Wing Hearts in Four-Winged Ultrabithorax-mutant Flies—the role of Hox genes in wing heart specification

Genetics ◽  
2021 ◽  
Author(s):  
Markus Tögel ◽  
Günther Pass ◽  
Achim Paululat
Keyword(s):  
Progenitor Cells ◽  
Hox Genes ◽  
Epidermal Cells ◽  
Thoracic Segment ◽  
Regulatory Activity ◽  
Evolutionary Novelty ◽  
Wild Type ◽  
Hox Gene ◽  
The Third

Abstract Wings are probably the most advanced evolutionary novelty in insects. The development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove haemolymph and apoptotic epidermal cells from the premature wing through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant Drosophila that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later thoracic segments T2 and T3. However, the adult dipterian fly harbors only one pair of wing hearts and also only one pair of wings located in thoracic segment T2. Here we show, that the specification of wing heart progenitors depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analysed the development of four wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment (T3) is transformed into a second thoracic segment (HT2). This results in a second pair of wings instead of the club-shaped halteres normally formed by T3. We show that a second pair of wild-type wing hearts is formed in the four-winged fly and that all wing hearts originate from the wild-type progenitor cells.

The role of lin-22, a hairy/enhancer of split homolog, in patterning the peripheral nervous system of C. elegans

Development ◽  
1997 ◽  
Vol 124 (15) ◽  
pp. 2875-2888 ◽  
Author(s):  
L.A. Wrischnik ◽  
C.J. Kenyon
Keyword(s):  
Stem Cell ◽  
Regulatory Gene ◽  
Epidermal Cells ◽  
Body Axis ◽  
Wild Type ◽  
Hox Gene ◽  
C Elegans ◽  
Cell Divisions ◽  
Body Regions ◽  
Stem Cell Divisions

In C. elegans, six lateral epidermal stem cells, the seam cells V1-V6, are located in a row along the anterior-posterior (A/P) body axis. Anterior seam cells (V1-V4) undergo a fairly simple sequence of stem cell divisions and generate only epidermal cells. Posterior seam cells (V5 and V6) undergo a more complicated sequence of cell divisions that include additional rounds of stem cell proliferation and the production of neural as well as epidermal cells. In the wild type, activity of the gene lin-22 allows V1-V4 to generate their normal epidermal lineages rather than V5-like lineages. lin-22 activity is also required to prevent additional neurons from being produced by one branch of the V5 lineage. We find that the lin-22 gene exhibits homology to the Drosophila gene hairy, and that lin-22 activity represses neural development within the V5 lineage by blocking expression of the posterior-specific Hox gene mab-5 in specific cells. In addition, in order to prevent anterior V cells from generating V5-like lineages, wild-type lin-22 gene activity must inhibit (directly or indirectly) at least five downstream regulatory gene activities. In anterior body regions, lin-22(+) inhibits expression of the Hox gene mab-5. It also inhibits the activity of the achaete-scute homolog lin-32 and an unidentified gene that we postulate regulates stem cell division. Each of these three genes is required for the expression of a different piece of the ectopic V5-like lineages generated in lin-22 mutants. In addition, lin-22 activity prevents two other Hox genes, lin-39 and egl-5, from acquiring new activities within their normal domains of function along the A/P body axis. Some, but not all, of the patterning activities of lin-22 in C. elegans resemble those of hairy in Drosophila.

scholarly journals The Role of Histone Demethylases and DNA Methyltransferases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3921-3921
Author(s):  
Katerina Rejlova ◽  
Alena Musilova ◽  
Martina Slamova ◽  
Karel Fiser ◽  
Karolina Skvarova Kramarzova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hox Genes ◽  
Fold Increase ◽  
Dna Methyltransferases ◽  
Histone Mark ◽  
Gene Promoters ◽  
Histone Demethylases ◽  
Atra Treatment ◽  
Hox Gene

Abstract Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous results showed that HOX gene expression differs among genetically characterized subtypes of pediatric acute myeloid leukemia (AML). Specifically, PML-RARa positive AML patients have overall lowest HOX gene expression which positively correlates with expression of histone 3 lysine 27 (H3K27) demethylases - JMJD3 and UTX and negatively with the expression of DNA methyltransferases - DNMT3a and DNMT3b. Interestingly, JMJD3 was already shown to be a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). From these findings we postulated a hypothesis that reduced levels of HOX genes in PML-RARa positive AML are a consequence of suppressed expression of histone demethylases resulting in increased H3K27 methylation and/or of elevated levels of DNMTs leading to de novoDNA methylation. We studied the role of histone demethylases and DNMTs in the regulation of HOX gene expression and the effect of treatment in PML-RARa positive cell lines (NB4 and ATRA-resistant clones NB4-LR2 and NB4-MR2). We treated NB4 cell line by all-trans retinoic acid (ATRA; 1uM), which was described to release the differentiation block caused by the presence of PML-RARa and to degrade the fusion protein. We observed that expression of particular HOX genes (HOXA1, HOXA3, HOXA4, HOXA5, HOXA7, HOXB4, HOXB6) measured by qPCR was significantly increased after ATRA treatment. While the level of JMJD3 was significantly increased upon ATRA treatment as well, the expression of UTX did not change. Furthermore, we detected significantly reduced expression of DNMT3b gene. To exclude a non-specific effect of ATRA, independent of PML-RARa, we used resistant clones LR2 and MR2 bearing mutations in retinoic acid-binding domain. HOX gene expression together with JMJD3, UTX and DNMT3b expression did not change upon ATRA treatment. These results confirm the PML-RARa-dependent regulation of HOX genes. To test the role of JMJD3 in the HOX gene expression regulation, we cultured NB4 cells with a specific inhibitor of histone demethylases, GSK-J4 (1 uM, 10 uM), in the presence of ATRA. The co-treatment caused significant decrease in the expression of studied HOX genes (HOXA1, HOXA3, HOXA5, HOXA7, HOXA10, HOXB4, HOXB6) in comparison to ATRA alone which supports the role of JMJD3 in the transcription regulation. Further, we performed chromatin immunoprecipitation (ChIP) to investigate if the changes of HOX gene expression upon ATRA and GSK-J4 treatment would correspond with changes of histone code on HOX gene promoter regions. ATRA treatment caused reduction of repressive histone mark (H3K27me3) on particular HOX gene promoters (HOXA1, HOXA3, HOXA5, HOXA7), by contrast, combinational treatment of ATRA and GSK-J4 reversed this effect. Accordingly, we detected that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2. Next we were interested if JMJD3 inhibition would interfere with the differentiation effect of ATRA. As shown previously, ATRA treatment alone caused differentiation of NB4 cell line whereas the combination with GSK-J4 did not reduce the effect. Interestingly, in addition to differentiation it led cells to apoptosis. Combination of drugs (ATRA - 1uM, GSK-J4 - 1, 2, 5uM) increased significantly the percentage of dead cells in comparison to ATRA or GSK treatment alone (GSK-J4 alone vs in combination with ATRA, 1uM - 1.8 fold, 2uM - 2.2 fold, 5 uM - 2.3 fold increase). Next we measured apoptosis in resistant clones LR2 and MR2. In both cases the highest concentration used of GSK-J4 (5uM) in combination with ATRA caused significant increase of dead cells as well (LR2 - 2.1 fold, MR2 - 2.0 fold increase). Our results indicate that JMJD3 is responsible for the regulation of HOX gene expression in PML-RARa positive leukemia since changes of HOX gene expression correspond with histone modifications on the regions of HOX gene promoters. We assume that DNA methylation driven by DNMT3b can also participate in this process. Moreover, our findings demonstrate potential therapeutic implications of GSK-J4 inhibitor in combination with ATRA in patients with acute promyelocytic leukemia who are not responsive to ATRA monotherapy. Supported by P304/12/2214 and GAUK 196616 Disclosures No relevant conflicts of interest to declare.

Gene loss and gain in the evolution of the vertebrates

Development ◽  
1994 ◽  
Vol 1994 (Supplement) ◽  
pp. 155-161
Author(s):  
Frank H. Ruddle ◽  
Kevin L. Bentley ◽  
Michael T. Murtha ◽  
Neil Risch
Keyword(s):  
Hox Genes ◽  
Gene Loss ◽  
Biological Function ◽  
Hox Gene ◽  
Step Process ◽  
Gene Expansion ◽  
Multiple Clusters ◽  
Adaptive Role ◽  
Hox Clusters

Homeobox cluster genes (Hox genes) are highly conserved and can be usefully employed to study phyletic relationships and the process of evolution itself. A phylogenetic survey of Hox genes shows an increase in gene number in some more recently evolved forms, particularly in vertebrates. The gene increase has occurred through a two-step process involving first, gene expansion to form a cluster, and second, cluster duplication to form multiple clusters. We also describe data that suggests that non-Hox genes may be preferrentially associated with the Hox clusters and raise the possibility that this association may have an adaptive biological function. Hox gene loss may also play a role in evolution. Hox gene loss is well substantiated in the vertebrates, and we identify additional possible instances of gene loss in the echinoderms and urochordates based on PCR surveys. We point out the possible adaptive role of gene loss in evolution, and urge the extension of gene mapping studies to relevant species as a means of its substantiation.

scholarly journals Anatomic Origin of Osteochondrogenic Progenitors Impacts Sensitivity to EWS-FLI1-Induced Transformation

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 313 ◽  
Author(s):  
Elise Pfaltzgraff ◽  
April Apfelbaum ◽  
Andrew Kassa ◽  
Jane Song ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Malignant Transformation ◽  
Ewing Sarcoma ◽  
Hox Genes ◽  
High Expression ◽  
Wild Type ◽  
Superficial Zone ◽  
The Impact

Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13, is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1-induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.

Differential Expression of Mll-AF9 Up-Regulated Genes Correlates with Enhanced Self-Renewal of Hematopoietic Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 733-733 ◽  
Author(s):  
Ashish R. Kumar ◽  
Wendy A. Hudson ◽  
Weili Chen ◽  
Rodney A. Staggs ◽  
Anne-Francoise Lamblin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Cell Types ◽  
Third Generation ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Third

Abstract In order to understand the pathophysiology of leukemia, we need to study the effects of leukemic oncogenes on the rare hematopoietic stem and progenitor cells. We investigated the self-renewal capabilities of the various hematopoietic cell types derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll-fusion gene under the control of the endogenous promoter present in every hematopoietic stem/progenitor cell. In methylcellulose cultures, we compared myeloid colony formation of Mll-AF9 cells to wild type progenitor populations over three generations of plating. In the first generation of plating, the Mll-AF9 common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) formed more colonies than the hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs). However, at the third generation of plating, colony numbers formed by Mll-AF9 HSCs and CLPs were significantly greater than those formed by CMPs and GMPs. By the third generation only occasional colonies were found in the wild type groups. These results demonstrate that while Mll-AF9 led to an increase in self-renewal of all 4 cell types studied, these effects were more pronounced in HSCs and CLPs. To identify the downstream genes that mediate the growth deregulatory effects of Mll-AF9, we compared gene expression profiles of Mll-AF9 derived cells to their wild type counterparts. To assess gene expression levels, we extracted RNA from wild type and Mll-AF9 HSCs, CLPs, CMPs and GMPs. We then amplified and labeled the RNA for analysis by Affymetrix murine 430 2.0 genome arrays. In an unsupervised analysis, the various Mll-AF9 cells clustered with their corresponding wild type counterparts, indicating that the expression of most genes was not significantly altered by Mll-AF9. To identify the genes that are differentially expressed in the Mll-AF9 derived cells, we performed a two-way ANOVA (with the genotype and cell type as the two variables) allowing for a false discovery rate of 10%. In this analysis, we found that 76 genes were up-regulated in all Mll-AF9 progenitor cells compared to their wild-type counterparts. This list included known targets of Mll-fusion proteins Hoxa5, Hoxa7, Hoxa9 and Hoxa10. Also included were Evi1 and Mef2c, two genes that have been implicated in promoting enhanced self-renewal of murine hematopoietic cells. Importantly, in wild type mice, these 6 genes were expressed at higher levels in HSCs and CLPs compared to CMPs and GMPs (average 3–25 fold). While we observed an average 2–10 fold increase in expression of these genes in all Mll-AF9 cell types compared to their respective wild type controls, the expression level was 3–8 fold higher in Mll-AF9 HSCs and CLPs compared to CMPs and GMPs. Thus, the expression of genes known to be intrinsically related to self-renewal is further enhanced as a result of the Mll-AF9 fusion gene. In conclusion, while activation of the Mll-AF9 genetic program and the resulting enhanced self-renewal occurs in all 4 cell types studied, these effects are greatest in HSCs and CLPs. Thus, HSCs and CLPs are likely to be more efficient than CMPs and GMPs in producing cellular expansion and targets for cooperating mutations resulting in leukemia.

The Role of Histone Demethylases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 876-876
Author(s):  
Katerina Rejlova ◽  
Karolina Kramarzova ◽  
Meritxell Alberich-Jorda ◽  
Karel Fiser ◽  
Marketa Zaliova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Histone Methylation ◽  
Hox Genes ◽  
Histone Demethylases ◽  
Atra Treatment ◽  
Hox Gene ◽  
Genes Expression ◽  
Methylation Mark

Abstract Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous findings described that HOX gene expression differs among genetically characterized subtypes of pediatric AML with PML-RARa+ patients having the lowest overall HOX gene expression. We observed that HOX gene expression positively correlated with expression of histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX and negatively with DNA methyltransferase DNMT3b. Interestingly, it has been shown that JMJD3 is a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). These findings led us to postulate the hypothesis that reduced levels of HOX genes in PML-RARa+ AML can be caused by the suppressed expression of histone demethylases, such as JMJD3 and UTX, resulting in increased H3K27 methylation and transcription inhibition. We chose PML-RARa+ NB4 cell line to study the role of PML-RARa fusion gene in the regulation of HOX gene expression. To inhibit the effect of PML-RARa we used all-trans retinoic acid (ATRA; 1 uM, 10 uM) which was described to release the block caused by this fusion protein. Expression of particular HOX genes (e.g., HOXA1, HOXA3, HOXA5, HOXA7) together with that of JMJD3 and UTX assessed by qPCR was significantly elevated after ATRA treatment, while gene expression of DNMT3b was decreased. To test whether the reduction in HOX gene expression is directly related to the levels of JMJD3 and UTX, we cultured NB4 cells with a specific inhibitor of these histone demethylases, GSK-J4 (1 uM, 10 uM), in combination with ATRA. This co-treatment led to inhibition of JMJD3 and UTX proteins, followed by significant reduction of HOX genes expression (e.g., HOXA1, HOXA3, HOXA5, HOXA7). This result supports our hypothesis that HOX genes expression is directly related to JMJD3/UTX activity. To determine the effect of ATRA and GSK-J4 on histone marks we have isolated histones by acid extraction and detected the levels of histones by western blot in NB4 ATRA or GSK-J4/ATRA treated cells. We observed that the level of repressive histone methylation mark (trimethylated H3K27; H3K27me3) was decreased after ATRA treatment (activation of JMJD3/UTX) and increased after GSK-J4/ATRA co-treatment (inhibition of JMJD3/UTX). The opposite effect was observed in active histone methylation marks where di- and tri-methylated H3K4 (H3K4me2, H3K4me3) increased after ATRA treatment and decreased after GSK-J4/ATRA co-treatment. H3K9 dimethylated (another repressive histone methylation mark) levels did not change. Next, to investigate the histone code directly in particular HOX genes regions we performed chromatin immunoprecipitation (ChIP) assays. We studied the presence of H3K27me3 and H3K4me2 in 5´UTR genomic region of particular HOX genes (HOXA1, HOXA2, HOXA3, HOXA5, HOXA7) in cells treated with ATRA alone or in the combination with GSK-J4. Preliminary results showed reduction in repressive marks (H3K27me3) upon ATRA treatment, whereas addition of GSK-J4 prevented this decrease. Accordingly, we observed that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2. To evaluate the role of DNA methylation in observed expression changes after ATRA treatment we performed bisulfite sequencing of particular promoter sites of HOX genes (e.g., HOXA7, HOXA5). Although we detected decreased DNMT3b gene expression after ATRA treatment there was no change in DNA methylation of CpGs in studied regions. Our results demonstrate that changes in chromatin activity correspond with changes in HOX gene expression. Moreover, ChIP data show direct binding of the modified histones and HOX 5´UTR sites. Our data implicate histone demethylases in regulation of HOX gene expression in PML-RARa+ leukemic blasts. DNA methylation in these particular HOX genes is not involved in the regulation. Elucidating the mechanism of regulation of HOX genes expression can help to understand their role in the leukemogenic process. Supported by GACR P304/12/2214 and GAUK 568213. Disclosures No relevant conflicts of interest to declare.

scholarly journals BAX regulates follicular endowment in mice

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 865-876 ◽  
Author(s):  
Chuck R Greenfeld ◽  
Melissa E Pepling ◽  
Janice K Babus ◽  
Priscilla A Furth ◽  
Jodi A Flaws
Keyword(s):  
Cell Viability ◽  
Germ Cell ◽  
Proliferative Activity ◽  
Primordial Germ Cell ◽  
Initial Endowment ◽  
Regulatory Activity ◽  
Wild Type ◽  
X Protein ◽  
Primordial Follicles

It is believed that the endowment of primordial follicles in mammalian ovaries is finite. Once follicles are depleted, infertility ensues. Thus, the size of the initial endowment has consequences for fertility and reproductive longevity. Follicular endowment is comprised of various processes that culminate with the incorporation of meiosis-arrested oocytes into primordial follicles. Apoptosis is prominent during follicular endowment, and apoptosis regulatory genes are involved in its regulation. Conflicting data exist with regard to the role of the proapoptotic Bcl-2 associated X protein (BAX) in follicular endowment. Therefore, we investigated the role of BAX during follicular endowment in embryonic and neonatal ovaries. We found that BAX is involved in regulating follicular endowment in mice. Deletion ofBaxyields increased oocyte numbers in embryonic ovaries and increased follicle numbers in neonatal ovaries when compared with wild-type ovaries. Increased follicular endowment inBax−/−ovaries is not due to enhanced germ cell viability. Further, it is not due to an increased primordial germ cell (PGC) allotment, a delay in the onset of meiosis, or altered proliferative activity of oogonia. Instead, our data suggest that the regulatory activity of BAX in follicular endowment likely occurs during PGC migration, prior to PGC colonization of the gonad.

scholarly journals Hepatic progenitor cells promote the repair of schistosomiasis liver injury by inhibiting IL-33 secretion in mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Beibei Zhang ◽  
Xiaoying Wu ◽  
Jing Li ◽  
An Ning ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Liver Injury ◽  
Progenitor Cells ◽  
Normal Liver ◽  
Protective Role ◽  
Dependent Manner ◽  
Hepatic Progenitor Cells ◽  
Wild Type ◽  
Mice Model

Abstract Background Hepatic schistosomiasis, a chronic liver injury induced by long-term Schistosoma japonicum (S. japonicum) infection, is characterized by egg granulomas and fibrotic pathology. Hepatic progenitor cells (HPCs), which are nearly absent or quiescent in normal liver, play vital roles in chronic and severe liver injury. But their role in the progression of liver injury during infection remains unknown. Methods In this study, the hepatic egg granulomas, fibrosis and proliferation of HPCs were analyzed in the mice model of S. japonicum infection at different infectious stages. For validating the role of HPCs in hepatic injury, tumor necrosis factor-like-weak inducer of apoptosis (TWEAK) and TWEAK blocking antibody were used to manipulate the proliferation of HPCs in wild-type and IL-33−/− mice infected with S. japonicum. Results We found that the proliferation of HPCs was accompanied by inflammatory granulomas and fibrosis formation. HPCs expansion promoted liver regeneration and inhibited inflammatory egg granulomas, as well as the deposition of fibrotic collagen. Interestingly, the expression of IL-33 was negatively associated with HPCs’ expansion. There were no obvious differences of liver injury caused by infection between wild-type and IL-33−/− mice with HPCs’ expansion. However, liver injury was more attenuated in IL-33−/− mice than wild-type mice when the proliferation of HPCs was inhibited by anti-TWEAK. Conclusions Our data uncovered a protective role of HPCs in hepatic schistosomiasis in an IL-33-dependent manner, which might provide a promising progenitor cell therapy for hepatic schistosomiasis.

scholarly journals Novel Evidence That the Mannan-Binding Lectin (MBL) Pathway of Complement Activation Plays a Pivotal Role in Triggering Mobilization of Hematopoietic Stem/Progenitor Cells By Activation of Both the Complement and Coagulation Cascades

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3371-3371
Author(s):  
Mateusz Adamiak ◽  
Malwina Suszynska ◽  
Ahmed Abdel-Latif ◽  
Ahmed Abdelbaset-Ismail ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Complement Activation ◽  
Classical Pathway ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Mannan Binding Lectin ◽  
Binding Lectin ◽  
Poor Mobilizers ◽  
Mbl Deficiency

Abstract Background . The complement cascade (ComC), which is part of the innate immune system, exerts several pleiotropic effects, and, as we have demonstrated, it is required for mobilization of hematopoietic stem/progenitor cells (HSPCs) during infection or tissue/organ injury as well as in response to administration of pharmacological mobilizing agents, such as G-CSF or AMD3100 (Blood 2004, 103, 2071-2078). The ComC is activated by three pathways: the classical, mannan-binding lectin (MBL), and alternative pathways. Activation of the ComC and generation of cleavage fragments of the fifth component of the ComC (C5), such as C5a, desArgC5a, and C5b, by classical C5 convertase initiates events that are required for egress of HSPCs from bone marrow (BM) into peripheral blood (PB) (Leukemia 2009, 23, 2052-2062). Recent results indicate that the coagulation cascade (CoaC) is activated in parallel with activation of the ComC during the mobilization process and plays a supportive role, because thrombin has "C5 convertase-like activity" (Leukemia 2014, 28, 2148-2154). While a requirement for ComC activation and the pivotal roles of the distal part of complement activation and the generation of C5 cleavage fragments have been previously demonstrated (Leukemia 2009, 23, 2052-2062), mice with mutations in components of the classical pathway (C1q-/- mice), in which the distal pathway of C5 activation remained intact, do not show impairment of HSPC mobilization (Leukemia 2010, 24, 1667-1675). Aim of the study. Since no studies have yet been performed to address the role of the MBL pathway in triggering the mobilization of HSPCs, we became interested in its involvement in both ComC and CoaC activation after administration of G-CSF or AMD3100. The MBL pathway is homologous to the classical pathway but contains a soluble MBL receptor instead of C1q, and MBL functions as activator of the MBL-associated serine proteases, MASP-1 and MASP-2, which are activated downstream of both the ComC and the CoaC. Hypothesis. We hypothesized that the MBL-initiated ComC and CoaC activation pathways are involved in triggering mobilization of HSPCs and that MBL deficiency may result in poor mobilization efficiency.Materials and Methods. In our experiments, 2-month-old, MBL-deficient (MBL-/-) and MASP-1-deficient (MASP-1-/-) mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. Following mobilization, we measured i) the total number of white blood cells (WBCs), ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors, and iii) the number of Sca-1+c-kit+lineage- (SKL) cells in PB. In parallel, we evaluated activation of the ComC after administration of G-CSF and AMD3100 in experimental animals by employing C5a ELISA. To address the role of the CoaC in MBL-MASP-1- and MBL-MASP-2-induced mobilization, MBL-/- mice were treated with inhibitors of the CoaC (refludan) in some of the experiments. Results. We found that the MBL-MASP ComC activation pathway is involved in pharmacological G-CSF- and AMD3100-induced mobilization of HSPCs. As predicted, MBL-/- and MASP-1-/- mice were found to be poor mobilizers. Furthermore, inhibition of the CoaC by refludan inhibited mobilization in wild type animals but did not generate the additional defects seen in MBL-/- mice. Conclusions. We identified a previously unrecognized role for the MBL-MASP-1 pathway in triggering ComC and CoaC activation in the HSPC mobilization process. This finding explains the pivotal role of the MBL pathway in triggering activation of the proximal part of the ComC and explains why, even with a deficiency in activation of classical pathway components (C1q), mobilization of HSPCs proceeds normally as long as the MBL pathway is intact. Taking into consideration that ~10% of normal people are poor activators of the MBL pathway and that this percentage corresponds with the ~10% of the normal healthy population that are poor mobilizers, we are currently investigating whether MBL deficiency correlates with poor mobilization status in patients. MBL could be an important predictive parameter for identifying poor mobilizers. Disclosures No relevant conflicts of interest to declare.

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