Dual Role for CIB1 in Thrombopoiesis: CIB1 Suppresses Megakaryocyte Quantities but Supports Adhesion, Migration, and Proplatelet Formation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2384-2384
Author(s):  
John C Kostyak ◽  
Ulhas P Naik
Keyword(s):  
Conflicts Of Interest ◽  
Dual Role ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Proplatelet Formation ◽  
And Migration ◽  
Platelet Depletion ◽  
Defective Clearance

Abstract Abstract 2384 Megakaryocytes (MKs) are large polyploid cells that produce platelets through a process known as thrombopoiesis. Thrombopoietin (Tpo) is the major cytokine that regulates a variety of steps in this process, including hematopoietic stem cell (HSC) differentiation to MKs, proplatelet formation, and platelet release into the circulation. However, the molecular mechanism of thrombopoiesis is poorly understood. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in thrombopoiesis, we utilized a Cib1−/− mouse model. We observed that Cib1−/− mice have a slightly elevated number of platelets and bone marrow (BM)-derived MKs than wild-type (WT) controls (p<0.05). Rate of platelet clearance was comparable in Cib1−/− and WT mice, suggesting that the defective clearance is not the cause of the observed elevated platelet number. In order to determine if the HSC differentiation is dysregulated by the ablation of Cib1, we analyzed MK-colony forming unit production, which revealed an increase in the colony forming cells with Cib1 deletion compared to WT (p<0.05). Additionally, BM from Cib1−/− mice, cultured with Tpo for 24 hours, produced more highly polyploid MKs than WT BM (p<0.05). These results suggest that Cib1 may negatively regulate initial steps of megakaryopoiesis. Subsequent analysis of Tpo signaling revealed that activation of FAK, a known suppresser of Tpo signaling, is attenuated, as indicated by reduced FAKY925 phosphorylation in Cib1−/− BM-derived MKs treated with Tpo. Consequently, Akt and ERK1/2 activation downstream of Tpo was enhanced. These results suggested that Cib1 inhibits Tpo signaling by augmenting FAK activation. Interestingly, platelet recovery in Cib1−/− mice following platelet depletion by experimental immunothrombocytopenia was attenuated compared to WT (p<0.05). This could be due to impaired adhesion and migration of MKs on the extracellular matrix. Consistent with this notion, adhesion to fibrinogen and fibronectin and migration towards an SDF-1α gradient were significantly reduced in Cib1−/− MKs compared to WT (p<0.05). Additionally, Cib1−/− MKs formed fewer proplatelets compared to WT (p<0.05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in thrombopoiesis, initially by negatively regulating Tpo signaling, and later by supporting MK migration and proplatelet production. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

Blood ◽  
2012 ◽  
Vol 119 (3) ◽  
pp. 838-846 ◽  
Author(s):  
John C. Kostyak ◽  
Meghna U. Naik ◽  
Ulhas P. Naik
Keyword(s):  
Mouse Model ◽  
Binding Protein ◽  
Dual Role ◽  
Wild Type ◽  
Platelet Recovery ◽  
Polyploid Cells ◽  
And Migration ◽  
Platelet Depletion

Abstract Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1−/− mouse model. Cib1−/− mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1−/− mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAKY925 phosphorylation was reduced in Cib1−/− megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1−/− mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1−/− megakaryocytes compared with WT (P < .05). In addition, Cib1−/− megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Platelets Protect From Lipopolysaccharide-Induced Lethal Endotoxemia by Inhibiting Macrophage-Dependent Inflammation Via the Cyclooxygenase 1 (COX1) Signaling Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 93-93
Author(s):  
Binggang Xiang ◽  
Guoying Zhang ◽  
Xiang-An Li ◽  
Andrew J. Morris ◽  
Alan Daugherty ◽  
...  
Keyword(s):  
Septic Shock ◽  
Platelet Transfusion ◽  
Conflicts Of Interest ◽  
Plasma Concentrations ◽  
Platelet Inhibition ◽  
Severe Thrombocytopenia ◽  
Wild Type ◽  
Activated Platelets ◽  
Platelet Depletion

Abstract Abstract 93 Sepsis is a tremendous burden for health-care systems. Patients with sepsis often have low platelet counts, and septic patients with severe thrombocytopenia have a poor prognosis and higher mortality. However, the role of platelets in the pathogenesis of sepsis has not been well elucidated. We investigated the role of platelets in septic shock using a mouse model of lipopolysaccharide (LPS)-induced endotoxemia. Depletion of platelets by intraperitoneal injection of a rat anti-mouse GPIb monoclonal antibody increased mortality and aggravated organ failure in endotoxemic mice as evident by increases in plasma aminotransferase (ALT), aspartate aminotransferase (AST), Lactate dehydrogenase (LDH), and Creatine kinase (CK) concentrations, while transfusion of platelets reduced mortality. Increases in mortality rate in thrombocytopenic mice by LPS challenge was not due to inflammatory hemorrhage, because there was no significantly hemorrhage observed in brains and lungs from mice pre-treated with either control IgG or the anti-GPIba antibody and blood RBC and Hb concentrations between IgG pre-treated mice and the anti-GPIba antibody pre-treated mice were similar. TNF-a, which is produced mainly by macrophages in vivo, plays critical roles in the development of disseminated intravascular coagulation, acute respiratory distress syndrome and shock in sepsis. Our data indicate that plasma concentrations of proinflammatory cytokines, TNF-a and IL-6, were markedly increased by platelet depletion and decreased by platelet transfusion in the mice challenged with LPS. Effects of platelet depletion on TNF-a production were eliminated in the mice that macrophages were pre-depleted. Furthermore, LPS- or thrombin-activated platelets or releasates from activated platelets inhibited TNF-a and IL-6 production in macrophages in vitro. Inhibition of TNF-a and IL-6 production in macrophages by activated platelets was prevented by pre-incubation of platelets with a COX1 inhibitor aspirin. Moreover, platelets from wild type mice but not COX1 deficient mice inhibited LPS-induced TNF-a and IL-6 production in macrophages. Transfusion of COX1 deficient platelets failed to protect against endotoxemia. Washed platelets from wild-type mice or platelet releasates from thrombin-activated wild-type mice inhibited LPS-induced TNF-a and IL-6 production in macrophages lacking TXA2 receptor, TP, suggesting that a metabolite other than TXA2 is responsible for platelet inhibition of macrophage function. We found that stimulation of platelets with thrombin or LPS induced PGE2 production and pre-incubation of macrophages with an antagonist of PGE2 receptor EP4 reversed platelet inhibition on TNF-a and IL-6 production in macrophages. Our results indicate that platelets protect against septic shock by inhibiting macrophage-dependent inflammatory response via the COX1/PGE2/EP4 dependent pathway. Thus, these findings demonstrate a previously unappreciated role for platelets in septic shock and suggest that platelet transfusion may be effective in treating septic patients. Disclosures: No relevant conflicts of interest to declare.

Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4344-4344
Author(s):  
Amanda Scholl ◽  
Kentson Lam ◽  
Alex Muselman ◽  
Tingdong Tang ◽  
Shinobu Matsuura ◽  
...  
Keyword(s):  
Family Member ◽  
Conflicts Of Interest ◽  
Protein Family ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Wild Type ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor

Abstract RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Mayuri Tanaka-Yano ◽  
Dahai Wang ◽  
Eleanor Meader ◽  
Melissa A. Kinney ◽  
Vivian Morris ◽  
...  
Keyword(s):  
Young Adult ◽  
Developmental Stage ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Polycomb Group ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Developmental Maturation

Abstract Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis. First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7. Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels. As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold). To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors. Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals C-CBL Is a Critical Negative Regulator of Megakaryopoesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1581-1581
Author(s):  
Sebastian J. Saur ◽  
Melanie Märklin ◽  
Alexandra Poljak ◽  
Manuela Ganser ◽  
David E. James ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Platelet Factor ◽  
Physiological Level ◽  
Hematopoietic Stem ◽  
Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors in order to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis modulating megakaryocyte differentiation, promoting endomitosis and proplatelet formation and as such supports the self-renewal and survival of hematopoietic stem cells. To allow proper proliferation and differentiation of different hematopoetic lineages, TPO signal transduction must be tightly regulated. Several mechanisms negatively modulating hematopoiesis and differentiation of the megakaryocytic lineage have previously been identified. Among those are suppressors cytokine signaling, protein phosphatases as well as a multitude of negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation via lysosomes or proteasomes. In this study, we investigated the mechanisms that regulate TPO-mediated MPL degradation in primary mouse cells. Previous studies have identified CBL as an E3 ligase responsible for the ubiquitination of MPL in cell lines. In order to determine the potential role of c-CBL in murine thrombopoiesis, we used Cre/loxP technology to specifically delete c-CBL in the megakaryocytic lineage. Mice expressing two floxed c-CBL alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter. This yielded progeny with the desired genotype of c-CBLfl/fl PF4-Cre (CBL ko) after two generations of breeding. The desired cohort exhibited a quantitative absence of c-CBL in megakaryocytes and platelets as assessed by western blotting compared with wild type C57/BL6 mice. The expression of CBL in other hematopoietic cells such as B cells, T cells, neutrophils, monocytes and dendritic cells remained unaffected in this conditional ko strain. The experimental cohort showed significantly higher numbers of megakaryocytes in the bone marrow and of platelets in the peripheral blood as compared to wild type mice (1.2 mio vs. 1.8 mio cells/µl, p<0.0001). In addition, the platelets from the mutant mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). To evaluate the role of c-CBL in mature megakaryocytes, total bone marrow was collected from 12 wk old CBL ko mice and grown in TPO-containing culture medium for 72 h. Megakaryocytes derived from the bone marrow of wild type mice served as controls. Mature megakaryocytes were eventually isolated on a BSA-density gradient. Subsequent Western Blot analysis revealed a significant reduction of MPL ubiquitination in the CBL ko mice as compared to wild type mice, thereby identifying c-CBL as a critical negative regulator of megakaryopoesis. Taken together, we have successfully ablated c-CBL specifically from the megakaryocyte lineage and could demonstrate that this has profound effects on platelet counts and platelet size. In addition, we were able to show that c-CBL ablation leads to reduced ubiquitination of MPL and a consecutively longer half life of this protein culminating in substantially increased megakaryopoiesis in the c-CBL ko cohort. In summary, these data enhance our understanding of the regulation of TPO signaling and the physiological role of CBL in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 221-221
Author(s):  
Xun Shang ◽  
Lina Li ◽  
Jose Concelas ◽  
Fukun Guo ◽  
Deidre Daria ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Rac Gtpase ◽  
Hematopoietic Stem ◽  
And Migration ◽  
Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p&lt;0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p&lt;0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cell Function and Promotes G-CSF Mediated HSC Mobilization

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4334-4334
Author(s):  
Angela Herman ◽  
Molly Romine ◽  
Darlene Monlish ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function. To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2-/- + Tlr2+/+ marrow transplanted into Tlr2+/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r-/-) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs. Disclosures No relevant conflicts of interest to declare.

Angiopoietin-Like 3 Affects Homing Ability of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2632-2632
Author(s):  
Masato Umikawa ◽  
Junke Zheng ◽  
HoangDinh Huynh ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Physiological Role ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Intrinsic Effect

Abstract Abstract 2632 Angiopoietin-like proteins (Angptls) are a seven-member family of secreted glycoproteins that share sequence homology with angiopoietins. It is known that several members of the Angptl family including Angptl3 support ex vivo expansion of hematopoietic stem cells (HSCs). However, the physiological role of Angptls in the hematopoietic system is not well known. Here we show that Angptl3 is expressed by both bone marrow stromal cells and HSCs. To study the intrinsic effect of Angptl3 in mouse HSCs, we isolated the same number of HSCs from wild-type and Angptl3-null mice and performed reconstitution analysis. Adult bone marrow Angptl3-null HSCs showed decreased repopulation compared to wild-type HSCs, suggesting that Angptl3 has cell-autonomous effect on HSC activity. By contrast, HSCs isolated from liver of the null mice had enhanced HSC repopulation activity than their wild-type counterparts. To study whether this effect is caused by difference in homing, we injected CFSE labeled wild-type HSCs and Angptl3 null HSCs into lethally irradiated mice, and checked the homing to bone marrow, spleen, and liver. While homing of these two types of cells to bone marrow or spleen was not significantly different, Angptl3 null HSCs homed better to the liver than the wild-type HSCs. Our result suggests that Angptl3 is important for the retention of HSCs in the bone marrow, and the absence of Angptl3 leads HSCs to move to extramedullary organs such as liver. Disclosures: No relevant conflicts of interest to declare.

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