Deficiency of Bone Marrow Stromal Cx43 Expression Induces Hematopoietic Progenitor Homing Deficiency and Cell-Cycle Arrest of Hematopoietic Stem Cells and Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 349-349
Author(s):  
Lina Li ◽  
Cynthia A. Presley ◽  
Bryan Kastl ◽  
Jose A. Cancelas
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression ◽  
Hematopoietic Stem ◽  
M Phase ◽  
Deficient Mice

Abstract Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be critical in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. BM cells are directly connected through gap junctions (GJs) which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. Due to the perinatal death of Cx43 germline null mice, an interferon-inducible, conditional genetic approach (Mx1-Cre), expressed by both hematopoietic and stromal BM cells, was used to study the role of Cx43 in stem cell function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration (Presley C, et al., Cell Comm. Adh., 2005). Here, we observed that after 5-FU administration, Cx43 expression is predominantly located in the endosteum. To study the role of stroma-dependent Cx43 in hematopoiesis, we developed hematopoietic chimeras by BM transplantation of wild-type Cx43 HSC into stromal Cx43-deficient mice. Stromal Cx43 deficiency induced a severe impairment of blood cell formation during the recovery phase after 5-FU administration compared to stromal Mx1-Cre-Tg wild-type controls (Table 1), as well as a significant decrease in BM cellularity (~60% reduction) and progenitor cell content (~83% reduction). Cell cycle analysis of 5-FU-treated BM progenitors from stromal Cx43-deficient mice showed an S-phase arrest (S phase: 63.5%; G2/M phase: <1%) compared to wild-type chimeric mice (S phase: 38.6%, G2/M phase: 7.8%, p=0.01) suggesting a cell division blockade. Unlike Cx43-deficient primary mice, a differentiation arrest at the HSC compartment was observed in 5-FU-treated, stromal Cx43-deficient mice, since the content of competitive repopulating units (CRU) at 1 month, of 14-day post-5-FU BM of stromal Cx43-deficient mice was increased (27.7 ± 0.67) compared to recipients of HSC from stromal wild-type counterparts (26.5 ± 0.92 CRU, p < 0.01). Interestingly, wild-type hematopoietic progenitor homing in stromal Cx43-deficient BM was severely impaired with respect to wild-type BM (5.1% vs10.4 %, respectively, p < 0.01), while hematopoietic Cx43-deficient BM progenitors normally homed into the BM, suggesting a differential role for Cx43 in stromal and HSC. In conclusion, expression of Cx43 in osteoblasts and stromal cells appears to play a crucial role in the regulation of HSC homing in BM and hematopoietic regeneration after chemotherapy. Peripheral blood counts of WT and stromal Cx43-deficient chimeric mice after 5-FU administration (150 mg/Kg) Neutrophil counts (×10e9/L) Reticulocyte count (%) Day post-5-FU WT Cx43-deficient WT Cx43-deficient * p < 0.05 Day +8 2.89 ± 0.06 0.81 ± 0.02* 2.0 ± 0.6 3.0 ± 0.9 Day +11 9.11 ± 2.5 3.13 ± 0.8* 6.1 ± 0.6 2.7 ± 0.3* Day +14 6.22 ± 5.7 7.58 ± 8.2 7.5 ± 0.5 2.5 ± 0.5*

Connexin-43 Regulates the Cell Cycle Entry of Hematopoietic Stem Cells within the Stem Cell Niche.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1500-1500
Author(s):  
Daniel Gonzalez-Nieto ◽  
Gabriel Ghiaur ◽  
Lina Li ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Marked Reduction ◽  
Connexin 43 ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 1500 Poster Board I-523 Bone marrow (BM) osteoblasts and stromal (O/S) cells are crucial in the establishment of the hematopoietic niches in the BM. Connexin 43 (Cx43) is expressed by BM stromal cells and by hematopoietic stem cells and progenitors (HSC/P) and is overexpressed in the BM endosteal space upon administration of chemotherapy or radiotherapy. We have previously reported that Cx43 is critical in fetal liver and in BM hematopoiesis. Since Cx43 is expressed by both HSC and the hematopoietic microenvironment, we dissected out the cellular mechanisms responsible for Cx43 function in the BM. We analyzed the hematopoiesis of mice deficient in Cx43 in the O/S cells (Collagen 1α-Creflox/flox; O/S-Cx43-deficient) or in the hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). Upon basal conditions, analysis of the HSC compartment of H-Cx43-deficient mice showed a ∼30% decreased content of immunophenotypically defined long-term HSC (LT-HSC) in BM of H-Cx43KO mice compared with their WT littermates, whereas there was not significant variation in the ST-HSC population content. The reduced LT-HSC population in H-Cx43KO mice was associated with a modest increased quiescence (∼12% increase of LT-HSC in G0). Interestingly, the expression of cyclin D1 and p21cip1 in the H-Cx43KO LT-HSC were 50% reduced and 4-fold increased, respectively, suggesting a decreased ability to enter cell cycle. While we found no significant engraftment difference in primary recipients of competitive repopulation assays, we found a marked reduction (>50%) in the engraftment ability of LT-HSC Cx43-deficient cells when transplanted into secondary recipients. When submitted to stress by 5-fluorouracil (5-FU) administration, H-Cx43KO mice showed a severely decreased hematopoietic recovery of peripheral blood (PB) counts for neutrophils and platelets accompanied with a marked reduction in the BM cellularity and hematopoietic progenitor content on day +14 after treatment. This defect was associated with a dramatic decreased (∼75 %) in the proliferation of the Cx43-deficient, LT-HSC population by 48 hours post-5-FU administration and a relative decrease of the expansion of the ST-HSC/MPP pool as early as 6 days post-5-FU administration. Interestingly, O/S-Cx43-deficient mice also showed severely delayed hematological recovery after 5-FU administration, with reduction in cellularity and hematopoietic progenitor content, suggesting that the increased hematopoietic toxicity induced by 5-FU in the context of Cx43 deficiency may depend on HSC-to-O/S Cx43 homotypic communication. This communication would be responsible of control of the G1 restriction checkpoint in LT-HSC. In summary, our findings suggest that Cx43 expression plays a crucial role controlling the LT-HSC pool size and fitness in response to stress. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Dissection of the Role of Connexin-43 in Hematopoietic Progenitors and Stromal Cells Unveils Distinct Hematopoietic Intrinsic and Extrinsic Regulatory Functions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 607-607
Author(s):  
Lina Li ◽  
Bhuvana Murali ◽  
Dealma N. Worsham ◽  
Susan K. Dunn ◽  
Jose A. Cancelas
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Connexin 43 ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression

Abstract Bone marrow (BM) stromal cells seem to be crucial in the establishment of the hematopoietic niches in bone marrow. BM stromal cells can communicate through gap junctions, which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM stromal cells and upon adhesion to stroma, by hematopoietic stem cells and progenitors (HSC/P). Cx43 has been shown to be essential in controlling osteoblast and fibroblast function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC/P in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration in Mx1-Cre-Tg;Cx43KO mice (Presley C, et al., Cell Comm. Adh., 2005). We have also previously shown that after 5-FU administration, Cx43 is predominantly expressed in the endosteum and the deficiency of Cx43 in stroma of Collagen I (ColI)-Cre;Cx43KO and chimeric mice impairs their hematopoiesis by impairing the homing of wild-type (WT) hematopoietic progenitors and after 5-FU administration, the hematopoietic progenitor cycling inducing a ∼30% expansion of the long-term stem cell compartment in BM (Li L et al., ASH 2006). Interestingly, stromal Cx43-deficient mice contain around twice as many CFU-F as wild-type (WT) mice. Now, we have further investigated the role of stromal Cx43 expression in the regulation of hematopoietic progenitor adhesion to stroma, trans-stromal migration and mobilization. Cx43-deficient stromal cells display complete absence of intercellular communication as assayed by calcein dye transfer which can be reverted by retroviral transduction of Cx43. Trans-stromal migration of hematopoietic progenitors through Cx43-deficient irradiated stroma is impaired (7.8% vs 13.8% in WT stroma, p=0.015) but primary adhesion to Cx43-deficient irradiated stroma and in vivo mobilization response to G-CSF in ColI-Cre;Cx43KO mice were similar to WT controls, suggesting that stromal Cx43 plays a role in the regulation of the post-adhesion migration of HSC/P. On the other hand, Cx43-deficient HSC/P from Vav1-Cre;Cx43KO primary and chimeric mice show severe impairment of blood cell formation during the recovery phase after 5-FU administration (day +14) compared to wild-type controls (ANC: 0.23±0.12 vs 1.40±1.25 x 109 neutrophils/L; Platelet count: 135±91 vs 572±205 x 109 platelets/L; p < 0.05). Cx43 deficiency in hematopoietic progenitors did not significantly impair their homing ability in wild-type mice. Taken together, these studies indicate that Cx43 expression plays distinct roles in the regulation of hematopoietic intrinsic and extrinsic mechanisms. While Cx43 expression in stroma seems to be crucial in the regulation of the stromal progenitor and HSC pool content as well as HSC/P trans-stromal migration and homing, deficiency of Cx43 in either hematopoietic cells or stromal cells independently induce a significant impairment in the post-chemotherapy blood formation in vivo, suggesting that, under stress, hematopoietic regeneration depends on complete Cx43 channels communicating HSC/P and stromal cells.

scholarly journals Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity

2000 ◽  
Author(s):  
Gideon Grafi ◽  
Brian Larkins
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Storage Protein ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Cyclin B ◽  
Regulatory Subunit ◽  
Maize Endosperm ◽  
M Phase

The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

scholarly journals The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage.

2021 ◽  
Author(s):  
Katheeja Muhseena N. ◽  
Shankar Prasad Das ◽  
Suparna Laha
Keyword(s):  
Dna Damage ◽  
Budding Yeast ◽  
S Phase ◽  
Wild Type ◽  
Yeast Protein ◽  
Replication Checkpoint ◽  
Bud Emergence ◽  
Checkpoint Pathway ◽  
M Phase

Abstract Background: The helicase Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This budding yeast protein is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results: G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with a faster kinetics in chl1 mutants compared to wild-type cells. This role of Chl1p in G1 phase is Rad9p dependent and independent of Rad24 and Rad53. rad9chl1 shows similar bud emergence as the single mutants chl1 and rad9 whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24 , rad53 and chl1 . In case of damage induced by genotoxic agent like hydroxyurea, Chl1p acts as a checkpoint at G1/S. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further we have observed that the checkpoint defect is synergistic with the replication checkpoint Sgs1p and functions in prallel to the checkpoint pathway of Rad24p. Conclusion: Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint, confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1 thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p as well as Rad53p activation when damaging agents perturbs the DNA.

Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2221-2221
Author(s):  
Cyrus Khandanpour ◽  
Ulrich Duehrsen ◽  
Tarik Möröy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

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.

HDAC8 Regulates Long-Term Hematopoietic Stem Cell Quiescence and Maintenance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1468-1468
Author(s):  
Wei-Kai Hua ◽  
Jing Qi ◽  
Qi Cai ◽  
Emily Carnahan ◽  
Ling Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Poly I:C ◽  
Control Group ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Polycytidylic Acid ◽  
Deficient Mice

Abstract Long-term (LT) hematopoietic stem cells (HSC) are responsible for life-long production of mature blood cells of all lineages through tightly concerted cell fate decisions including quiescence, self-renewal, differentiation and apoptosis. Histone deacetylase 8 (HDAC8) is a member of class I HDAC enzymes that remove acetyl moieties from lysine residues on histones and a variety of non-histone proteins. Specifically, HDAC8 has been shown to modulate the acetylation cycle of cohesin complex protein SMC3. Loss-of-function mutations in HDAC8, located on the X chromosome q13, have been found in patients with Cornelia de Lange Syndrome (CdLS) and those with CdLS-like features. These HDAC8 mutations are associated with severely skewed X-inactivation (100% wild type allele) in the peripheral blood of female patients, possibly due to selection against the mutant alleles. However, the expression and function of HDAC8 in normal HSCs and hematopoiesis remain unknown. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin-cKit+Sca1+CD150+CD48-) population in adult mice. To determine the function of HDAC8 in adult hematopoiesis, we generated conditional Hdac8 deficient mice using the Mx1-Cre and a floxed Hdac8 allele (Mx1-Cre/Hdac8f/f(y)) andconfirmed that Hdac8 is successfully deleted by polyinosinic-polycytidylic acid [poly (I:C)] treatment. Phenotypic analysis of Hdac8 deficient mice showed increased LT-HSC population compared to similarly treated control mice. However, largely normal steady state hematopoietic profile was found in Hdac8 deficient mice at 6 weeks and 1 year after induction. To further track Hdac8-deleted cells, we generated Cre/Hdac8f/f(y) mice with a dual fluorescence Rosa26mT/mG (mT/mG) Cre reporter allele, which expresses dTomato prior to Cre induction and becomes GFP+ after Cre-mediated recombination. We assessed hematopoietic repopulation by transplanting bone marrow cells from Cre/Hdac8f/f(y)/mTmG+mice (2 x 105) together with wild type support cells (2 x 105) into lethally irradiated CD45.1+ congenic recipients. Hdac8 deletion was induced by treating the recipients with 7 does (14 m▢g/kg/dose) of poly (I:C). We found that Hdac8 deletion did not affect CD45.2+ or GFP+ donor-derived overall engraftment or lineage repopulation up to 16 weeks. There was also no change in the frequency or number of GFP+ donor-derived phenotypic LT-HSCs in the bone marrow. Serial transplantation was performed to further assess long-term repopulating activity of HSCs. Hdac8 deficient cells were significantly (p=0.019; n=3) compromised in multi-lineage repopulation in secondary transplant recipients. Except a modest reduction in Pre-GM, there was no change in the overall composition of Hdac8 deficient CD45.2+-derived populations. Upon tertiary transplantation, no donor engraftment was observed for Hdac8 deficient cells (0 out of 4) compared to 50% positive engraftment in control group (4 out of 8). These results indicate that HDAC8 is crucial for maintaining long-term serial-repopulating activity over time. Cell cycle analysis revealed that Hdac8 deficient LT-HSCs display reduced quiescence and increased cycling, consistent with the increased number of phenotypic LT-HSC seen in Hdac8 deleted mice. Therefore, we further tested the sensitivity of Hdac8 deficient mice to serial ablation with 5-fluorouracil (5FU), an S phase-specific cytotoxic chemotherapeutic agent. Impaired hematopoietic recovery and increased lethality (p<0.001; n=23) was seen in Hdac8 deficient mice treated with 5-FU (100 mg/kg) every 7 days, indicating that Hdac8 deletion renders hypersensitivity to serial ablation. There were significnatly less phenotypic LT-HSCs in Hdac8 deficient mice 6 days after 5-FU treatment (p<0.01; n=4). In parallel, we observed increased DNA strand beaks as indicated by γ-H2AX staining and comet assays (p<0.001; n>100 cells). Analysis of p53 activation, cell cycle regulators and DNA dmage response are ongoing. Collectively, our study indicates that HDAC8 plays a pivotal role in LT-HSC quiescence and maintenance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Higher Vertebrate Specific Gene Ribonuclease Inhibitor (RNH1) Is Essential for Adult Hematopoietic Stem Cell Function and Cell Cycle Regulation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 273-273
Author(s):  
Nicola Daniele Andina ◽  
Mayuresh Sarangdhar ◽  
Aubry Tardivel ◽  
Giuseppe Bombaci ◽  
Mahmoud Hallal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Regulation ◽  
Cell Function ◽  
Wild Type ◽  
Ribonuclease Inhibitor ◽  
Hematopoietic Stem ◽  
Dominant Phenotype ◽  
Cycle Regulation ◽  
Deficient Mice

Hematopoietic stem cells (HSC) in higher vertebrate species, especially in mammals, maintain hematopoiesis throughout adult life and require critical cell cycle regulation for their self-renewal and cell fate decisions. Although cell cycle pathways are quite conserved across animal species, it is unknown whether a higher vertebrate specific cell cycle regulation exists in adult mammalian HSCs. Recently, we have published that Ribonuclease inhibitor (RNH1) regulates erythropoiesis by controlling GATA1 mRNA translation. Here, we report that RNH1, which is present only in higher vertebrates regulates HSC cell cycle and HSC function. To study the role of RNH1 in hematopoiesis, we generated hematopoietic-specific knockout mice by backcrossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Rnh1-deficiency (Rnh1FL/FLVav1-iCre mice) resulted in hematopoietic alterations resembling emergency myelopoiesis. At 15 weeks of age Rnh1-deficient mice had reduced hemoglobin levels (144.4 ± 2.6 vs 165.0 ± 4.2 g/L, p = 0.005), decreased lymphocytes (4.1 ± 0.8 vs 9.6 ± 1.6 K/µL, p = 0.023), increased neutrophils (3.2 ± 0.6 vs 1.5 ± 0.2 K/µL, p = 0.046) and monocytes (0.65 ± 0.05 vs 0.09 ± 0.02 K/µL, p = 0.0001) in the peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type andRnh1-deficient mice, however the number of erythroid cells and lymphoid cells (T and B cells) was significantly decreased, whereas myeloid cells were significantly increased. Rnh1-deficient spleens were significantly larger than wild type controls and showed extramedullary hematopoiesis. Surprisingly, although Rnh1-deficient mice showed myeloproliferation they survived normally and did not show progression to leukemia. However, they did not tolerate even little stress, such as 35 µg LPS administration, which lead to early mortality. We analysed the progenitor populations in the BM. In line with the myelopoiesis dominant phenotype granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Cell extrinsic factors such as growth factors and the bone marrow niche play a critical role in shaping lineage choice. To exclude this, we performed bone marrow transplantation experiments (BMT) by transplanting wild type (Rnh1FL/FL) and Rnh1-deficient (Rnh1FL/FLMx1-Cre+) bone marrow into lethally irradiated CD45.1 congenic mice. After reconstitution Rnh1 was deleted by administration of polyinosinic:polycytidylic acid (polyI:C). We observed a similar myelopoiesis dominant phenotype in Rnh1-deleted mice. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-deficient mouse BM, suggesting that RNH1 could affect HSC function. Supporting this Rnh1-deficient HSCs failed to engraft lethally irradiated mice in competitive BMT experiments. Furthermore, Rnh1-deficient HSCs produced significantly less and smaller colonies in in-vitro colony forming cell (CFC) assays. Transcriptome analysis showed increased expression of genes related to cell cycle, kinetochore, DNA damage and decreased expression of genes related to stem cell function in Rnh1-deficient LT-HSCs and ST-HSCs. Corroborating this, Rnh1-deficient LT-HSCs and ST-HSCs showed increased S/G2/M phase in cell cycle analysis. In line with this, at the molecular level, we found that RNH1 directly binds to cell-cycle related proteins such as cyclin-dependent kinase 1 (CDK1), cell-division cycle protein 20 (CDC20) and mitotic checkpoint protein BUB3, suggesting direct involvement of RNH1 in cell cycle regulation. Confirming this, pharmacological inhibition of CDK1 (RO-3306, 10 µM) in Rnh1-deficinet ST-HSCs restored colony size in CFC assays, suggesting that RNH1 and CDK1 inhibition have a synergistic effect in ST-HSCs. In summary, our results demonstrate that RNH1, which is present only in higher vertebrates, is essential for HSC cell cycle regulation and steady state hematopoiesis. 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 Requirement of U2AF1 in the Maintenance and Function of Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1195-1195
Author(s):  
Avik Dutta ◽  
Yue Yang ◽  
Bao Le ◽  
Golam Mohi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Rna Sequencing ◽  
Marked Decrease ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
And Function ◽  
Deficient Mice

Mutations in the RNA spliceosome genes have been frequently found in myelodysplastic syndromes (MDS). U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. U2AF1 mutations have been identified in ~11% cases of MDS and are associated with poor prognosis. However, the role of wild type U2AF1 in normal hematopoiesis has remained unknown. To determine the role of U2AF1 in hematopoietic stem/progenitor cell (HSPC) function and normal hematopoiesis, we have generated a conditional U2AF1 knockout (floxed) mouse. We crossed the U2AF1 floxed mouse with Mx1Cre mouse and the expression of Cre recombinase was induced with pI-pC injection at 4 weeks after birth. All induced Mx1Cre;U2AF1fl/fl (U2AF1-deleted) mice became moribund or died between 11-12 days after pI-pC induction. U2AF1-deleted mice exhibited marked decrease in bone marrow (BM) cellularity and significantly reduced numbers of WBC, neutrophil, RBC and platelet counts in their peripheral blood compared with control animals. Flow cytometric analyses revealed a dramatic decrease in myeloid, erythroid and megakaryocytic precursor cells in U2AF1-deficient mice compared with control animals. Hematopoietic progenitor colony assays showed a marked decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2AF1-deficient mice. Histopathologic analysis revealed severe BM aplasia in U2AF1-deficient mice. Together, these data suggest that deletion of U2AF1 results in profound defects in hematopoietic development. The fatal BM failure in U2AF1-deficient mice prompted us to examine the HSPC compartments in the BM of these animals. We observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as early progenitors including common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2AF1-deficient mice, indicating a defect at the earliest stage of adult hematopoietic development. To determine whether the loss of HSCs in U2AF1-deficient animals is cell autonomous, BM cells from uninduced control (U2AF1fl/fl; no cre) and Mx1Cre;U2AF1fl/fl mice were transplanted into lethally irradiated WT C57BL/6 mice. Six weeks after transplantation, recipients were injected with pI-pC to induce the deletion of U2AF1. All the recipients of U2AF1-deficient BM became moribund within 14 days after pI-pC induction. Deletion of U2AF1 in the recipient animals resulted in pancytopenia and marked decrease in HSC/progenitors, myeloid, erythroid and megakaryocytic cells similar to that observed in the primary U2AF1-deficient mice, suggesting that the hematopoietic defects in U2AF1-deficient HSCs is cell intrinsic. We performed competitive repopulation assays to further evaluate the function of U2AF1-deficient HSCs. BM cells from uninduced control (U2AF1fl/fl; no cre) and Mx1Cre;U2AF1fl/fl mice (CD45.2+) were mixed with CD45.1+competitor BM cells at a ratio of 1:1 and then transplanted into lethally irradiated congenic recipient animals (CD45.1+). Chimerism analysis in the transplanted animals revealed that U2AF1-deficient mice BM cells were completely unable to compete with WT BM cells. The percentages of U2AF1-deficient CD45.2+(donor-derived) LSK, myeloid, B and T cells were markedly reduced in the recipient animals compared with wild type U2AF1 BM donor at 16 weeks after transplantation, indicating that U2AF1-deficiency impairs the repopulation capacity of the HSCs. To gain insights into the mechanism by which U2AF1controls HSC maintenance and function,we performed RNA-sequencing on purified LSK cells from control and U2AF1-deleted mice. Analysis of RNA-sequencing data revealed significant down regulation of genes related to HSC maintenance, cell cycle and JAK-STAT pathway in U2AF1-deficient LSK cells compared with control LSK. RNA sequencing also identified significantly altered splicing events in several important genes in U2AF1-deficient LSK cells. The most commonly altered splicing events were exon skipping/inclusion. We also observed increased phospho-H2AX and DNA damage in U2AF1-deficient BM cells. Overall, our results suggest an essential role for U2AF1 in the maintenance and function of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

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