scholarly journals Sox4 Is Required for the Formation and Maintenance of Multipotent Progenitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1577-1577 ◽  
Author(s):  
Hong Zhang ◽  
Min Ye ◽  
Robert S. Welner ◽  
Daniel G. Tenen
Keyword(s):  
Progenitor Cells ◽  
Absolute Number ◽  
Brdu Incorporation ◽  
Binding Motif ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Genetic Ablation ◽  
Qpcr Analysis ◽  
Multipotent Progenitors ◽  
Deficient Mice

Abstract Introduction Hematopoiesis is maintained by a hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of many diseases. Within the hematopoietic hierarchy, hematopoietic stem cells (HSCs) give rise to multipotent progenitors that have lost their self-renewal capacity but remain multipotent to differentiate into mature blood cells. However, the precise molecular mechanisms that modulate this transition are not fully understood yet. Results We recently discovered that genetic ablation of SRY sex determining region Y-box 4 gene (Sox4) in the murine hematopoietic system resulted in dramatic loss of multipotent progenitor population (CD48+CD150-Lin-kit+Sca1+, or CD48+CD150-LSK) both relatively (to the total LSK population) and in absolute number. Interestingly, the absolute number of HSCs (CD48-CD150+Lin-kit+Sca1+, or SLAM+LSK) in these conditional Sox4-deficient mice was comparable to their wild-type counterparts. Transcriptional factor Sox4 belongs to the high-mobility group (HMG) domain superfamily which also includes other Sox proteins, TCF-1 (T-cell factor 1) and LEF-1 (lymphoid enhancer factor 1). Sox4 has been implicated in leukemogenesis and may potentially contribute to stem cell properties. Nevertheless, the precise roles of Sox4 in hematopoietic stem/progenitor cells and the underlying mechanisms have not been defined yet. Further analysis of stem/progenitor compartment defined by Flt3 and CD34 expression demonstrated a major loss in lymphoid-primed multipotent progenitors (LMPPs) (CD34+Flt3+LSK) with relatively normal formation of LT-HSCs (CD34-Flt3-LSK) and ST-HSCs (CD34+Flt3-LSK) upon the loss of Sox4, suggesting that Sox4 is essential for the development from HSCs to multipotent progenitors. Such observation is in line with the expression pattern of Sox4. Quantitative PCR (qPCR) analysis of wild-type mice revealed that expression of Sox4 increased from HSCs to multipotent progenitors which expressed Sox4 at the highest level among all the hematopoietic compartments. Studies of biological behaviors further indicateed that disruption of Sox4 had no effect on proliferative capacity of HSCs and multipotent progenitors, as evidenced by BrdU incorporation assay. However, Annexin V/propidium iodide staining revealed an increased frequency of apoptotic multipotent progenitors, but not that of HSCs upon the ablation of Sox4. In a transplantation setting, although Sox4-deficient LSKs homed appropriately to the bone marrow, they exhibited severely impaired ability to give rise to multipotent progenitors, but contributed normally to HSCs compared to the wild-type donors. Among a set of genes crucial to the biological properties of stem/progenitor cells, qPCR analysis revealed that upon the loss of Sox4, only the levels of Ikaros1 and Ikaros2, the two major Ikaros isoforms in stem/progenitor cells, were downregulated specifically in multipotent progenitors, but remained normal in HSCs. Intriguingly, in a reminiscent manner of Sox4-deficient mice, mice lacking both Ikaros 1 and Ikaros 2 proteins, also exhibited disrupted B cell development and selectively impaired LMPPs. Previous study identified an enhancer of Ikaros locus as the only cis-regulatory element that was capable of stimulating reporter expression in the LMPPs. Our sequence analysis revealed a highly conserved Sox4 binding motif within this enhancer, therefore potentially connecting Sox4 with the known regulatory networks that modulate the differentiation of HSCs. Currently, we are working on (1) confirming the direct transcriptional regulation of Ikaros by Sox4; (2) assessing whether Ikaros mediates the functions of Sox4 in the formation or maintenance of the multipotent progenitors population in vivo; and (3) delineating the downstream regulatory network of Sox4 in stem/progenitor cells. Conclusion In summary, out study reveals a novel role for Sox4 gene in early hematopoiesis and brings important insights into the regulatory mechanisms underlying the commitment of HSCs toward multipotent progenitors. Disclosures No relevant conflicts of interest to declare.

Role of the Cell Cycle Regulator Cdh1 in Physiology and Pathology of Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2419-2419
Author(s):  
Jo Ishizawa ◽  
Eiji Sugihara ◽  
Norisato Hashimoto ◽  
Shinji Kuninaka ◽  
Shinichiro Okamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Differentiation ◽  
Progenitor Cells ◽  
Genotoxic Stress ◽  
Absolute Number ◽  
Wild Type ◽  
Deficient Mice ◽  
Oncogenic Stress

Abstract Abstract 2419 Various key molecules for cell cycle, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators for anaphase-promoting complex/cyclosome, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Because there are only few reports about Cdh1 from this point of view, we focused on Cdh1 functions on the hematopoietic system, in which distinct populations of cells can be precisely identified by their cell surface markers, in physiology and pathology. For this purpose, we generated Cdh1 conditional gene-trap (GT) mice, by overcoming the embryonic lethality of Cdh1 homozygous GT mice. We introduced the Cdh1 cDNA replacing vector into ES cells derived from Cdh1 heterozygous GT mice. The resulted construct contains the floxed Cdh1 cDNA allele which is cleaved under the existence of Cre recombinases. We crossed mice carrying this Cdh1 transgene in homozygous (Cdh1f/f) with Mx1-Cre transgenic mice to obtain Mx1-Cre (+) / Cdh1f/f mice, in which Cre recombinases are induced in vivo by administration of pIpC. In this system, we found that the Cdh1-deficient mice 4 months after pIpC treatment, compared to Cdh1-intact mice (Mx1-Cre (-) / Cdh1f/f mice), exhibited a subtle but significant decrease in absolute number of mature lineage progenitor cells (4.3 ± 0.31 × 107 vs 3.2 ± 0.10 × 107 /femurs and tibiae; p=0.009). Furthermore, this phenomenon was conspicuous by irradiation as short as 7 days after pIpC treatment. In 48 hours post-irradiation, the absolute number of mature lineage progenitor cells decreased markedly in the Cdh1-deficient mice (7.4 ± 0.82 × 106 vs 3.6 ± 0.46 × 106; p=0.0023) and in addition, both of CD34+ and CD34- LSK cells were also decreased (absolute number of CD34- cells: 905 ± 194 vs 344 ± 223; p= 0.03). These results indicate that the loss of Cdh1 induces genotoxic fragility especially in these two subpopulations, the mature lineage progenitors and the stem cells. We also confirmed that the increased cell loss induced by irradiation in Cdh1-deficient mice is the result of mitotic catastrophe following G2/M checkpoint slippage due to loss of Cdh1 by DNA content analysis. We next focused on how oncogenic stress, as another genotoxic stress, effects on the cell fragility by Cdh1 loss. We performed retroviral transduction of N-myc into Cdh1-intact and Cdh1-deficient bone marrow mononuclear cells (BM-MNCs) and transplanted those into irradiated wild type mice. In this system, which our laboratory has established recently, the transplanted mice develop precursor B cell lymphoblastic leukemia (pre-B ALL) phenotype in high frequency (more than 80%) when wild type BM-MNCs were used as cell source. Our hypothesis at that time was that oncogenic stress due to N-myc induces the loss of stem/progenitor cell function, and in result, that Cdh1 loss reveals negative effects on leukemogenesis or changes its lineage phenotype by affecting pseudodifferentiation due to N-myc. However, against our speculation, 70% (7 out of 10) of mice transplanted with N-myc transduced Cdh1-deficient BM-MNCs developed pre-B ALL, which was the same frequency and the same phenotype as in Cdh1-intact cell sources. Of note, Cdh1 loss did not have a great impact on the prognosis of these pre-B ALL mice (median survival: 80 days in Cdh1-intact group vs 95 days in Cdh1-deficient group; p= 0.049). In conclusion, our results suggest that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells both physiologically and pathologically; especially under irradiation stress. In contrast, Cdh1 is dispensable for B cell leukemogenesis and does not have a great impact on the natural prognosis of non-treated pre-B ALL. It is interesting that oncomine mRNA microarray database and other few reports indicate that human pre-B ALL cases are also divided into two groups according to the expression level of Cdh1, and it is the matter remained to be solved whether Cdh1 expression level affects the prognosis of treated patients. We propose that our Cdh1-deficient pre-B ALL mice have a potential as promising mouse model in order to assess this proposition and to prove that Cdh1 affects the sensitivity of pre-B ALL to treatments which causes the genotoxic stress, such as radiotherapy and genotoxic agents. Disclosures: Saya: Kyowa Hakko Kirin, Co., Ltd.: Research Funding.

Identification of Sox4 As Key Oncogene in Leukemias with Mutated or Silenced C/EBPα

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 114-114
Author(s):  
Hong Zhang ◽  
Min Ye ◽  
Meritxell Alberich-Jordà ◽  
Giovanni Amabile ◽  
Philipp B. Staber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Model Systems ◽  
Myeloid Differentiation ◽  
Wild Type ◽  
Down Regulation ◽  
Hematopoietic Stem ◽  
Gene Expression Signatures ◽  
Deficient Mice

Abstract Abstract 114 Transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) controls cell proliferation and myeloid differentiation. In 7∼10% of patients with acute myeloid leukemia (AML) C/EBPA is either mutated or epigenetically silenced. C/EBPA mutated leukemias differ from C/EBPA silenced leukemias in prognosis and phenotype, yet both leukemias cluster together based on genome wide gene expression signatures, indicating a unifying mechanism of disease. So far, the key molecular downstream events required for C/EBPA loss to trigger leukemogenesis are still unclear. Based on microarray gene expression analysis, we here used a shRNA screening platform to search for mediators of leukemic outgrow of C/EBPα-deficient progenitor cells. In our screen, oncogene Sox4 was identified as a gene that was up-regulated in C/EBPα-deficient hematopoietic stem cells (HSCs, both lineage−c-kit+ScaI+ HSCs and SLAM+ HSCs) and whose down-regulation abrogated aberrant self-renewal ability and restored myeloid differentiation of C/EBPα-deficient stem/progenitor cells, as demonstrated by in vitro serial-replating and differentiation assays. Chromatin immunoprecipitation confirmed the endogenous binding of C/EBPα at the proximal promoter of Sox4 in the stem/progenitor cells enriched population (lineage−c-kit+) and the mature myeloid population (Mac1+Gr1+). In vitro promoter reporter assay demonstrated that wild-type human C/EBPA, but none of the C/EBPA mutants identified from AML patients, repressed Sox4 transcription through its binding to a highly conserved C/EBPα binding site. C/EBPα and Sox4 showed reciprocal expression patterns in both HSCs and various hematopoietic compartments during myeloid maturation of wild type mice. Furthermore, expression of Sox4 was up-regulated in HSCs of C/EBPα-deficient mice as well as in leukemia-initiating cells (LICs) of a murine C/EBPα mutant AML model. To further genetically dissect the role of Sox4 in driving leukemia in the absence of functional CEBPα, we generated Sox4, C/EBPα double deficient mice and observed that loss of Sox4 alleviated the abnormal stem/progenitor cell expansion and defective myeloid programming caused by C/EBPα deficiency. In addition, comparisons of the murine C/EBPα mutant AML model with a Sox4-induced AML model revealed that leukemia initiating cells of both leukemia models were enriched in immunophenotypically similar populations and exhibited comparable gene expression signatures. Similar to the C/EBPα knockout model, down-regulation of Sox4 by shRNA in LICs from murine C/EBPα mutant AML was also sufficient to abolish their augmented serial-replating ability. Importantly, enhanced expression of SOX4 in AML patients with either mutated or epigenetically silenced C/EBPA compared to other AML subtypes confirmed the findings in our mouse model systems. Our data demonstrate that failure to suppress Sox4 expression is the underlying mechanism of leukemias with mutated or silenced C/EBPα. These data also uncover a promising rationale for a therapeutic target in these leukemias. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Critical role for Gimap5 in the survival of mouse hematopoietic stem and progenitor cells

2011 ◽  
Vol 208 (5) ◽  
pp. 923-935 ◽  
Author(s):  
Yuhong Chen ◽  
Mei Yu ◽  
Xuezhi Dai ◽  
Mark Zogg ◽  
Renren Wen ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Critical Role ◽  
Hematopoietic Progenitor Cell ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
T Cell Lymphopenia ◽  
Stem And Progenitor Cells ◽  
Deficient Mice

Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1—which is essential for HSC survival—and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.

Homeostasis and regeneration of the hematopoietic stem cell pool are altered in SHIP-deficient mice

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3541-3547 ◽  
Author(s):  
Cheryl D. Helgason ◽  
Jennifer Antonchuk ◽  
Caroline Bodner ◽  
R. Keith Humphries
Keyword(s):  
Negative Regulator ◽  
Wild Type Littermate ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
Multipotent Progenitors ◽  
Important Negative Regulator ◽  
Total Body ◽  
Deficient Mice

AbstractSH2-containing inositol 5-phosphatase (SHIP) is an important negative regulator of cytokine and immune receptor signaling. SHIP-deficient mice have a number of hematopoietic perturbations, including enhanced cytokine responsiveness. Because cytokines play an important role in the maintenance/expansion of the primitive hematopoietic cell pool, we investigated the possibility that SHIP also regulates the properties of cells in these compartments. Primitive hematopoietic cells were evaluated in SHIP-deficient mice and wild-type littermate controls using the colony-forming unit-spleen (CFU-S) and competitive repopulating unit (CRU) assays for multipotent progenitors and long-term lympho-myeloid repopulating cells, respectively. Absence of SHIP was found to affect homeostasis of CFU-S and CRU compartments. Numbers of primitive cells were increased in extramedullary sites such as the spleen of SHIP-deficient mice, although total body numbers were not significantly changed. In vivo cell cycle status of the CRU compartment was further evaluated using 5-fluorouracil (5-FU). SHIP-deficient CRUs were more sensitive to 5-FU killing, indicating a higher proliferative cell fraction. More strikingly, SHIP was found to regulate the ability of primitive cells to regenerate in vivo, as CRU recovery was approximately 30-fold lower in mice that received transplants of SHIP-deficient cells compared with controls. These results support a major role for SHIP in modulating pathways important in homeostasis and regeneration of hematopoietic stem cells, and emphasize the importance of negative cytokine regulation at the earliest stages of hematopoiesis. (Blood. 2003;102:3541-3547)

scholarly journals The switch of intestinal Slc26 exchangers from anion absorptive to HCO3−secretory mode is dependent on CFTR anion channel function

2010 ◽  
Vol 298 (5) ◽  
pp. C1057-C1065 ◽  
Author(s):  
Anurag Kumar Singh ◽  
Brigitte Riederer ◽  
Mingmin Chen ◽  
Fang Xiao ◽  
Anja Krabbenhöft ◽  
...  
Keyword(s):  
Anion Channel ◽  
Transport Activity ◽  
Wild Type ◽  
Regulatory Relationship ◽  
Genetic Ablation ◽  
Anion Transporters ◽  
Absolute Requirement ◽  
Heterologous Expression Systems ◽  
Deficient Mice ◽  
Nhe3 Inhibitor

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO3−) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO3−secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl−/HCO3−exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO3−secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO3−secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na+/H+exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl−absorptive mode and Cl−exit via CFTR to operate in the HCO3−secretory mode.

The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 32-32
Author(s):  
Lei Wang ◽  
Linda Yang ◽  
Marie–Dominique Filippi ◽  
David A. Williams ◽  
Yi Zheng
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Brdu Incorporation ◽  
Low Density ◽  
Actin Assembly ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

Sign in / Sign up

Export Citation Format

Share Document