Murine Stem Cell Factor Research Articles

Early exposure of murine embryonic stem cells to hematopoietic cytokines differentially directs definitive erythropoiesis and cardiomyogenesis in alginate hydrogel three-dimensional cultures.

HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and β-major globin), and proteins (Gata-1 and β-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

GW24-e1023 Advanced Glycation End Product Modified Low Density Lipoprotein Activates Mast Cells via Toll-Like 4 Receptor Pathway

ObjectivesTo investigate the effects of AGE-LDL, in murine bone marrow-derived mast cells (mBMMCs), on mast cell degranulation and their possible signal pathways.MethodsCarotid plaques were excised during Carotid Endarterectomy of diabetic...

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Ectopic Expression of the Leukemogenic Protein E2A-PBX1 in Early Hematopoietic Progenitors Blocks B-Lymphoid Commitment

Abstract 1393The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis.We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development.In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing. Disclosures:No relevant conflicts of interest to declare.

Hematopietic Stem Cells with Defects in the NF-κB Alternative Pathway Show a Deficient Repopulation Capacity

Abstract 1269Hematopoietic stem cells (HSCs) maintain the production of all blood cells through the lifespan of an organism, and regenerate the whole hematopoietic system after stressful episodes such as high dose chemotherapy or upon transplantation. The functions of HSCs in these 2 situations, steady-state and under stress, are controlled by a variety of molecules, which may provide different contribution to each process. We investigated whether the NF-kB alternative pathway might have a role in HSCs functions, using mice deficient for two components of this pathway: NF-kB-inducing kinase (NIK) or p52. The activation of NIK is generally known as the alternative (or non-canonical) NF-kB pathway, and drives the post-translational processing of p100 to mature p52, which results in the translocation to the nucleus of p52-containing complexes such as p52/RelB. Apart from the already reported defects in B-cell maturation, both NIK- and p52-deficient mice did not present major disturbances in blood cells numbers. The absolute numbers of marrow cells were not different among the knocked-out and the wild-type mice. We first studied the compartment of marrow cells known to be enriched for HSCs, either lineage-depleted Sca1-positive ckit-positive cells (LSK), or CD150 positive CD48 negative cells. The proportions of marrow cells with the immunophenotype of HSCs in either NIK-deficient or p52-deficient mice were similar to those in control mice. The amount of clonogeneic progenitor cells in the marrow was assessed in standard CFU-GM cultures, and gave no differences in output in any of the mice studied. We set up in vitro liquid cultures with murine stem cell factor and human interleukin-11, and determined the cellular production weekly. Cultures started with NIK-deficient marrow cells produced significantly less numbers of cells and CFU-GM, compared with those started with wild type marrow. This deficit in hematopoietic capacity was further confirmed in a more stringent assay of HSC function, the in vivo competitive repopulation assay. Equal numbers of lineage-depleted (Lin-) CD45.2 marrow cells of either NIK-deficient, p52-deficient or wild-type mice, were mixed with Lin- CD45.1 marrow cells of syngeneic mice, and transplanted into lethally irradiated CD45.1 recipients. Four months after transplant, the chimeric status and the hematopoietic lineage repopulation of CD45.2 cells was assessed in peripheral blood (PB). NIK- or p52-deficient HSCs repopulated the B-, T- and myeloid-lineages but at significantly lower levels when compared to wild type HSCs. Total donor CD45.2 cells and total CD45.2 LSK cells were also significantly lower in the marrows of mice transplanted with NIK- or p52-deficient HSCs versus those of controls. We used the marrows of the repopulated mice for secondary transplants, and confirmed the defect in the repopulating capacity of NIK- and p52-deficient HSCs. Our results suggest that the NF-kB alternative pathway plays a role in the function of HSCs, and this role may be important under stress conditions. Disclosures:No relevant conflicts of interest to declare.

Structural Specificity of the Biological Activity of Human and Murine Stem Cell Factor in Stress Erythropoiesis,

Abstract 3395Stem cell factor (SCF) is a cyokine produced by bone marrow stromal cells that upon engagement of its cognate receptor cKit and in combination with other hematopoietic growth factors initiates downstream phosphorylation events that generate hematopoietic progenitors and precursors of all types. SCF and cKit are encoded by highly conserved genes. The human (hSCF) and murine (mSCF) forms are highly homologous and are 82% identical at the amino acid levels. Both have been reported to sustain differentiation of human mast cells from cord blood (CB) but mast cells generated with mSCF remain immature (Mitzu et al, PNAS 90:735–739). In addition, anecdotic reports indicate that hSCF is 100-fold less active than mSCF on mouse cells while mSCF and hSCF are equally active on human cells.To clarify if structural differences between the two growth factors have biological significance, we compared cord blood (CB) mononuclear cells and CD34pos cells as the stem cell source for in vitro production of colony forming cells, CFU-GM and BFU-E, using established techniques, and for ex-vivo expansion of megakaryocytes (MK) (SCF + IL-3 + TPO) and erythroid cells (EB) under standard conditions (IL-3 and EPO) and conditions of stress (IL-3 + EPO + dexamethasone).In semisolid assay, hSCF and mSCF (20 ng/mL) sustained growth of similar numbers of CFU-GM (51±5 vs 44±5/2×105 MNC and 15.4±0.9 vs 13.3±2.0/500 CD34pos cells)- and BFU-E (8.5±0.5 vs 8.7±1.2/500 CD34pos cells)-derived colonies. By day 12 of MK expansion assay, hSCF and mSCF induced similar 2-1.6-fold increases over those observed with IL-3 and TPO alone. In addition, hSCF and mSCF-stimulated cultures contained similar number of MK as identified by morphology and CD41a/CD42b staining (∼40–60% CD41aposCD42bpos cells) (Figure 1). By day 13 of EB expansion culture without steroids, hSCF and mSCF generated similar numbers of EB (FI= ∼6–8 with both MNC and CD34pos cells), ∼60% of which had a mature CD36posCD235ahigh phenotype (Figure 1), but, by day 15 of EB expansion with steroids, hSCF induced significantly greater numbers of EB that mSCF (FI=154±45 vs 54±15 from MNC, and 13,200±740 vs 2,000±550 for CD34pos cells, respectively, p<0.01 in both cases). In addition, day 8 EB generated with mSCF were more mature than those generated with hSCF (86% vs 31% of CD36posCD235ahigh EB, Figure 1). Similar results were obtained in EB expansion cultures of AB MNC. [Display omitted] To clarify the difference in biological activity of hSCF and mSCF in cultures containing steroids, expression of glucocorticoid receptor α (GRα) and hSCF and mSCF signaling in adult blood (AB) and CB EB generated with the two growth factors were compared. hSCF-generated EB expressed 2-times more GRα than those generated with mSCF (Figure 2) and the GRα content of hSCF-generated EB was reduced by 50% after 24 h exposure with mSCF. In addition, both transient (within 15 m) and sustained (after 2 h) ERK phosphorylation were observed only in EB stimulated with hSCF while both hSCF and mSCF induced low levels of STAT-5 phosphorylation but neither factor induced AKT phosphorylation. [Display omitted] In summary, hSCF and mSCF have overlapping biological activities in culture conditions which mimic steady-state hematopoiesis (semisolid cultures, MK and EB expansion without dexamethasone) but hSCF was uniquely capable to sustain GRα expression and was more effective than mSCF in cultures which mimic stress erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.

BCR-ABL but Not JAK2 V617F Inhibits Erythropoiesis through the Ras Signal by Inducing p21CIP1/WAF1

BCR-ABL is a causative tyrosine kinase (TK) of chronic myelogenous leukemia (CML). In CML patients, although myeloid cells are remarkably proliferating, erythroid cells are rather decreased and anemia is commonly observed. This phenotype is quite different from that observed in polycythemia vera (PV) caused by JAK2 V617F, whereas both oncogenic TKs activate common downstream molecules at the level of hematopoietic stem cells (HSCs). To clarify this mechanism, we investigated the effects of BCR-ABL and JAK2 V617F on erythropoiesis. Enforced expression of BCR-ABL but not of JAK2 V617F in murine LSK (Lineage(-)Sca-1(hi)CD117(hi)) cells inhibited the development of erythroid cells. Among several signaling molecules downstream of BCR-ABL, an active mutant of N-Ras (N-RasE12) but not of STAT5 or phosphatidylinositol 3-kinase (PI3-K) inhibited erythropoiesis, while N-RasE12 enhanced the development of myeloid cells. BCR-ABL activated Ras signal more intensely than JAK2 V617F, and inhibition of Ras by manumycin A, a farnesyltransferase inhibitor, ameliorated erythroid colony formation of CML cells. As for the mechanisms of Ras-induced suppression of erythropoiesis, we found that GATA-1, an erythroid-specific transcription factor, blocked Ras-mediated mitogenic signaling at the level of MEK through the direct interaction. Furthermore, enforced expression of N-RasE12 in LSK cells derived from p53-, p16(INK4a)/p19(ARF)-, and p21(CIP1/WAF1)-null/wild-type mice revealed that suppressed erythroid cell growth by N-RasE12 was restored only by p21(CIP1/WAF1) deficiency, indicating that a cyclin-dependent kinase (CDK) inhibitor, p21(CIP1/WAF1), plays crucial roles in Ras-induced suppression of erythropoiesis. These data would, at least partly, explain why respective oncogenic TKs cause different disease phenotypes.

Combined Effects of Interleukin-7 and Stem Cell Factor Administration on Lymphopoiesis after Murine Bone Marrow Transplantation

The decreased ability of the thymus to generate T cells after bone marrow transplantation (BMT) is a clinically significant problem. Interleukin (IL)-7 and stem cell factor (SCF) induce proliferation, differentiation, and survival of thymocytes. Although previous studies have shown that administration of recombinant human IL-7 (rhIL-7) after murine and human BMT improves thymopoiesis and immune function, whether administration of SCF exerts similar effects is unclear. To evaluate independent or combinatorial effects of IL-7 and SCF in post-BMT thymopoiesis, bone marrow (BM)-derived mesenchymal stem cells transduced ex vivo with the rhIL-7 or murine SCF (mSCF) genes were cotransplanted with T cell-depleted BM cells into lethally irradiated mice. Although rhIL-7 and mSCF each improved immune reconstitution, the combination treatment had a significantly greater effect than either cytokine alone. Moreover, the combination treatment significantly increased donor-derived common lymphoid progenitors (CLPs) in BM, suggesting that transplanted CLPs expand more rapidly in response to IL-7 and SCF and may promote immune reconstitution. Our findings demonstrate that IL-7 and SCF might be therapeutically useful for enhancing de novo T cell development. Furthermore, combination therapy may allow the administration of lower doses of IL-7, thereby decreasing the likelihood of IL-7-mediated expansion of mature T cells.

Pimecrolimus Induces Apoptosis of Mast Cells in a Murine Model of Cutaneous Mastocytosis

Background: Cutaneous mastocytosis (CM) is a common type of mastocytosis. Current treatment of CM is generally symptomatic. Pimecrolimus has been demonstrated as an effective anti-inflammatory drug for the treatment of inflammatory skin diseases, but whether it treats CM remains unknown. Methods: The murine model of CM was induced by subcutaneous injection of 100 µg/kg recombinant murine stem cell factor (rmSCF) for a total of 17 days in Balb/c mice. Beginning on the 8th day, treatment with pimecrolimus 1% cream or vehicle was performed topically and daily for 10 days. The clinical signs of CM were scored, and pathological analysis was performed with toluidine blue staining and hematoxylin and eosin staining. The in situ apoptotic mast cells (MCs) were studied by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The cutaneous histamine level was measured by ELISA. Results: In the rmSCF-treated mice, the clinical signs of CM, including erythema, wheal after rubbing lesion skins, and increased thickness of skin, were obvious compared to control mice, and were reduced after pimecrolimus treatment. The numbers of cutaneous MCs and neutrophils were significantly greater in mice with CM than in control mice, and pimecrolimus treatment decreased the numbers of MCs but not neutrophils. Extensive apoptosis of cutaneous MCs was observed in pimecrolimus-treated mice. The cutaneous histamine level was elevated in the mice with CM compared with healthy controls, and was lowered after treatment with pimecrolimus. Conclusions: Pimecrolimus effectively treats CM by reducing the density of cutaneous MCs and the subsequent histamine production through inducing MCs apoptosis.

PU.1 Directly Regulates cdk6 Gene Expression, Linking the Cell Proliferation and Differentiation Programs in Erythroid Cells

Cell proliferation and differentiation are highly coordinated processes during normal development. Most leukemia cells are blocked from undergoing terminal differentiation and also exhibit uncontrolled proliferation. Dysregulated expression of transcription factor PU.1 is strongly associated with Friend virus-induced erythroleukemia. PU.1 inhibits erythroid differentiation by binding to and inhibiting GATA-1. PU.1 also may be involved in controlling proliferation of erythroid cells. We reported previously that the G(1) phase-specific cyclin-dependent kinase 6 (CDK6) also blocks erythroid differentiation. We now report that PU.1 directly stimulates transcription of the cdk6 gene in both normal erythroid progenitors and erythroleukemia cells, as well as in macrophages. We propose that PU.1 coordinates proliferation and differentiation in immature erythroid cells by inhibiting the GATA-1-mediated gene expression program and also by regulating expression of genes that control progression through the G(1) phase of the cell cycle, the period during which the decision to differentiate is made.

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.

CCR1 expression and signal transduction by murine BMMC results in secretion of TNF-alpha, TGFbeta-1 and IL-6.

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.

CCR1 expression and signal transduction by murine BMMC results in secretion of TNF-α, TGFβ-1 and IL-6 (98.10)

Abstract Chemokine receptors are important co-stimulatory molecules found on many blood cells. Chemokine receptor 1 (CCR1) is one of the important chemokine receptor identified in different immune cells known for its function in various diseases. The expression and functions of chemokine receptors on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express mRNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine interleukin-3 expressed CCR1 after 5-6 weeks. Co-stimulation of CCR1 by its ligand macrophage inflammatory protein 1α (MIP-1α) and the IgE receptor FceRI enhanced BMMC degranulation compared to FceRI stimulation alone (85% vs. 54%, p &amp;lt; 0.0001) and Ca2+ influx. We report for the first time that mBMMCCCR1+ cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1- FceRI co-stimulation. These factors include transforming growth factor β-1, tumor necrosis factor-α, ciliary neurotrophic factor and the cytokine interleukin-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.

Bcl2 enhances induced hematopoietic differentiation of murine embryonic stem cells

Bcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to "knock-down" endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G 0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 "silencing" or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications.

The Early Growth Response Factor-1 Is Involved in Stem Cell Factor (SCF)-induced Interleukin 13 Production by Mast Cells, but Is Dispensable for SCF-dependent Mast Cell Growth

The stem cell factor (SCF) plays a central role in the regulation of mast cell function and growth. However, roles of transcription factors involved in these processes remain incompletely defined. The early growth response factor-1 (Egr-1) is a member of the zinc finger transcription factor family. A role for Egr-1 in SCF-induced mast cell activation and growth was investigated in mouse bone marrow-derived mast cells (BMMC). The stimulation of BMMC with SCF induced a strong expression of Egr-1 mRNA. SCF-induced Egr-1 nuclear translocation and DNA binding were demonstrated by electrophoretic mobility shift assay (EMSA) and immunofluorescence assay. SCF-induced IL-13 expression was significantly reduced at both mRNA and protein levels in Egr-1-deficient BMMC. In addition, the synergy between IgE and SCF on IL-13 and IL-4 production was reduced in Egr-1-deficient mast cells. Interestingly, Egr-1 deficiency had little effect on SCF-induced mast cell growth. SCF-induced Egr activation likely requires tyrosine phosphorylation because a tyrosine kinase inhibitor PP2 blocked SCF-induced nuclear protein binding to Egr probe as determined by EMSA. Thus, Egr-1 is required for SCF-induced IL-13 expression, but not mast cell growth. Egr-1 represents a novel mechanism for SCF-induced mast cell activation.

Dynamic localization and persistent stimulation of factor-dependent cells by a stem cell factor / cellulose binding domain fusion protein

The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.