The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

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Absence of the Wnt Antagonist Sclerostin Adversely Affects B Cell Development in the Bone Marrow Niche

Blood ◽

10.1182/blood.v118.21.220.220 ◽

2011 ◽

Vol 118 (21) ◽

pp. 220-220 ◽

Cited By ~ 1

Author(s):

Corey J Cain ◽

Randell Rueda ◽

Bryce T McLelland ◽

Nicole Collette ◽

Gabriela Loots ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Wnt Signaling ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Niche ◽

Self Renewal ◽

Hematopoietic Stem ◽

Osteoblast Activity

Abstract Abstract 220 Hematopoietic cell fate decisions are dependent on their localized microenvironmental niche. In the bone, endosteal osteoblasts have been shown to support hematopoietic stem cells (HSC) self-renewal, as demonstrated by transgenic and knockout mouse models in which osteoblast populations were increased or decreased. In addition, Wnt signaling and the Wnt antagonist Dkk-1 have been implicated in various aspects of hematopoiesis and HSC self-renewal. Sclerostin (Sost) is a secreted protein that is primarily expressed by fully mature osteocytes and acts on osteoblasts as a negative regulator of bone growth, by antagonizing Wnt signaling by its binding to the Wnt co-receptors Lrp4, Lrp5, and/or Lrp6. Here, we investigated the role of Sost on hematopoiesis in the bone marrow niche. Increased osteoblast activity in sclerostin-knockout (Sost−/−) mice results in hypermineralized bones with small bone marrow cavities. As such, Sost−/− mice contain markedly reduced numbers of CD45+hematopoietic cells in the bone marrow. Since hematopoietic stem cell activity is dependent on osteoblast function, we examined whether the hyperactive osteoblast activity in Sost−/− mice influences the numbers of hematopoietic stem cells, lymphoid progenitor cells and myeloid progenitor cells in the bone marrow. Surprisingly, no differences were observed in hematopoietic stem and progenitor cell frequency and cell number. However, we found the bone marrow of Sost−/− mice to be depleted of B cells, and this reduction can be attributed to premature apoptosis beginning at the pre-pro-B cell stage. Examination of Sost expression showed that no hematopoietic cells expressed Sost, however, pre-pro, immature and recirculating B cells expressed Lrp5 and Lrp6. These gene expression patterns suggested that the defect in B cell development in Sost−/− mice is non-cell autonomous and that absence of Sost could affect Wnt signaling in these populations. We observed that the expression of Wnt target genes CCND1 and Lef-1 were not affected by the absence of Sost, but c-Myc was significantly upregulated in recirculating B cells in the bone marrow. We also observed a significant decrease in CXCL12 expression in the bone marrow stroma in Sost−/− mice, consistent with their inability to adequately support B cell development. Taken together, our results indicate that the B cell developmental defects in Sost−/− mice are non-cell autonomous, and we are currently performing reciprocal bone marrow transplantation experiments to further support this hypothesis. Our studies demonstrate a novel role for Sost in the regulation of B cell development in the bone marrow, and demonstrate that distinct Wnt antagonists play specific roles in the regulation of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽

10.1182/blood.v114.22.1465.1465 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1465-1465

Author(s):

Jason Mullenix ◽

Kimi Y Kong ◽

Kristin Severns Owens ◽

Jason Rogers ◽

Shannon FitzPatrick ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Mature Mirnas ◽

Cell Development ◽

B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

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Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo

Blood ◽

10.1182/blood.v89.2.518 ◽

1997 ◽

Vol 89 (2) ◽

pp. 518-525 ◽

Cited By ~ 34

Author(s):

Shunichi Takeda ◽

Takeyuki Shimizu ◽

Hans-Reimer Rodewald

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Fetal Liver ◽

Critical Role ◽

Cell Development ◽

B Cell Development ◽

Wild Type ◽

Hematopoietic Stem

Abstract The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.

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Sclerostin Deficiency Alters Peripheral B Lymphocyte Responses in Mice

10.1101/357772 ◽

2018 ◽

Cited By ~ 2

Author(s):

Arthur Chow ◽

Jourdan Mason ◽

Larrisha Coney ◽

Jamila Bajwa ◽

Cameron Carlisle ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Response ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Protein Antigens ◽

Hematopoietic Stem ◽

B Cell Response

AbstractUnderstanding how changes in bone physiology and homeostasis affect immune responses will inform how to retain strong immunity in patients with bone disease and in aged individuals. We previously identified sclerostin (Sost) as a mediator of cell communication between the skeletal and the immune system. Elevated bone mineral density in Sost-knockout (Sost-/-) mice contributes to an altered bone marrow microenvironment and adversely affects B cell development. B cells originate from hematopoietic stem cells within the bone marrow and mature in peripheral lymphoid organs to produce antibodies in response to infection and/or vaccination. In this study, we investigated whether the aberrant B cell development observed in the bone marrow of Sost-/- mice extends to peripheral B cells in the spleen during immune challenge, and if these changes were age-dependent. Concomitant with more severe changes in bone architecture, B cell development in the bone marrow and in the spleen worsened with age in Sost-/- mice. B cell responses to T-independent antigens were enhanced in young Sost-/- mice, whereas responses to T-dependent antigens were impaired. Our results support the hypothesis that the adverse effects of B cell development in the Sost-deficient bone marrow microenvironment extends to the peripheral B cell immune response to protein antigens, and suggest that the B cell response to routine vaccinations should be monitored regularly in patients being treated with sclerostin antibody therapy. In addition, our results open the possibility that Sost regulates the T-independent B cell response, which might be applicable to the improvement of vaccines towards non-protein antigens.

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Gas7 Induces The Proliferation Of Ph+ ALL Cells and Prevents The Differentiation Of Early B Cell Progenitors Into CD25high Small Pre-B Cells

Blood ◽

10.1182/blood.v122.21.2506.2506 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2506-2506 ◽

Cited By ~ 1

Author(s):

Jaewoong Lee ◽

Maike Buchner ◽

Huimin Geng ◽

Srividya Swaminathan ◽

Eugene Park ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Surface ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Cell Development ◽

B Cell Development ◽

Specific Gene

Abstract Background Growth arrest-specific gene 7 (Gas7) first discovered in growth-arrested NIH3T3 cells possesses WW, Fes/CIP4 homology (FCH), and coiled-coil domains, which can act as an adaptor for SH2 or 3-containing proteins. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Recently, Gas7-deficient mouse transiently expressing truncated form of Gas7 mutant protein shows motor activity defects due to reduced motor neuron number (Huang BT et al., PLoS One 2012). Interestingly, MLL-GAS7 resulting from t(11;17)(q23;p13) has been reported in a treat-related acute myeloid leukemia (AML) and in a pediatric acute lymphoblastic leukemia (ALL). However the specific role of Gas7 in an area of hematology has not been determined yet. Results We found that Gas7-deficient bone marrow (BM)-derived progenitor B cells, grown in vitro in the presence of interleukin 7 (IL-7), display two distinct populations of CD43highCD25- and CD43lowCD25high representing the small pre-B cells compared to their normal counterparts which show only CD43highCD25- population. As expected, CD43lowCD25high population showed reduced IL7R expression on the cell surface and increased expression of intracellular µHC, Igα and surface Igβ compared to CD43highCD25- population. Consistent with the high expression of CD25, Gas7 deficient B cell progenitors showed significantly increased expression of κ light chains on cell surface with decreased levels of P-AktS473 as well as increased levels of P-Erk T202/Y20, p53 and p21. Moreover, Gas7 mRNA expression was specifically upregulated by >13-fold in pre pro-B (Hardy fraction A) and small pre-B (Hardy fraction D) subsets compared to other subsets of B cell progenitors, suggesting that Gas7 may be involved in the regulation of early B-cell development. To elucidate the function of Gas7 in Ph+ B cell lineage leukemia, we transformed bone marrow B cell progenitors from Gas7-deficient mice with BCR-ABL1. Gas7 deficient Ph+ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis through the inhibition of Stat5Y694 phosphorylation as well as increased levels of p21. We found that Imatinib-mediated suppression of Stat5Y694 phosphorylation dramatically upregulates the expression of Gas7. In agreement with effects of Stat5 on the sensitivity of Ph+ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph+ ALL cells showed high susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 leads to loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Conclusions Here we show that Gas7 may play critical roles in early B-cell development and BCR-ABL1-driven leukemia cell survival. Pathways affected by Gas7 include Stat5, AKT and Erk signaling which is known as a downstream of BCR-ABL1 and as major regulators of B-cell development. Disclosures: No relevant conflicts of interest to declare.

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Nonpermissive bone marrow environment impairs early B-cell development in common variable immunodeficiency

Blood ◽

10.1182/blood.2019003855 ◽

2020 ◽

Vol 135 (17) ◽

pp. 1452-1457 ◽

Cited By ~ 1

Author(s):

Arianna Troilo ◽

Claudia Wehr ◽

Iga Janowska ◽

Nils Venhoff ◽

Jens Thiel ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Immature B Cells ◽

Cell Repopulation

Abstract Common variable immunodeficiency (CVID) is a disease characterized by increased susceptibility to infections, hypogammaglobulinemia, and immune dysregulation. Although CVID is thought to be a disorder of the peripheral B-cell compartment, in 25% of patients, early B-cell development in the bone marrow is impaired. Because poor B-cell reconstitution after hematopoietic stem cell transplantation has been observed, we hypothesized that in some patients the bone marrow environment is not permissive to B-cell development. Studying the differentiation dynamics of bone marrow-derived CD34+ cells into immature B cells in vitro allowed us to distinguish patients with B-cell intrinsic defects and patients with a nonpermissive bone marrow environment. In the former, immature B cells did not develop and in the latter CD34+ cells differentiated into immature cells in vitro, but less efficiently in vivo. In a further group of patients, the uncommitted precursors were unable to support the constant development of B cells in vitro, indicating a possible low frequency or exhaustion of the precursor population. Hematopoietic stem cell transplantation would result in normal B-cell repopulation in case of intrinsic B-cell defect, but in defective B-cell repopulation in a nonpermissive environment. Our study points to the importance of the bone marrow niche in the pathogenesis of CVID.

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The Dual Bromodomain Protein Brd2 Controls Primary B Cell Mitogenesis and Cell Cycle in Mice Reconstituted with Lentivirus-Transduced HSCs

Blood ◽

10.1182/blood.v112.11.2465.2465 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2465-2465

Author(s):

Wanda P. Blanton ◽

Fangnian Wang ◽

Hongsheng Liu ◽

Paul Romesser ◽

Douglas Faller ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Brdu Incorporation ◽

Cell Compartment ◽

Hematopoietic Stem

Abstract Transcriptional control of cellular proliferation and differentiation is critically important in hematopoiesis; specifically, the role of chromatin-dependent regulatory processes in this context is poorly understood. The human BRD2 proto-oncogene encodes a double bromodomain protein that binds to acetylated histone H4 in chromatin and is located within the MHC class II locus, suggesting Brd2 plays a role in immunity. However, BRD2 shares no sequence similarity with other MHC genes, nor is Brd2 involved in antigen processing, but rather it plays a role in mitogenic signal transduction. We have previously found that whole-body knockout of Brd2 is lethal to mice. However, when Brd2 was expressed constitutively in the B cells of transgenic mice, Brd2 binds E2F proteins, histone acetylases and Swi/Snf complexes, and co-activates cyclin A leading to B cell lymphoma and leukemia. Importantly, elevated levels of Brd2 have been reported in primary malignant B cells from human and mouse. We therefore hypothesize that Brd2 multiprotein complexes, working through chromatin modification, are crucial in the control of the cell cycle and in the mitogen responsiveness and proliferation of the B cell compartment. To study the effects of Brd2 in B cell development and proliferation, we performed bone marrow transplants of hematopoietic stem cells in a chimeric mouse model. Hematopoietic stem cells were sorted from CD45.1 donor mice with the characteristic ‘side population’ profile by flow cytometry and transduced with lentivirus containing vectors for Brd2 overexpression, shRNA knockdown, or control vectors. Recipient CD45.2 mice were lethally irradiated and a functional immune system was successfully reconstituted with donor cells and CD45.2 competitor BM cells. Mice were immunophenotyped and functional B cell mitogenic capacity was examined by BrdU incorporation into LPS-stimulated B cells. We found that in the spleen, Brd2 expression dramatically expands the CD45.1 (but not CD45.2) B cell compartment at the expense of T cells and renders B cells mitogenically hypersensitive. Compared with control, there was an increase in BrdU incorporation at 24 and 48 hours (29.8% v. 43.5% at T=24 h; 56.9% v. 66.7% at T=48 h). Preliminary results also suggest that B cell development was skewed in the bone marrow and periphery towards B1a phenotype. Moreover, downregulation of Brd2 via shRNA blocked cyclin A transcription and completely arrested B cell development and proliferation. Taken together, these data suggest that Brd2, through epigenetic regulation of the cell cycle, plays an important role in B-lymphopoiesis, proliferation, and stimulation.

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Precursor B Cell Acute Lymphoblastic Leukemia Induces Pro-Leukemic Changes in the Bone Marrow Microenvironment That Contribute to Therapeutic Resistance

Blood ◽

10.1182/blood.v120.21.1466.1466 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1466-1466

Author(s):

Christopher D Chien ◽

Elizabeth D Hicks ◽

Paul P Su ◽

Haiying Qin ◽

Terry J Fry

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Therapeutic Resistance

Abstract Abstract 1466 Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although cure rates for this disease are approximately 90%, ALL remains one of the leading causes cancer-related deaths in children. Thus, new treatments are needed for those patients that do not respond to or recur following standard chemotherapy. Understanding the mechanisms underlying resistance of pediatric ALL to therapy offers one approach to improving outcomes. Recent studies have demonstrated the importance of communication between cancer cells and their microenvironment and how this contributes to the progression and therapeutic resistance but this has not been well studied in the context of ALL. Since the bone marrow is presumed to be the site of initiation of B precursor ALL we set out in our study to determine how ALL cells utilize the bone marrow milieu in a syngeneic transplantable model of preB cell ALL in immunocompetent mice. In this model, intravenously injected preB ALL develops first in the bone marrow, followed by infiltration into the spleen, lymph node, and liver. Using flow cytometry to detect the CD45.2 isoform following injection into B6CD45.1+ congenic recipients, leukemic cells can be identified in the bone marrow as early as 5 days after IV injection with a sensitivity of 0.01%-0.1%. The pre-B ALL line is B220+/CD19+/CD43+/BP1+/IL-7Ralpha (CD127)+/CD25-/Surface IgM-/cytoplasmic IgM+ consistent with a pre-pro B cell phenotype. We find that increasing amounts of leukemic infiltration in the bone marrow leads to an accumulation of non-malignant developing B cells at stages immediately prior to the pre-pro B cell (CD43+BP1-CD25-) and a reduction in non-malignant developing pre B cells at the developmental stage just after to the pre-pro B cell stage (CD43+BP1+CD25+). These data potentially suggest occupancy of normal B cell developmental niches by leukemia resulting in block in normal B cell development. Further supporting this hypothesis, we find significant reduction in early progression of ALL in aged (10–12 month old) mice known to have a deficiency in B cell developmental niches. We next explored whether specific factors that support normal B cell development can contribute to progression of precursor B cell leukemia. The normal B cell niche has only recently been characterized and the specific contribution of this niche to early ALL progression has not been extensively studied. Using a candidate approach, we examined the role of specific cytokines such as Interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP) in early ALL progression. Our preB ALL line expresses high levels of IL-7Ralpha and low but detectable levels of TLSPR. In the presence of IL-7 (0.1 ng/ml) and TSLP (50 ng/ml) phosphSTAT5 is detectable indicating that these receptors are functional but that supraphysiologic levels of TSLP are required. Consistent with the importance of IL-7 in leukemia progression, preliminary data demonstrates reduced lethality of pr-B cell ALL in IL-7 deficient mice. Overexpression of TSLP receptor (TSLPR) has been associated with high rates of relapse and poor overall survival in precursor B cell ALL. We are currently generating a TSLPR overepressing preBALL line to determine the effect on early ALL progression and are using GFP-expressing preB ALL cells to identify the initial location of preB ALL occupancy in the bone marrow. In conclusion, or model of early ALL progression provides insight into the role of the bone marrow microenvironment in early ALL progression and provides an opportunity to examine how these microenvironmental factors contribute to therapeutic resistance. Given recent advances in immunotherapy for hematologic malignancies, the ability to study this in an immunocompetent host will be critical. Disclosures: No relevant conflicts of interest to declare.

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Dlk-1 Regulates B Cell Development Through Altered Bone Marrow Stromal Microenvironment, Not by Affecting Hematopoitic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v120.21.3465.3465 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3465-3465

Author(s):

Edyta Pawelczyk ◽

Heba A Degheidy ◽

Allison L Branchaw ◽

Kenn Holmbeck ◽

Steven R Bauer

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Methyl Cellulose ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Wild Type ◽

Mineral Density ◽

Hematopoietic Stem

Abstract Abstract 3465 Introduction: DLK-1(delta-like 1) is a member of the EGF-like homeotic protein family whose expression is known to influence cell fate decisions through cell-cell interactions. It is also known to influence the differentiation of bone marrow stromal cells (BMSC) and hematopoietic stem cells (HSC) in bone marrow. Recently, we reported the essential role of DLK-1 in B cell development, which showed that the absence of DLK-1 led to accumulation of the earliest B cell progenitors (pre-pro B cells or Fraction A (Fr A)) in bone marrow, an altered pattern of B cell development in the spleen, and an altered humoral immune response. The objective of this study was to determine whether alterations in the HSC compartment or the BMSC microenvironment contributed to Fr A accumulation in mdlk1−/− mice. Methods: The mdlk1−/− and wild type bone marrow osteoblast and HSC compartments were analyzed by multicolor flow cytometry and in vitro methyl-cellulose colony forming cell assays. Bone marrow harvested from mdlk1−/− and wild type mice was assessed for BMSCs colony forming efficiency (CFU-F) and cultured. Supernatants from cultured BMSCs were analyzed by protein arrays. Since osteoblasts are an important component of the bone marrow microenvironment, OPN+CD45-TER119-ALP+ osteoblasts were identified in the bone marrow and quantified by flow cytometry. Finally, the femurs of mdlk1−/− and wild type mice were analyzed by micro-computed tomography (uCT) scanning. Results: Using flow cytometry, we observed no statistically significant changes in the HSC and progenitor populations in the absence of DLK-1 in mice at 4 and 16 weeks of age. The results of methyl-cellulose assay confirmed the findings of flow cytometry experiments and showed no statistically significant differences in the number of CFU-G, CFU-GM, and CFU-M of 4 and 16 week old mdlk1−/− mice as compared to wild-type control mice. However, significant alterations in the microenvironment of the mdlkl −/− were observed. CFU-F efficiency of mdlk1−/− bone marrow BMSC isolated from 4 week old mice was significantly decreased when compared to age-matched controls. Furthermore, the uCT scans showed the mineral density of the femoral bone significantly decreased in 4 week old mdlk1−/− mice and the number of osteoblast cells analyzed by flow cytometry was decreased by 10%. The analysis of BMSC supernatants revealed a striking down regulation of factors associated with osteoblast function and differentiation such as osteoactivin, PF-4, Follstatin-like 1, Frizzled-6, IGF-1, M-CSF, DKK-1 and others. Conclusions: Our results indicate that accumulation of the earliest B cell progenitors with DLK-1 ablation is the result of multiple defects in the bone marrow microenvironment including decreased CFU-F, decreased number of osteoblasts, decreased bone mineral density or alterations in factors important for osteoblast function but not from increase in numbers of hematopoietic stem or progenitors cells. Our laboratory is investigating this further. Disclosures: Pawelczyk: Baxter Inc.: currently employed by Baxter Inc. Other.

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RNAi Screening Identifies A Novel Role for A-Kinase Anchoring Protein 12 (AKAP12) in B Cell Development and Function

Blood ◽

10.1182/blood.v120.21.855.855 ◽

2012 ◽

Vol 120 (21) ◽

pp. 855-855 ◽

Cited By ~ 1

Author(s):

Mutlu Kartal-Kaess ◽

Luisa Cimmino ◽

Simona Infantino ◽

Mehmet Yabas ◽

Jian-Guo Zhang ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Leukemia Cell ◽

Cell Development ◽

B Cell Development ◽

Leukemia Cell Line ◽

B Lymphopoiesis ◽

Catalytic Subunits ◽

B Cell Leukemia

Abstract Abstract 855 The cAMP signaling pathway has emerged as a key regulator of hematopoietic cell proliferation, differentiation, and apoptosis. Signal specificity is achieved through local activation of signaling enzymes that are anchored to subcellular organelles and membranes. In particular, A-kinase anchoring proteins (AKAPs) coordinate and control cAMP responsive events. AKAPs were originally classified based on their ability to bind cAMP-dependent protein kinase (protein kinase A; PKA). The activity of PKA is regulated by its two regulatory subunits, which from a dimer that binds to the two catalytic subunits. Binding of cAMP to the regulatory dimer dissociates the catalytic subunits and activates PKA. Anchoring of PKA by AKAPs constrains PKA activity to a relevant subset of potential substrates. Thus, AKAPs contribute to the precision of intracellular signaling events by directing anchored enzyme pools to a subset of their physiological substrates at specific subcellular localizations. Using an in vitro short hairpin RNA (shRNA) screen against potentially druggable targets, we have uncovered a requirement for AKAP12 in the proliferation of a cultured pre-B cell leukemia cell line. In the hematopoietic system of mice and humans, expression of AKAP12 is tightly restricted to the pro/pre/immature stages of B lymphopoiesis, suggesting a potential role in pre-B cell receptor (pre-BCR) or BCR signaling. We find that retroviral knockdown or germline knockout of AKAP12 in mice leads to an increase in pre B and immature B cells in the bone marrow. In contrast, B cell numbers in the spleen are significantly reduced, as are recirculating B cells in the bone marrow. Transplantation of AKAP12 null hematopoietic stem and progenitor cells from fetal liver into wildtype recipients demonstrates an autonomous defect in the development of AKAP12−/− B cells. Competitive bone marrow transplantations confirm that this defect is cell autonomous and not due to a defective bone marrow environment or secretion of a B cell inhibitory factor. To identify AKAP12 interaction partners, we overexpressed FLAG-epitope tagged AKAP12 in a pre-B cell leukemia cell line. Affinity purification of AKAP12 showed a repeated co-immunoprecipitation of poorly characterized RIO kinase 1 (RIOK1). Our current efforts are focused on investigating the interaction between RIOK1 and AKAP12 and their role in the control of B cell development and cell cycle progression. Further, we are focusing on a likely role for AKAP12 in the scaffolding of PKA, PKC and phosphodiesterases by analyzing the activation of signaling cascades in cultured primary wildtype and AKAP12−/− pre B cells. Additionally, we are investigating the role of the BCR in vivo by testing if enforced expression of BCR components rescue B cell development in a AKAP12−/− BCR transgenic mouse model (SWHEL mouse). In summary, we have confirmed a novel role for AKAP12 in B lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

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