense system of thymus-independent Ab responses against foreign pathogens and therefore share a number of functional properties. Recently, development of B-1a cells was shown to be controlled by the transcription factor NFATc1. We show here that mice deficient for NFATc2 and c3 display a distinct lower representation of MZ B cells, which is correlated with a reduced capturing of trinitrophenyl-Ficoll. In contrast, mature follicular B cells from NFATc2/c3−/− mice are strongly increased in number. NFATc2/c3−/− B cells exhibit a marked increase in BCR-induced intracellular Ca2+ mobilization and proliferation. However, trinitrophenyl-Ficoll-specific IgM and IgG3 responses of NFATc2/c3-deficient mice are intact, and chimeric mice reconstituted with NFATc2/3-deficient B cells show a normal number of MZ B cells and normal BCR responses. These observations suggest that the strongly elevated Th2 cytokine milieu in NFATc2/c3-deficient mice leads to a hyperactivation of mature, follicular B cells, whereas MZ B cells are less responsive to these signals.